Laser Flash Spectroscopy of Zinc/Ruthenium Myoglobins: An Investigation of Distance and Medium Effects on Photoinduced Long-Range Intraprotein Electron Transfers Citation Axup, Andrew William (1987) Laser Flash Spectroscopy of Zinc/Ruthenium Myoglobins: An Investigation of Distance and Medium Effects on Photoinduced Long-Range Intraprotein Electron Transfers. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/1a3m-hb31. https://resolver.caltech.edu/CaltechTHESIS:04052019-103258317 Abstract An experimental investigation of the distance dependence of long-range intramolecular electron transfer in ruthenium-modified zinc myoglobins has been performed. The zinc/ruthenium-modified metalloproteins were prepared by substitution of zinc-mesoporphyrin IX diacid (ZnP) into four previously characterized pentaammineruthenium(III) (a 5 Ru) derivatives of sperm whale myoglobin (Mb). The derivatives are a 5 Ru(His-48)Mb, a 5 Ru(His-12)Mb, a 5 Ru(His-116)Mb, and a 5 Ru(His-81)Mb. Pulsed laser excitation of the zinc myoglobin produces the long-lived and highly reducing triplet excited state ( 3 ZnP * ). Electron transfer from this triplet to the ruthenium, 3 znP * -Ru 3+ → ZnP + -Ru 2+ (ΔE° ≅ 0.8 V), was measured by time-resolved transient absorption techniques. The observed electron-transfer rates are 7.0 x 10 4 , 100, 89, and 85 s -1 for the His-48, -12, -116, and -81 derivatives, respectively, at 25°C The electron-transfer distances were evaluated using computer modelling in which rotation about the C α -C β bond of the imidazole side chain in the ruthenium-modified histidines is restricted by nonbonded repulsions with atoms at the protein surface. Recent crystallographic results for a 5 Ru(His-48)Mb indicate that the histidine has considerable rotational flexibility. The estimated accessible distances, both heme edge to inner coordination sphere ligand (e-e) and metal-to-metal (m-m), are as follows. For the His-48 derivative, the e-e range is 13.4-16.6 Å and the m-m range is 18.6-24.1 Å; His-12 ranges are 22.1-22.4 Å and 28.8-30.4 Å; His-116 ranges are 18.9-20.4 Å and 23.1-27.8 Å; and His-81 ranges are 19.0-19.4 Å and 26.3-26.9 Å. In addition, the orientation angles (θ) of the electron-transfer pathways with relation to the heme plane at a position of closest approach are 25° (His-48), 20° (His-12), 35° (His-116), and 25° (His-81). Fitting the rate data to an exponential distance dependence yields the expression k et = 8 x 10 9 exp(-β(R-4)) s -1 , where β = 1.2 Å -1 and R - 4 ≥ 0 (R is the minimum e-e distance in Å). The electron-transfer rate in a 5 Ru(His-12)Mb(ZnP) is anomalously high (100 vs. 2 s -1 predicted by the rate-distance equation), thereby indicating that the 3 ZnP * -Ru 3+ electronic coupling may be enhanced by an intervening tryptophan residue that lies parallel-planar to the heme along the reaction pathway. Activation enthalpies calculated from the temperature dependences of the electron-transfer rates over the range 5-40°C are 1.7 ± 1.6 (His-48), 4.7 ± 0.9 (His-12), 5.4 ± 0.4 (His-116), and 5.6 ± 2.5 (His-81) kcal mol -1 . Dynamic flexibility of the protein region containing His-48 may reduce the activation enthalpy with respect to the other more rigidly located derivatives. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: Chemistry Degree Grantor: California Institute of Technology Division: Chemistry and Chemical Engineering Major Option: Chemistry Thesis Availability: Public (worldwide access) Research Advisor(s): Gray, Harry B. Thesis Committee: Bercaw, John E. (chair) Gray, Harry B. Marcus, Rudolph A. Chan, Sunney I. Defense Date: 19 November 1986 Funders: Funding Agency Grant Number Fannie and John Hertz Foundation UNSPECIFIED Record Number: CaltechTHESIS:04052019-103258317 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:04052019-103258317 DOI: 10.7907/1a3m-hb31 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 11439 Collection: CaltechTHESIS Deposited By: Mel Ray Deposited On: 10 Apr 2019 14:34 Last Modified: 16 Apr 2021 22:16 Thesis Files Preview PDF - Final Version See Usage Policy. 24MB Repository Staff Only: item control page

LASER FLASH SPECTROSCOPY OF ZINC/RUTHENIUM MYOGLOBINS: AN INVESTIGATION OF DISTANCE AND MEDIUM EFFECTS OF PHOTOINDUCED LONG-RANGE INTRAPROTEIN ELECTRON TRANSFERS Thesis by Andrew William Axup In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 1987 (Submitted 19 November 1986) -11- ACKNOWLEDGMENT: I would like to extend my sincerest assisted me through would like to thank opportunity to participate my graduate years. With my advisor, Harry Gray, in research his appreciation to especial for group. those who pleasure, allowing Open me I th e interaction and boundless enthusiasm are among the hallmarks of the ever-memorable Gray Group. I wish also to thank the Fannie and John Hertz Foundation for its generous financial support throughout my graduate career. Of the many daily contributions from the members of the bio and photo groups, I would like to particularly thank Mike Albin, Jenny Karas, and Charlie Lieber for their time and comments during the composition of technical proposals expertise of and this Tom thesis. Dunn, I by greatly whose value and admire the efforts, even the most serious equipment failure was soon rectified. Finally, I would like to thank my parents for be written. their unending faith my friends, family, and above all, that this thesis would indeed ever -iii- To Nannie -iv- ABSTRACT: An range experimental investigation intramolecular electron myoglobins has talloproteins were diacid been (ZnP) by four derivatives derivatives are a 5 Ru(His-48)Mb, 100, derivatives, techniques. 85 respectively, at evaluated Ca-Cf> bond histidines is protein surface. indicate that The whale excitation the by Recent the the m-m range 30.4 A; His-116 is ligand His-48 3 znP*- ruthenium, are s· 1 for the His-48, -12, -116, and -81 The electron-transfer distances in chain are which rotation in ruthenium-modified the repulsions about the with atoms for a 5 Ru(His-48)Mb results at the fie xi bili ty. has considerable distances, both (e-e) metal-to-metal (m-m), are as e-e 13.4-16.6 A and and derivative, 18.6-24.1 ranges the state rates crystallographic accessible For to excited electron-transfer non bonded histidine follows . triplet myoglobin observed side sphere zinc The imidazole coordination of triplet the estimated myoglobin reducing modelling the pentaammineruthe- and computer restricted IX a 5 Ru(His-116)Mb, using of zinc-mesoporphyrin = 0.8 V), was measured by time-resolved trans- and were me- a 5 Ru(His-12)Mb, this 89, zinc/ruthenium-modified The from transfer zinc (Mb). highly absorption 7.0xlO.c, sperm and Ru3+ -+ ZnP+-Ru 2 + (.0.£ 0 ient of long-lived Electron ruthenium-modified of dependence characterized laser (3ZnP'\ long- substitution Pulsed the of in The previously (a 5 Ru) produces distance transfer nium(III) a 5 Ru(His-8 I )Mb. the performed. prepared into of the rota tiona! heme range edge is inner to A; His-12 ranges are 22.1-22.4 A and 28 .818.9-20.4 A and 23.1-27.8 A; and His-81 -v- ranges are 19.0-19.4 angles (9) of plane at 12), 35° a A and the electron-transfer position of (His-116), exponential closest and 25° e-e minimum distance In 12)Mb(ZnP) is anomalously distance equation), coupling may be (His-81). reduce the (His- Fitting the rate the and that -4 R an ~ 0 (R is the rate in a 5 Ru(His- predicted by the 3 znP*-Ru 3+ the electronic calculated from the temperature dependences of rates over the range 5-40°C are 1.7 ± (His-48), ± 0.4 (His-116), 5.6 ± 2.5 flexibility of activation enthalpy rigidly located derivatives. the the reaction and protein with tryptophan rate- along 5.4 intervening to expression 2 s·l VS. data heme (His-12), Dynamic 20° the enthalpies 0.9 heme to tion ± the (His-48), A-1 indicating to 25° electron-transfer (100 an relation orientation are yields The high with the by parallel-planar electron-transfer thereby A). addition, enhanced lies may approach dependence distance In pathways exp(-,B(R-4)) s-1 , where ~ = 1.2 8x 10 9 4.7 A. 26.3-26.9 region respect residue pathway. 1.6 Activa- (His-81) containing to the that other the kcal His-48 more -viTABLE OF CONTENTS: Acknowledgment ii Abstract iv List of Figures viii List of Tables X Chapter I Introduction 1 Chapter II Experimental 10 Materials and Apparatus 11 Preparations and Purifications 13 Instrumentation and Methods 23 Chapter III Results and Discussion 28 Reconstitution 29 Emission Spectroscopy 29 Kinetics 31 Activation En thalpies 47 Distance Evaluation 59 Through-Bond 60 Through-Space 62 Orientation Angles 73 Medium Effects 73 Chapter IV Implications 77 -viiAnother a 5 Ru(His)protein(ZnP) System 78 [Zn, Fe] Hybrid Protein-Protein Complexes 79 Rigid Organic Linkers 86 Photosynthetic Reaction Center 90 Directions of Future Work 91 Appendix A Calculation of Model System Coordinates 95 Appendix B Kinetic Analysis 103 Appendix C Evaluation of Nonbonded Repulsions References and Notes 106 111 -viiiLIST OF FIGURES: 1. Structure of accessible porphyrin sperm whale histidines are edge to the myoglobin (a-carbon indicated. imidazole ring chain). Distances are 13 Surface- from A (His-48), (His-81), 20 A (His-116), and 22 A (His-12). 2. Structure of porphyrin IX. Absorption spectra Differing of groups are indica ted mesoporphyrin IX (top) Spectrum of and for zinc-mes- Mb(ZnP)(aq) (bottom) is included for comparison. Computer-genera ted A 18 oporphyrin IX (center) in DMF. 4. 19 7 proto and meso forms . 3. the structure 20 of a 5 R u(His) from His-48 crystallo- graphic data and calculated a 5 Ru coordinates. 27 5. Transient absorption data for Mb( 3ZnP\ 35 6. Analysis (fit and residuals) of Mb( 3 ZnP*) data (monophasic first- order nonzero endpoint). 36 7. Transient absorption data for a 5 Ru(His-48)Mb( 3ZnP\ 40 8. Analysis (fit and residuals) of a 5 Ru(His-48)Mb( 3ZnP*) data (monophasic first-order nonzero endpoint). 41 9. Transient absorption data for a 5 Ru(His-81)Mb( 3ZnP\ 43 10. Analysis (fit and residuals) of a 5Ru(His-8l)Mb( 3 ZnP•) 44 (monophasic first-order nonzero endpoint). 11. Analysis (fit and residuals) of data a 5 Ru(His-8l)Mb( 3ZnP*) data (biphasic first-order zero endpoint). 45 12. Plot of ln(kd/T) versus 1/T for Mb( 3ZnP\ 50 13. Plot of In(kr/T) versus 1/T for a 5 Ru(His-48)Mb( 3ZnP\ 52 -ix14. Plot of ln(kf/T) versus 1/T for a 5 Ru(His-81)Mb( 3ZnP\ 54 15. Plot of ln(kf/T) versus 1/T for a 5 Ru(His-116)Mb( 3ZnP\ 56 16. Plot of ln(kf/T) versus 1/T for a 5 Ru(His-12)Mb( 3ZnP\ 58 17. Plot of were ln(kf) versus determined Metal-to-metal distance a 5 Ru(His)Mb( 3 ZnP\ for from the native protein separations are designated by Distances crystal triangles structure. and edge- to-edge distances by diamonds. 6 5 18. Plot of ln(kf) versus distance a 5Ru(His)Mb( 3 ZnP\ for Distances were determined from optimized rotations of the Ccx - Cf> bond in the labelled residues. Metal-to-metal separations are designated 69 by triangles and edge-to-edge distances by diamonds. 19. Analysis (k k0 = exp(-f>(R - R 0 ))) of ln(kf) versus distance Rate-distance data for His-48, 116, and 81 are included. 20. Intervening residues on through-space pathways 72 between ruthenium- 75 labelling sites and porphyrin in myoglobin. 21. Plot of ln(kf) versus distance for four a 5Ru(His)protein(ZnP) Structures naphthyl A), c) ex81 periments. 22 . data. of (R donor-spacer-acceptor = 10 A), b) systems, distances are indicated. (R biphenyl-steroid- naphthalene-bridge-cyanoethylene dimethylaniline-phenyl-anthracene porphyrin-linker-quinone a) = 10 A). (R = 7.5 Edge-to-edge A), (R = 9 and d) donor-acceptor 89 -xLIST OF TABLES: 1. Comparison of absorption data for metMb, apoMb, and Mb(ZnP). 30 2. Rate constants for Mb(ZnP) and a 5Ru(His-48)Mb(ZnP). 37 3. Rate constants for a 5Ru(His)Mb(ZnP), His-81, 116, 12. 46 4. Eyring plot data for a 5Ru(His)Mb(ZnP), His-81, 116, 12. 48 5. Through-bond distances from the axial histidine to the ruthenium modification sites in sperm whale myoglobin. 6. Intersite and closest ligand distances 61 evaluated from the modi- fied native myoglobin crystal structure. 7. Distance ranges evaluated from 63 nonbonded repulsions at 67 protein surface. 8. Observed and the calculated rate data for Zn / Fe-hybrid protein- protein complexes. 85 C-1. Selected covalent and van der Waals radii of atoms. 108 C-2. Nonbonded repulsion der Waals radii. distances calculated from covalent and van 109 -1- CHAPTER I Introduction -2- Electron transfers cally significant Many of contain the important processes these several are such protein-bound steps in biologi- respiration. 1 - 4 photosynthesis and are catalyzed by metalloenzymes that metal centers. The metal centers may are separ- electronic inter- reactions as mechanistic function as electron-transfer sites and/or substrate-binding loci. ated In these multisite proteins, the metal centers by long distances (>10 A). 6 At such distances, be weak and electron-transfer these long distances, observed, notably actions are expected correspondingly tron-transfer to slow. rates At have been often rates however, in the should rapid be elec- photosynthetic reaction centers. 6 These somewhat unexpected results, as well as the fundamental importance of stimulated ate electron-transfer both the theoretical factors and govern thermodynamic driving reorganization, the force spatial in biological experimental of the the orientation processes, investigations electron-transfer between distance the include that reactions kinetics. have to elucid- These factors electron-transfer sites, the sphere overall reaction, the inner of donor and acceptor, the and the nature of the intervening medium. Several experimental approaches elucidate tron the contributions of transfer. Early work involved between were various observed solution. rates until donors by donor Increasing a and plateau these have been taken parameters to the study of acceptors. 7 fluorescence The in an the attempt to rates of elec- bimolecular quenching electron-transfer quenching by the driving force produced was reached. The plateau acceptors increased kinetics in fluid quenching was attributed to dif- -3fusion limiting control the effects reactions in fer randomly factors in of the bimolecular diffusion a rigid distributed at involved matrix. 8 •9 fixed processes. studying The donors distances, Attempts the electron-trans- and requiring to acceptors rather were elaborate calculations to determine the electron transfer rates. Intermolecular talloproteins have periments, a the agreed been complex to reasonably the well site-to-surface distance con tact was point description studied. mechanistic of From the distance model that protein redox with complexes rates was in determined by featured close site. 1 0 • 1 1 estimates of ex- utilizing of the results metalloprotein redox models, the actual information, a reliable the molecular known. Without this orientation, these approach Though me- the from distance, with observed evaluated not the inorganic of electron-transfer bimolecular in organic reactions and intervening medium associated with the reaction pathway is lacking. In systems tion order to and allow of factors molecular linked of to the greater that systems disadvantages control influence in electron sought. porphyrin-quinone system established investigations ing .I 2 Similar between two redox and work a has involved cen ters. 1 3 • 1 • definitive of In Initial the the the transfer, been in of governing have structure nonrigid, circumvent intermolecular selective varia- well-defined uni- studies utility involving of this intramolecular fluorescence a chain polypeptide as class quencha bridge both cases, the linking frame determination of separation a distance was and orientation was not obtained. Numerous fixed-site systems have recently been synthesized. -4- Rigid linkers have systems. 1 5 - 1 9 A in many of these that a network been in corpora ted several proposed has indicated facilitates electron transfer investigations have provided the through- bond and through-space pathway lengths are nearly the same. In many biological processes, an insight into fer systems electron-transfer donor-acceptor was through through-bond in and recent of carbon-carbon aliphatic bridge. 2 0 the mechanisms a-bonds These in systems long-range appears to occur derstanding of the protein medium intraprotein electron transfer during theoretical operating however, through a mechanism where intraprotein noncovalently and work linked the electron trans- medium. mechanisms requires the been employed An that utilization un- operate of me- obtain two- talloproteins in further studies. Several site successful meta lloprotein Knowledge strategies with systems fixed crystallographic of the characterization of the ning One approach medium. have distances and orientations. structure of protein allows distance, orientation, in tersi te has involved physiologically matched complex. 2 1 - 2 4 This method generate docked complex's protein-protein structure is the long-range intraprotein the study of two-site systems in specific location on proteins which a to to form the the a and association multisite the interveof two protein-protein may require computer-graphics simulation not a three-dimensional redox single-site if the alternative approach to electron transfer centered on active species known. An structure to metalloprotein. has is attached The latter to a strat- egy is the focus of this work. The selection of the inorganic redox agent is based on two -scriteria. First, protein surface. inert in the the And relevant eaquoruthenium(II) histidine residue modified derivatives aeruginosa azurin. 2 7 -s 0 mined tryptic the by resulting with employed of horse The site of fragments. in these of native proteins allowed orientations. In been successfully ruthenium only the important kinetic data, but as the modification site, they are unsuitable employed m in ruthenium- and Pseudomonas subsequent analysis characterized proteins the site for of was structures intersite medium systems contain deter- computer-modelled Crystallographic intervening These con- was of a protein histidine evaluation of attachment with one proteins. addition, and and agreement the c chain on transfer cytochrome the pentaammin- side has a mined . ible well-defined of studies protein two reaction imidazole at substitutionally earlier In Ia belled The electron the predictions, and be in of accessibility tances must heart surface the complex the intraprotein of specificity states. digestion each some oxidation methodology peptide with the been This react second, long-range of must (a 5 Ru(H 2 0)) has formation. 2 5 • 2 6 studies complex was have only ex- provided one studying dis- access- distance and orientation effects. By histidines, more For reason, this modification. than As meta lloprotein a selecting one sperm for ruthenium labelling. been prepared and molecular rate-distance whale indicated with relation myoglobin tn Figure The four I, electron -transfer rate could be determined . was chosen for ruthenium four histidines singly-modified characterized. 3 1 •3 2 between In surface-accessible multiple previous the are available derivatives studies, ruthenium the label have intra- at the -6- Figure 1. Structure of sperm Surface-accessible the porphyrin whale histidines edge to the myoglobin are (a-carbon indicated. imidazole ring 19 A (His-81), 20 A (His-116), and 22 A (His-12). Distances are 13 chain). from A (His-48), -8histidine-48 residue and the heme perimental determination of the at the overall three longer reaction, Fe 2 + distances -+ Ru 3+, been determined. 3 2 ,s 8 Ex- intramolecular electron-transfer rates iron has has not been possible because the is very slow at low driving force (ll.E 0 = 0.02 V). In this work, the "ruthenated" myoglobin driving so order to increase the rates to all four ruthenium-labelled The excited triplet and has V). an electron Substitution of that The can reduce porphyrin the cation was the modified m electron-transfer could be determined. is a donor m long-range electron- zinc porphyrin for the myoglobins systems. (multidistance) triplet 0.8 as that histidines ( 3 ZnP•) porphyrin ruthenium-modified the In multisite lived utilized studies. 2 1 - 2 4 transfer heme been zinc force system good reducing produces the agent the desired Photoexcita tion generates a long- histidine-bound ruthenium(III) (ll.E 0 radical (ZnP+) then can oxidize the ruthenium, returning the system to its initial state (Scheme I). Scheme 1. Utilizing transfer transient these rates myoglobins, zinc/ru theni um-modif ied over absorption the distance methods. range Changes 22 13 in A other were the electron- measured factors, such by as -9driving force and inner sphere reorganization, are minimized by utilizing the same donor ( 3ZnP•) and acceptor (a 5 Ru) in the four dif- ferent derivatives. Having obtained myoglobins, an kinetic accurate to complete the the surface regions ate the data the zinc/ruthenium-modified of the site separation relation. A computer-graphics assessment rate-distance degree for of each modified histidine was was image examined of to evalu- of rotational flexibility accessible the histidine residues. Nonbonded repulsions of the ruthenium-modified imidazole side with protein surface limit the chain residue. From estimated. The the these interactions, intervening a medium rotational the description tion of data can the electron-transfer change in parameter can within well-defined variations. be correlated can be on a this pathway with various now distance electron-transfer each the each the of Based of of approach plane. porphyrin range minimum was examined as well as the orientation of each to to needed information, respect a detailed and an evalua- presented. The kinetic of distance parameters be pathway system -10- CHAPTER II Experimental -11- Materials and Apparatus: Distilled purification used in water, system the filtered (No. through a specific resistance 2794, preparation of (Mallinckrodt), nitric (Baker), sulfuric acid (Mallinckrodt) solutions were prepared hydroxide (Baker) acid and all aqueous were from Barnstead 18 was Hydrochloric acid (Baker, 60%), and Aqueous base perchloric acid used as received. grade sodium bicarbonate (Baker). and potassium Sodium chloride (Baker) and sodium dithionite (MCB) were used as obtained. (MCB) ates) was at 4°C solvents, (U.S. stored to over aluminum prevent the Industrials, absolute), (Baker, absolute), methanol (Woelm accumulation (Baker), chloroform oxide of acetate Waters Associ- peroxides. Other organic (MCB), ethanol and Jackson), (Burdick (Mallinckrodt), pyridine 2-Butanone neutral, N,N-dimethylformamide ethyl water MQ-cm), > solutions. reagent sodium Nanopure and toluene were prepared (Burdick and Jackson), were used as received. Tris(hydroxymethyl)aminomethane buffer solutions from reagent grade Trizma hydrochloride and base (Sigma). M, pH 7.2 (25°C), the manufacturers Trizma base, 7.8 A J.1 = 0.05 (5°C) buffer solution was prepared according specification 4 L of water). (28.08 A J.l g = 0.1 of · Trizma HCI, 2.68 g to of M buffer solution was obtained by doubling the quantities of Trizma salts used as described above. Phosphate buffer solutions were prepared from reagent grade monoand dibasic sodium 7.0 (25°C) buffer phosphate solution salts was (Mallinckrodt). prepared by A general J.l = 0.1 reference M, pH specifi- cations (10.85 g of sodium phosphate monobasic (NaH 2 Po 4 •H 2 0), 15.20 g of sodium phosphate dibasic (Na 2 HP0 4 (anhydrous), 4 L of water). 8 4 A -12- JJ 0.01 = (1:9, pH M, 7.0 (25°C) buffer/water) a buffer = 1J 0.1 solution was pH M, 6.7 prepared solution by diluting (18.08 g of NaH 2 Po 4 •H 2 0, 12.73 g of Na 2 HP0 4 anhyd., 4 L of water). Carboxymethyl pre-swollen, microgranular), (six ufacturer cellulose aliquot settled resin volume slurry, poured into was buffer was a cation exchange equilibrated column, and by CM-52 indicated as changes). increased resin, Following 20% allowed with to (Whatman, by the roan- equilibration, the buffer to for several settle form a hours. Five column volumes of buffer were passed to pack the column prior to CM-52 resins were cleaned after use. solution bead (-1-3 size NaCI). M 20-80 Sephadex ion equilibrated was JJ) use by washing with a exchange in the high-salt gel, G-25-80 (Sigma, desired buffer, slurried and poured in a fashion similar to that described for CM-52. Sephadex gels were cleaned by multiple washings with buffer or water. Samples through line. a At were degassed purged manganese oxide least vacuum/purge five samples. Transfers were 20 stainless, non-coring ga. and column) done with purified argon a dual manifold vacuum-argon cycles were used to deoxygenate with a cannula on anaerobically tips). Other air sensitive (passed (Aldrich, manipulations were performed under argon in a Vacuum Atmospheres Co. HE-43-2 Dri Lab inert atmosphere box. Concentration of protein of small molecules were achieved with an (YM-5 filter, 5000 molecular weight cutoff). solutions Amicon and removal ultrafiltration system A Gilson Minipuls 2 per- istaltic pump with 0.32 mL/m tubing was used to regulate buffer flow through some chromatographic flow rate of about 30 mL/h. columns. A setting of 400 provides a -13Sample washings and with spectrophotometric water. Periodically, hydrochloric acid/nitric acid, and then rinsed cells cells followed with water until were were by cleaned washed by with concentrated constant pH multiple 3:1 (v jv) sulfuric acid, was reached. Cells were either air or vacuum dried . Preparations and Purifications: General Materials: perch lora te 8 6 The chloride 8 6 and nan throline)cobal t(III), Co(phen)~ +, methods. solution saturated A salts were tris-(1, I 0-phe- prepared by was used to synthesized by by amalgam. 8 7 Co(phen)~ + of of literature oxidize the reduction of protein samples. Pentaammineaquoruthenium(II) (Strem) chi oropen taa mmi neru theni u m(III) amalgam was aqueous mercuric precipitated as prepared from chloride the was zinc metal zinc (Mallinckrodt) (Mallinckrodt) hexafluorophosphate The solution. salt and and a Zinc saturated product stored under was vacuum dessication or used directly in the labelling reaction with protein. Purification of sperm whale skeletal muscle ferrimyoglobin: In 20 mL of Tris (~ dissolved. ferrimyoglobin, metMb, were centrifuged to separate the applied a to CM-52 = 0.05 buffer column insoluble (4 em x M, of Sigma protein solution was material. The supernatant was 80 equilibrated The em) pH 7.2), 6 g with Tris -14- buffer (IJ ,.. 0.05 M, pH 7.2) at 4°C. with Tris typically buffer -= (IJ observed, and The sample was eluted at 50 mL/h 0.05 M, pH 7.2). Band IV (the richest Four in separable metMb) bands was were used for these studies. Preparation of pentaammineruthenium(III)ferrimyoglobin:8 8 A solution containing 600 mg (25 mL of 1.3 mM metMb) of ferrimyoglobin in Tris buffer (IJ = 0.05 M, pH 7.2) was degassed and purged with argon in a septum-stoppered the protein solution the protein. 130 afluorophosphate when mg were of 120 mL bottle. degassing was degassed pentaammineaquoruthenium(II) dissolved in avoided Excessive foaming because 15 mL of argon-purged pentaammineaquoruthenium(II) was transferred it of denatures Tris hexbuffer (IJ = 0.05 M, pH 7.2). The the ferrimyoglobin reduced to the ferro- brown to deep red. room solution. temperature The ferrimyoglobin and the species solution under was changed argon to immediately color from After the mixture had reacted without agitation at for thirty minutes, the reaction was quenched by elution on a Sephadex G-25-80 column (phosphate buffer, 1-1 = 0.1 M, pH 7.0). The protein fraction was collected, oxidized with Co(phen)~ +, and stored at 4°C. Purification of pentaammineruthenium(III)ferrimyoglobin: 8 2 ,s 8 The mixture of ruthenated myoglobins was desalted by five cycles of Amicon concentration and dilution with water, and then reduced to a final volume of 6 mL (600 mg, 5.6 mM modified metMb). The modified -15myoglobins (2117 were separated Multiphor, 2197 by isoelectric Electrofocusing focusing. Power Supply) gels were prepared by standard procedures, 3 8 An LKB system was used. Two except that the ampholine solutions were passed through an Amicon YM-5 filter to remove any high molecular of weight ampholytes. the removal the protein To each gel, 3 mL of protein solution were applied, and the gels run 10 W). observing the the remaining low This molecular procedure weight facilitated ampholytes from solution following focusing. were Progress at of 4°C the separation of tion of native, the gels were scooped from disposable the (power supply settings: 1500 focusing was monitored visually modified myoglobin singly-modified stopped. The and band bands. The ampholytes 22 by When of were rnA, sufficient doubly-modified the trays and eluted from frit. V, bands singly-modified resolu- occurred, derivatives was the gel with water through a removed from the collected protein solution by Amicon ultrafiltration. The mixture of singly ru thena ted myoglobin derivatives was con- = 0.1 M centrated and loaded onto a CM-52 column (equilibrated with IJ Tris buffer, pH was eluted 7.2, 4 ern x 70 ern) at 4°C. with Tris buffer at 50 mL/h . Four bands were collected and each was concentrated and at short-term storage or frozen 4°C for rechrornatographed, Tris buffer. Co(phen)~+ for storage. The order of elution was His-116, His-81, and His-48.3 1 •3 2 The protein if added to from necessary. Fractions . were kept at -60°C (0.1 mM protein) all protein modified the CM-52 column in solutions was His-12, -16Preparation of zinc-mesoporphyrin IX diacid: The (Figure porphyrin 2). 250 dimethyl mg of ester was mesoporphyrin first IX saponified dimethyl to ester the diacid (Sigma) were dissolved in 5 mL of pyridine in a 50 mL three-neck flask. was wrapped in foil and placed under an argon flow. The flask 20 mL of 1% potassium hydroxide in methanol (0.29 g KOH (89%) in 25 mL MeOH) were added with refluxed for tate the 3 mL of water. The two hours. 6 N hydrochloric porphyrin diacid. The reaction mixture acid reaction was heated was added mixture to and precipi- was refrigerated literature methods. 3 9 overnight. The sample was centrifuged and decanted. Zinc(II) was inserted into the free base by The diacid was dissolved in 15 mL of N,N-dimethylformamide and to reflux. minutes, base 0.5 an zinc chloride (Mallinckrodt) absorption spectrum was remained, g an aliquot from a was heated added. After 15 any free taken (Figure 3). If saturated solution of zinc chloride in dimethylformamide was added. The remove precipitated all traces of dried under vacuum. solid was pyridine. If the the porphyrin in hot The with water several zinc-mesoporphyrin IX times diacid to was porphyrin did not dry completely, residual pyridine was the likely cause. ing washed The pyridine was removed N,N-dimethylformamide soluble material on a medium frit. The solution and by redissolv- filtering the in- was cooled, ice water was added, and the sample was stored overnight at -20°C. Evaluation of the completeness of the reaction and purity of the product was done by thin layer chromatography (EM Reagents, silica gel 60 F 2 6 • ). Samples were spotted from chloroform solution and an -17- Figure 2. Structure of porphyrin for proto and meso forms. IX . Differing groups are indicated -18- R R ' '' '' , . M ... '' Proto: R = Meso: R "" , '' C2 H3 = C2 Hs M (metal): Fe, Zn -19- Figure 3. Absorption spectra of mesoporphyrin oporphyrin IX (center) in DMF. included for comparison. IX (top) and zinc-mes- Spectrum of Mb(ZnP)(aq) is -20- UJ u z<( co 0::: 0 (/) co c::::( 500 600 WAVELENGTH (NM) 700 -21(vjvjv) 85:13.5:1.5 the developer." 0 used as by toluene/ethyl acetate/methanol sample luminescence Progress of the under UV solvent system chromatography light. The was was followed zinc-mesoporphyrin IX diacid was stored in a foil-wrapped vial at -60°C. Preparation of apomyoglobin: The heme was removed from the myoglobin by the acidic 2-butanone method." 1 •4 2 ex traction bath. All solutions were 20 mg of salt-free myoglobin (10 acidified to pH 2.9 with added in equal volume to twice to assure adequate mixing. minutes to accelerate layers. The organic was repeated apoprotein dialysis N the protein was (Spectrum weight separation was icejwa ter mM metMb) were acid. solution, of removed On removed Medical cutoff, hydrochloric an 2-Butanone and gently the and organic the was inverted The sample was centrifuged two or three times. solution bag molecular layer in mL of 0.11 0.1 the cooled for five and aqueous extraction procedure the final ex traction, the aqueous and transferred Industries, 20.4 3.2 mL/cm) that had twice dialyzed against to a Spectrapor mm diameter, 6-8000 been previously cleaned by standard methods." s The sample was L of 10 mM sodium bicarbonate (3.36 g NaHC0 3 in 4 L H 20) followed by three times against 1 L of ued IJ for six 0.1 M phosphate buffer, pH 7.0. to twelve hours. The concentration of the peak porphyrin. at 424 Typically, nm indicated heme the removal in apoprotein 44 15.8 mM" 1 cm" 1 incomplete removal of the excess 99% was determined from the absorption spectrum (£ 2 8 0 minor Each dialysis was contin- = of ). was A iron achieved . -22- Apoprotein prepared in this fashion was immediately used for recon- stitution with the zinc mesoporphyrin. Preparation of reconstituted myoglobin:• 6 The apoprotein solution was maintained at 4°C or ice bath temperatures for of zinc the duration mesoporphyrin of the IX diacid NaOH(aq) in a 5 mL beaker. 7.0) were added insertion were process. dissolved to the porphyrin solution. dropwise to the apoprotein with the dark at stirred was then in made. turbed overnight. After in 10 drops 3 mg of 0.1 N 2 mL of phosphate buffer (IJ = 0.1 M, pH added was Approximately 4°C for twelve more The gentle twelve hours, resulting solution swirling. The mixture second insertion hours. A the solution was left was undis- The sample was centrifuged for one to two hours and the supernatant was decanted and saved for purification. Purification of reconstituted myoglobins: The a zinc-mesoporphyrin Sephadex 0.01 M, pH band was G-25-80 IX column, reconstituted equilibrated myoglobin with was phosphate buffer 7.0), and eluted with the same buffer at 4°C. collected and concentrated by Amicon applied to (1-l The protein ultrafiltration for loading on the phosphate (IJ .., 0.01 M) equilibrated CM-52 column (3 em x 22 em). elute An ionic strength gradient, 1-1 "" 0.01 the sample at 30 mL/h. A two-flask to 0.1 M, was used to reservoir with 1 L of 1-1 0.01 M and 1 L of 1-1 -= 0.1 M phosphate buffer was used to generate the gradient. elute, and Unmodified 72 84 zinc h myoglobin were typically required for required the 30 ruthenated - 36 h to derivatives. -23Additional 0.1 1-1 Zinc-substituted M buffer myoglobins was needed to complete were refrigerated in recorded with an emission 0.1 M, phosphate some elutions. foil-wrapped vials and used as quickly as possible. Instrumentation and Methods: Emission spectra were described previously." 6 A4 1 4 1.0) were degassed in a vacuum cell with a - cuvette side-arm. Samples (IJ Spectra were = recorded at spectrophotometer buffer pH 7.0, em fluorescence ambient temperature. Ex- citation of the sample was by a 200 W HgXe lamp (Oriel) with a Oriel model 6240 Arc Lamp Power Supply at 404 nm (interference filter). Ex- citation 1.0 was cut off at 446 nm and the monochromator slits were mm. Emission lifetime measurements of the Mb(ZnP)'s were made with a pulsed laser system described previously. 8 0 Excitation was at 532 nm (50 mJ / pulse at 10 Hz with 63 J/pulse lamps) with a repetition rate of Hz. filter Fluorescence at 583 and emissions 635 nm were with monitored through a monochromator slits of Corning 3-67 0.4 The mm. output signal from a Hamamatsu model R 955 photomultiplier tube was amplified by amplifier. 200 a LeCroy model VVJOIATB pulses were collected and PDPII / 03-L computer. Data were analyzed high-speed averaged on wideband pulse Digital model a by executing a least squares first-order fitting routine. Transient absorption employed the same lifetime instrumentation lifetime excitation except and measurements detection where noted. of apparatus Samples the as (IJ Mb(ZnP)'s the = emission 0.1 M, -24phosphate buffer pH 7.0, A 4 1 4 with a em fluorescence 1.25) were degassed in a vacuum cell = cuvette side-arm. A 500 W continuous wave tungsten lamp with an Infrared Industries Model 518 Lamp Power Supply served as the probe beam source. The beam was collimated (f em, diam. = 7.5 em) in the lamp housing and focused 15.2 diam. = em, 10.0 0-51, 5-57, and 5-58. passed through em) through a series of = 17.7 by a lens (f Corning filters, = 0-52, The beam was cropped by a 0.8 em aperature and the sample cell. The beam was refocused (f = 7.0 em, diam. = 3.8 em) onto the slit of the monochromator (0.8 mm) through a A Tektronix FET probe amplifier with a 5 kQ res- Corning 5-57 filter. istor was used for all transients system, in which case the LeCroy 6500 digital stituted quired waveform recorder derivatives except 100 timebase. a ns / pt except the amplifier was was the set at least order fits squares pulses were taken with zero and Both The ~s/pt modification. 40 routines. used. His-48 data were averaged on the PDP computer. linear 100 a 5 Ru(His-48)Mb(ZnP) for Biomation all The at zinc-sublatter re- Hz and the Data were analyzed with non- monophasic and biphasic nonzero endpoints were made. first- Residuals were evaluated. Computer transfer studies modelling distances in crystallographic data Brookhaven database.• 8 •4 9 coordinates described crystallographic data relative in Ruthenium to Appendix the A. for were were ruthenium data assumptions from using the coordinates were hexaammine. 6 0 The were as electron- performed obtained pentaammine Brookhaven Three intra protein the myoglobins Protein from evaluate ruthenated the PCMODEL.'' 7 evaluated to to calculated the as ruthenium's -25spatial relation to the thenium was attached (Figure the 4). Second, imidazole imidazole ring to N £' the distal that ring. the And ruthenium third, reflex angle defined by C 0 -N£-C£. ually rotating the The allowable rotation was with atoms the protein at were that First, that the ru- nitrogen in the imidazole ring lies in the the N £ -Ru plane defined by vector bisects the The distance was evaluated by man- imidazole side chain limited made. by surface. residue about nonbonded These distances estimated from van der Waals contact radii. the repulsive interactions Ca-Cf> bond. interactions occurred at -26- Figure 4. Computer-generated structure of a 5 Ru(His) from tallographic data and calculated a 5 Ru coordinates. His-48 crys- -30- Table l. Comparison of absorption data for metMb, apoMb, and Mb(ZnP). uv Protein metMb 280 31.2 apoMb 280 15.8 Mb(ZnP) 280 15.8 a. A (nm) b. E (m~ c. this work 1 complex a So ret 409 157 505 ref 9.4 7 635 3.55 54,55 44 414 250 541 11.7 583 6.1 c cm- 1 ) absorption bands Aa Eb A E ref a 5 R u(His) 3+ 303 2.1 450 0.29 56 a 5R u(His) 2+ 260 3.26 280 3.16 56 -31symmetrical band at 586 nm and a weaker system composed of two broad peaks at 635 wavelength and range 643 nm. 550 to reports of fluorescence No nm. 6 7 800 emissions were observed These data accord with Mb(ZnP) 6 8 from hemoglobins.• 6 zinc-substituted other and Singlet the emission emission in the literature properties of lifetimes were not resolved with the nanosecond laser system (8 ns FWHM pulse), implying an upper limit of 10 ns for these lifetimes. Singlet other zinc porphyrins in aqueous solution have been the order of m yo globin at 3 ns. 6 9 Zinc temperature 6 0 low lifetimes reported to be on triplet emission has and metalloproteins, 6 1 in other of been observed in but room temperature solutions of Mb(ZnP) do not display phosphorescence. Kinetics: Transient flash quenching tron -transfer kinetic kinetic methods have been used analysis of a Ru(His)Mb(ZnP) 5 the system in zincjru theni um-modif ied mechanism is to study the elec- proposed presented in myoglobins. A for the in Scheme Appendix B. The electron- transfer rates were determined by observing the quenching of the 3 znP* decay. The first-order forward, rate expression components, kf, one electron-transfer derived containing rate (Equation the constants I) consists of two intrinsic decay, kd, and and other containing the the reverse rate, kb, where A 0 = [a Ru(His)Mb( 3ZnP.)] at t = 0 s and K 1 5 and K 2 are preex- -32ponentials as given in Appendix B. In addition to electron-transfer quenching, as shown in Scheme 1, the possibility of 3 znP• deactivation by Forster energy transfer was considered. The rate of Forster or dipole-dipole energy transfer can be calculated using the equation 6 2 (2) In the case of a 5 Ru(His)Mb(ZnP), the most important term in the equation is the overlap integral, fF (A.)t:A(A.)A. 4 dA.. This integral repres0 ents the extent absorption spin between the 3 znP• emission and mechanism of energy transfer populates overlap spectra. lowest-lying while of This ruthenium allowed, energy involves level, two The spin forbidden emission and Forster energy transfer the quenching should Other rate. dipole-quadrupole 2 Ru the interaction, transitions, 3 znP* and Overlap between the absorption is negligible, not a significant make energy transfer the overall 1znP (14300 cm- 1 ) 60 and 2 Ru-+ 4 Ru (16700 cm- 1 ). 68 3znP * ruthenium and contribution mechanisms, electric dipole/magnetic dipole, therefore for are to example expected to contribute even less than the dipole-dipole interaction. 6 4 Electron-transfer to be the system. 6 5 studies quenching, dominant deactivation Preliminary the conclusion is by electron stituted cytochrome c (horse transfer accounts for Jess described pathway that heart) than for Scheme the dominant the transfer; in bimolecular from results support quenching as and 0.01% assumed a 5 Ru(His)Mb( 3 ZnP *) transfer electron of between a Ru 3+ 6 indicates of total the is mechanism reaction the 1, that triplet triplet zinc-subenergy quench- -33ing.6 6 a Under samples (<I o/o) to pulsed laser quite stable. are even about after 500 of 10% purity samples. 6 7 impurities lD modified restored absorption pulses. transient conditions, A slow optical The minor component similar investigations c. 6 6 b source, contrast, remain unchanged reversible decay amounting the sample using a has was by observed. using been attributed to high protein of zinc-su bsti tu ted, ruthenium- the initial density optical pulse (8 ns FWHM, broad undergoes change was minimized Regardless, when a 5 Ru(His)Mb(ZnP) spectra within one second of the excitation In the density this slow component cytochrome repetition). tion The laser the The contribution of experimental band significant is I Hz microsecond excita- decomposition (-50%) within five flashes. The native Mb(ZnP) endpoint expression The rate constant the intrinsic at 25°C (Table 2) indicating for data a monophasic least four half -lives by this fit, 40 rate, kd. All rate The decay rate ± at determined triplet decay fit the triplet 4 2 s- 1 , ± 6). corresponds to constants Mb( 3ZnP*) no temperature from 38 ± s- 1 (7.6°C) to that within experimental error, there over a concentration Values of kd determined s· 1 , respectively, zinc excited range of that occurs. In no bimolecular addition, when quenching of Mb(ZnP) was reduced with sodium dithionite, 6 8 kd remains unchanged (39 demonstrating and/or that oxidized protein 3 2.37, b = 0.1 em) are 41 ± 2 s- 1 and "" implying state is reported of ranges 42 5 are indicated. 5 nonzero and otherwise 1-1M (A 4 1 4 == 0.75) to 90 1-1M (A 4 14 ± (Figures unless dependence. 46 first-order porphyrin :!: impurites 3 s· 1 ), do not -34- Figure 5. Transient absorption data for Mb( 3ZnP•). Figure 6. Analysis (fit and residuals) first-order nonzero endpoint). of Mb( 3ZnP•) data (monophasic -35- - 0 C> <j I 0.0 TIME <xlO -3 s ) -36- 4 (/') _I <t :::> Cl (/') 0 w 0::: ~ -4 0 Cl <I TIME <xlD- 3 s) -37- Table 2. Rate constants for Mb(ZnP) and a 5 Ru(His-48)Mb(ZnP). Temp. oc Mb(ZnP) kobs(=kd)x 1o-1 a 5 R u(His-48)Mb(ZnP) kobs(=kr)x1o-" (± 0.1) (s- 1 ) ln(kd/T) (s - 1 ) ln(kr /T) 7.6 3.8± 0.5 -2.00± 0.13 5.4± 0.9 5.26± 0.16 11.8 3.9± 0.4 -1.99±0.11 6.2± 0.8 5.38±0.13 16.2 3.9± 0.3 -2.00± 0.08 6.2:!: 0.6 5.37± 0.10 20.6 4.0± 0.3 -1 .99± 0.07 6.3± 1.3 5.37± 0.18 25.0 4.0± 0.2 -2.01 ± 0.05 7.0± 0.8 5.46±0.10 29.4 4.1± 0.2 -2.00± 0.05 7.7± 1.4 5.54± 0.19 34.0 4.2± 0.2 -1.99± 0.04 8.0± 1.0 5.56± 0.12 38.6 4.2± 0.1 -2.00± 0.03 8.1± 1.4 5.56± 0.19 -38significantly quench 3znP•. The a 5 Ru(His-48)Mb(ZnP) half -lives (Figures x 10 4 s- 1 was obtained. Sub- rate (40 ± 2 s- 1 ) does not this rate constant traction of the intrinsic decay result, hence least affect The exhibits the a slight decay rate, dependence (Table a 5 Ru(His-48)Mb(ZnP) was of the ± s- 1 , is similar to a monophasic 5 2), ranging also a 5 Ru(His) 3+ reduction 50 7 rate temperature photoexcited dithionite observed four 0.8 A following first-order ± 8). of monophasic 7.0 and decay a fit at endpoint of also for nonzero constant expression data measured acceptor. The the intrinsic native first order nonzero data indicate severe and 10). These data zero endpoint ex- to decay rate, kd. The residuals corresponding endpoint fit of deviations from monophasic can be the a 5 R u(His-81 )Mb(ZnP) behavior satisfactorily fit pression (Figure II). Nine m analysis. The observed the of 86 :t I2 s- 1 . transient absorption half -lives. Similar 3 s- 1 89 ± I2 derivatives a biphasic half-lives of first the I26 ± rate, 9 order fast component 12 s- 1 , and was observed analyses of the a 5 Ru(His-II6)Mb(ZnP) data IOI exhibit yielded ± II moderate electron-transfer s- 1 • respectively. temperature for less rate two and kf, His-81, II6, and dependences over the 81 )Mb(ZnP) no kobs· kf The Over the concentration range 3 - 15 in than constants, 5 - 40°C (Table 3). variation used IO% to the measurement and showed were corresponds to a The second component contributes less than a 5 Ru(His-I2)Mb(ZnP) of to (Figures Following ~M range a 5 Ru(His- reduction of the -39- Figure 7. Transient absorption data for a 5 Ru(His-48)Mb( 3ZnP\ Figure 8. Analysis (fit and residuals) of a 5 Ru(His-48)Mb( 3ZnP*) (monophasic first-order nonzero endpoint). data -40- 0.15 - 0 0 <, I 0.0 TIME <xlO -6 s ) -41- 12 1/) w 0:::: N l::x -~ '· ,,....·''· ...... t.,.. '.,., 'I· ". ... " t y·· . ~ .l'.·. "v.. '" X. '../ 1 ...' ":\ .. ,. ..p,' . ),•,. .,.... ""l',, .. n•·1 ~· • ·1 ,, ·' "' " '!,.. X"' .. ,. ~ ,·I ' ' "· " •'\ ' ...... ' ,... '· . ..~,··.' .·.''" .,•t;i ' ... .. .. •' 'l ·· ~' ".. ,.,, ..., ., ... t·'· t <.:p,. ; ..-.:-:.( ' .·'!I)',I .J:' 1 ,"l,.n+".,'l• ··. tr.·•, ....' ",' 'J.·:-J.. : '' ,.,..,, j ))., . 0 't;,t;..:. .I'V\ ' 0 ...''!. .;·: ·• .•, ,. ,;• ·',, ··.•. · '/\. . .. . ,, . ,. ·x .,. · ··. ..'\- •-··:.-. ·:·_., .. . ~ "~' '... ~ • ,;~·.,, "'..I . !~..,..~ .'''. . ,..... ·,~· I .. ·0.."" ,..,1,...i· ,.1"":. \.'' ·x..I.,.• \,,,•..,.) ~ \. .'.., .,. .·\ ~'" . ,.. " ~ '· ·'t; ;,' \ '• ,\.. r..i' ,',Y.\. ~ ' "~' ~· ·~ .. ';l " ~· ~~ i'' \' .~ ~ (\ ): ~ ~~ I 't I 'I,' ' . L ' "' ,j_' -12 , . II,., • •: . ~ •:~ . .~ ~. ... I I \ . ,· ' ' .• . " I •• , ., '· ''1'1., AI ,, '· •' } ', A , , , X. \1 ' "/', 0.15 0 0 <: I 0 TIME (x10 -6 s ) x ·. ... '.' ' • v~· ._,)~ - ~-~·'· ' 11 ........ -~~ ~ ·~.~ ·J~ • .. ,', • l' -42- Figure 9. Transient absorption data for a 5 Ru(His-8l)Mb( 3ZnP\ Figure 10. Analysis (fit and residuals) of a 5 Ru(His-8l)Mb( 3ZnP*) data (monophasic first-order nonzero endpoint). Figure 11. Analysis (fit and residuals) of (biphasic first-order zero endpoint). a 5Ru(His-8l)Mb( 3 ZnP*) data -43- 0.20 . 0 0 <, I TIME <xlo- 3 s) -44- 4 C/) 0 w = ~ -4 0.20 \ "'I t::l C> 0.10 <I TIME <xl0- 3 s) -45- 4 (/) ...J c:t :::> 0 (/) 0 UJ 0:::: ~ -4 0 . 0 <I '.-· • ' . ~lit=.:A-~· .. ~,~ ·.!. .. 0 TIME Cxl0- 3 s> .. -46- Table 3. Rate constants for a 5 Ru(His)Mb(ZnP), His-81, 116, and 12. a 5 Ru(His)Mb(ZnP) Temp. His-81 oc kobs (:!: 0.1) a His-116 k b f kobs (s-1 ) (s·l) 7.6 7.4:!: 0.2 11.8 a His-12 a k b f kobs (s·l ) (s·l) (s·l ) (s·l) 3.6:!: 0.5 8.6:!: 0.6 4.8:!: 0.8 9.9! 0.7 6.1:!: 0.9 9.0:!: 1.3 5.1:!: 1.4 9.2:!: 0.4 5.4:!: 0.6 10.3:!: 0.3 6.4:!: 0.5 16.2 9.7! 0.8 5.8! 0.9 10.5! 0.2 6.6! 0.4 11.7:!: 0.3 7.8! 0.4 20.6 11.2! 1.0 7.3! 1.0 11.4! 1.2 7.5! 1.2 11.9! 1.6 8.0:!: 1.6 25.0 12.6! 1.2 8.6! 1.2 13.0! 0.2 8.9! 0.3 14.1! 1.1 10.1!1.1 29.4 13.8! 0.5 9.7:!: 0.5 14.4! 0.7 10.3! 0.7 15.7!1.9 11.6:!: 1.9 34.0 15.2! 2.2 11.0! 2.2 15.9±0.1 11.7! 0.2 17.4! 0.5 13.3! 0.5 38.6 17.2±2.1 13.0! 2.1 18.3:!: 2.2 14.1:!: 2.2 19.2:!: 1.7 15.0:!: 1. 7 a. k 0 b 5 x10" 1 b. krx1 o· 1 k b f -47- a 5 Ru(His-81)Mb(ZnP) sample, the observed rate, 45 4 s-1 , was found :!: to be within experimental error of the native decay rate, kd. Activation Enthalpies: The plots enthalpies versus (ln(kf/T) 4, are of presented in activation, f1H+. were 1/T). These results, Figures 12-16. The determined compiled f1H+ in from Eyring Tables 2 and in Mb(ZnP) is 0.0 :!: 0.9 is 1.7 :!: 1.6 of 5.6 kcal mol- 1 . The a 5 R u(His-48)Mb(ZnP) activation enthalpy kcal mol- 1 . The His-81, derivatives exhibit l1H*'s 0.4, and respectively. The activa- not readily :!: 2.5, 5.4 :!: 116, 4.7 and 12 0.9 kcal mor 1 :!: ' tion entropies, -31 to -34 eu, are similar for all four derivatives. The reason for two distinct activation possible protein effects modified histidine apparent. In considering enthalpies, the locations of the His-12 (A helix), be examined. corner), and A residue the E within helix playing a role channel for ature X-ray isotropic When His-116 (G the helix) 4 8 CO-corner (histidine-64 in and dioxygen lie is on the residues (CO-corner), in different (arginine-45), valine-68), along His-81 protein with in myoglobin that displacements of nonhydrogen interactions, the any in flexible CO-corner activation the region. 7 1 The may contribute to enthalpies between the assignment CO-corner myoglobin then open a Additionally, low temper- mean-square of regions. implicated the mobility (EF- been permit greatest to of measurements atomic need elements have heme. 6 9 activation diffraction solvent in all binding to the for ence His-48 the conformational changes in corrected the enthalpies His-48 the atoms. 7 0 exhibits Ioca tion of the of His-48 measured differ- modification and the -48- Table 4. Eyring plot data for a 5 Ru(His)Mb(ZnP), His-81, 116, and 12. Temp. a 5 Ru(His)Mb(ZnP) oc His-81 His-116 His-12 (:!: 0.1) ln(kr/T) In(kr/T) In(kr/T) 7.6 -2.05:!:0.13 -1.77:!: 0.16 -1.53:!:0.13 11.8 -1.72:!: 0.27 -1.66:!: 0.10 -1.49:!: 0.12 16.2 -1.61:!: 0.15 -1.48:!: 0.06 -1.31:!:0.06 20.6 -1.39:!:0.15 -1.37:!:0.16 -1.30:!: 0.20 25.0 -1.24:!: 0.26 -1.21:!:0.03 -1.08:!:0.11 29.4 -1.14:!:0.06 -1.08:!: 0.07 -0.96:!: 0.17 34.0 -1.03:!: 0.20 -0.97:!: 0.02 -0.84:!: 0.04 38.6 -0.88:!: 0.16 -0.79:!:0.15 -0.73:!:0.11 -49- Figure 12. Plot of ln(kd/T) versus 1/T for Mb( 3ZnP\ -50- -1. 4 - -1.7 - ..... I ' 1' -I T "- '"' ,, ...... 'i' - -2.3 - -2.6 3.2 I I I I 3.3 3.4 3.5 3.6 -51- Figure 13. Plot of ln(kr/T) versus 1/T for a 5 Ru(His-48)Mb( 3 ZnP \ -56- -. 7 -1 1- .......... ..:¥:.4--1.3 -1. 6 -1.9~------------~-----------~--------------~-----------~ 3.2 3. 3 3.4 3.5 3.6 -57- Figure 16. Plot of ln(kf/T) versus 1/T for a 5 Ru(His-12)Mb( 3ZnP\ -58- -.8 1- ......... ..::.:.4- -1. 1 -1. 4 -1.7~---------r--------~----------r---------~ 3.2 3.3 3.4 3.5 3.6 -59other deri va ti ves. Distance Evaluation: The determination electron-transfer transfer the extent rates dynamics intersite of of is is interaction distance essential and distance the mechanisms. From theoretical considerations, expected have profound effect to the Experimental measurements tance necessary order in intramolecular understanding acceptor. are of the between to dependence to a of on the donor and electronic states of of rate dependence the evaluate the the mechanism electron- on of dis- electronic coupling. Theoretical categories treatments characterized of as electronic coupling through-bond and fall into two through-space. broad Both are concerned with the decay of the donor and acceptor wavefunctions over increasing distance. In general, the electronic coupling matrix element, HAB• is expected to decay exponentially with distance. (3) where HAB is the electronic interaction at van der Waals contact, R R .1 2 0 Both exponential through-bond distance and through-space The dependences. value mechanisms of ex, should lead to however, is ex- pected to be lower in the through -bond case, as the wavefunction decay is expected Some to treatments be affected predict bond transfer mechanism.7 3 a low by interactions power-law with dependence the for bond orbitals. the through- -60- Theoretical studies have shown that the through-bond and space pathways are tron tunneling. 7 4 distinct The limits limit of selected the general is determined of the donor, acceptor, and medium, as well as tion between adjacent incorporated rigid through-bond mechanism Recent tions.2 linkers theoretical hydrocarbon through-bond frame by of the elec- energetics the degree of interacFixed-site the donor and acceptor. 1 5 -I 9 been proposed in many of indicate that separate has is mechanism elements. to results through- facilitated by electron systems these transfer carbon-carbon have A systems. across a-bond a interac- ° In order to the assess contributions of through-bond and through-space mechanisms in protein-mediated systems, the electron- transfer obtained this work been examined in the measured from the proximal to the heme in the known to coordinate the rates in have light of myoglobin structural results. Through-bond: The histidine through-bond (His-93) native protein zinc porphyrin, nium in as (in correlation fact, corresponding the sites is is the distances. is zinc His-93 in on porphyrin iron(II) deoxy between based ligand residue presented between through-bond axial five-coordinate five-coordinate Angstroms) exists were The same intervening residues modification separation serves structure. with isostructural number of that distances and Table 4.7 each 5. of The A/residue. 7 6 seems virtually porphyrins. 7 5 electron-transfer It is likely rates that the The ruthe- through-bond No obvious and their a through- -61- Table 5. Through-bond distances from the axial histidine ruthenium modification sites in sperm whale myoglobin. residues distance a kf (#) (A) (s-1) His-12 81 380 101±11 4.62± 0.11 His-48 45 210 70± 8x10 3 11.16±0.10 His-81 12 56 86± 12 4.45± 0.26 His-116 23 110 89± 3 4.49± 0.03 a 5 Ru(His)Mb a. 4.7 A/residue ln(kf) to the -62- space mechanism is operative in the a 5Ru(His)Mb(ZnP) systems. Through-space: The through-space graphic data metal to between metal evaluated. distance selected (center Typically, to the was calculated atoms m the from the crystallo- protein structure; both center) and edge to edge distances were porphyrin ring edge and the imidazole ring are the locations of the a toms used to determine the edge to edge disA minimum distance of 4 A is incorporated into the data plots tances. to reflect the nonbonded inner sphere ligand presents these data. shows that the interactions that A rate between the porphyrin edge and discourage any closer contact. Table 6 of versus distance (R) (Figure 17) with distance, but at plot initially ln(kf) falls off moder- ately long ranges (24 - 29 A metal to metal, 19 - 23 A edge to edge), the electron-transfer error, the points rate plotted are so similar at that the distances appears in three to Figure very level 17 off. are different Within distinct. That long-range evaluated from the performed crystal structure experimental the rates distances suggests crystal structure are of pentaammineruthe- native in- correct. A recently nium(histidine-48)ferrimyoglobin 7 7 the histidine ruthenium-labelled supports (His-48) protein surface in the crystalline tance observed in the crystal calculated metal to metal separation structure. The fact that the is state. structure of histidine is the this hypothesis, greatly extended because from the The iron to ruthenium dis- -24 A, in contrast to the -18 A in the modified native residue can reorient itself by -63- Table 6. Intersite and closest ligand distances evaluated from 1n(kf) the modified native myoglobin crystal structure. metal-metal edge-edge kf (A) (A) (s-1) His-12 28 .8 22.1 101±11 4.62± 0.11 His-48 17.7 13.3 70±8x10 3 11.16±0.10 His-81 23.2 19.2 86± 12 4.45± 0.26 His-116 27.4 20.4 89± 3 4.49± 0.03 a 5 Ru(His)Mb -64- Figure 17. Plot of Distances structure. ln(kf) versus distance were determined from Metal-to-metal for the separations a 5 Ru(His)Mb( 3ZnP\ native protein are designated triangles and edge-to-edge distances by diamonds. crystal by -65- 24~------------------------------------------~ 18- 12- o4 I I I I I 9 14 19 24 29 R (~) -66rotating about the ca cf> bond indicates that the through-space to reassess separations should be reevaluated to optimize the distances. Computer-graphics imaging intramolecular through-space distances are quite structure (Table to-edge data similar Better 7). where the studies were used distances. The metal-to-metal to the values agreement, estimated however, distances of the fixed-site intramolecular is His-81 and from the minimum the native in the edge- His-116 are now given by seen nearly identical (Figure 18). The rate of a reaction is Equation 4, k = KV exp(-.6.G */RT). In the nonadiaba tic case (K << 1), when the (4) donor and acceptor are weakly coupled, KV may be approximated as, 7 2 (5) The electronic coupling matrix element, HAB• decreases exponentially with distance in many cases (Equation 3), so Kll where f> = 1x10 13 exp(-f>(R - R 0 )) s- 1 (6) 2a and 1x10 13 s- 1 is the assumed nuclear frequency at van der Waals contact, R = R0 • Under such conditions, the adiabaticity of the reaction approaches unity. The electron-transfer rate between -67- Table 7. Distance ranges evaluated from nonbonded repulsions at the protein surface. metal-metal edge-edge kf (A) (A) (s -I) His-12 28.8-30.4 22.1-22.4 101±11 4.62± 0.11 His-48 18.6-24.1 13.4- 16.6 70± 8x10 3 11.16±0.10 His-81 26.3-26.9 19.0-19.4 86± 12 4.45± 0.26 His-116 23.1-27.8 18.9-20.4 89± 3 4.49± 0.03 a 5 Ru(His)Mb ln(kf) -68- Figure 18. Plot of Distances ccx versus distance for were determined from optimized c~ separations a 5 Ru(His)Mb( 3ZnP\ ln(kf) bond are in the designated distances by diamonds. labelled by rotations residues. triangles and of the Metal-to-metal edge-to-edge -69- 24~----------------------------------------~ 18 s:: 12 01> 4 9 19 14 R (~) I> 24 I> 29 -70- fixed sites may then be expressed as k = 1x10 13 exp(-ll.G*/RT) exp(-~(R- R 0 )) s- 1 (7) or more simply, Fitting the Equation rate-distance data from the His-48, with 4 A (van der Waals contact at 10 R0 6x10 9 ~ 1 and several orders cept, k 0 (4 A), Since two different is activation zinc / ruthenium myoglobins, process made As can protein be enthalpies, calculated distances. through protein treatment in compares favorably carbon atoms in of A- 1 from any A to link a lower than allowance adjust between adjust with magnitude the 'l'f-bonding distance to The be inter- predicted. measured for in activated term's prefactor. may in the alter the and may require of k 0 (4 A). Nonetheless, estimate of 1.4 value configuration 7 8 the an reflected activation a to porphyrin distance theoretical electron-transfer been perturbations changes the the believed structural conformational order 19). direct are the (Figure have to 116 systems 1.2 A- 1 enthalpies no order of conf or rna tiona I changes activation 1.2 in ~ 81, and enthalpy more intricate A- 1 and experimental reactions between aroma tic Equation 8, for results molecules in rigid rna trices. 8 Given the rate expression in rate-distance the His-12 modification can be calculated. For a rate of 100 s- 1 , a values for -71- Figure 19. Analysis (k = k 0 data. eluded. Rate-distance exp( -~(R - R 0 ))) of ln(kf) versus distance data for His-48, 116, and 81 are in- -72- 24-r----------------------------------------~ 18 12 4 9 14 R (~) 19 24 -74- Figure 20. Intervening residues on through-space pathways thenium-labelling sites and porphyrin in myoglobin. between ru- -75- His 12 22. I A Hit 116 Hi.a 81 20.4 A 19.2 A His 48 13 . 3 A -76- and His-116 derivatives can now be understood media in their pathways. residues are expected coupling in no the pathways of His-48, terms through-space similarin tervening As in 81, of the electron-transfer to influence and 116, the these electronic three deriv- residue (Trp-14 ). Unlike atives are considered "medium neutral" with respect to each other. The His-12 medi urn contains a tryptophan His-82, Trp-14 contains planar to porphyrin, pathway the is Theoretical unique an thereby among calculations aromatic ring that providing those in the assuming a nearly is oriented parallel- that the His-12 myoglobin systems. evidence derivatized optimal orientation indi- cate that the Trp-14 may enhance the electronic coupling by the equivalent of 3 A. 7 9 edge to distance 19 A. Subtraction of this amount from the measured edge-to- reduces This is in the "effective" excellent distance agreement of with the the His-12 pathway predicted distance (18.8 A) based on the observed rate. In accordance substantial The in rate-distance with earlier work, 8 0 influencing relationship the effect intramolecular derived of medium can electron-transfer from the kinetics. myoglobin provides a direct determination of the "effective" pathway length. be data -77- CHAPTER IV Implications -78- In this long-range are section, electron discussed the implications transfer with in respect to recent data for the well as other metalloprotein systems this work. Second, through-space from extracted from in of light the results is proposed to from proceed rapid long-range thetic reaction centers tions myoglobin for are and considered edge future compared myoglobins First, recent cytochrome with the c results rei a tionship experiments is where mechanism. rates in of rate-distance systems through-bond to studies. myoglobin electron-transfer edge continuing are organic model by a dependence zinc/ruthenium-modified zinc/ruthenium ically thenium the distance zinc/ruthenium-modified other similar the kinetic as very the of electron Next, observed the context separation distances. investigations in discussed the the in of transfer kinet- photosyn- the zinc/ru- Finally, direc- field long- of range intraprotein electron transfer are discussed. Another a 5 Ru(His)protein(ZnP) System: The kinetic results for the zinc/ruthenium-modified cytochrome c may be utilized to extend the range of distances employed to fit Equation 8. This system is ideal to compare with the zinc/ruthenium-modi- fied myoglobins, because the same donor and acceptor are present. only major essentially distance change the is intervening I 1.7 that the intersite same. In a 5 Ru(His-33)Cyt A, medium as distance, determined by nonaromatic rate in system, predicted from this Equation 8 as the c(ZnP), computer contains electron-transfer with is the force is edge-to-edge modelling, residues. 8 1 and the The observed s- 1 , compares closely s- 1 ). 6 6 Inclusion of 7.0xi0 5 (5xi0 5 driving The the -79- a 5 Ru(His-33)Cyt c(ZnP) value yields a refined fit of the ln(kf) versus distance plot, k = 8x10 9 exp(-1.2A- 1 (R - 4)) s- 1 where, as before, - 4 R 0 (R 2:: in A) (Figure 3 znP* has (9) 21). This expression will be used in the discussion that follows. [Zn, Fe] Hybrid Protein-Protein Complexes: The highly reducing nature research groups to multisite metalloproteins and are study centers in these systems replace one (or two) of of intramolecular electron protein-protein hemes. the The hemes been by other transfer in both The metal complexes. general with utilized strategy zinc has been porphyrin. to Electron transfer is then studied from 3 znP* to the Fe 3+-heme. One well-characterized into stituted 1 hemoglobin of the two ' 1 have various the of f>Zn [ CXFe metal Fe]Hb). 2 1 ([Zn, hemoglobin CXFe 2 ' ' been system In of this used porphyrin system, hemoglobin f>; n] hybrid. to The the in pair the porphyrin producing distances The is submixed data for native and orientations distance IS cxie -f>; n distance of - 25 A. porphyrin are hybrid the site-to-site to edge distance between the porphyrins zinc / iron Crystallographic very long (35 A) compared with the shorter The edge is zinc subunits, determine pairs. type this rings favorably is about 20 A. oriented, that is, [ZnP+, Fe 2 +]Hb), they are nearly parallel-planar. The electron-transfer rate ([ 3ZnP*, Fe 3+]Hb -+ -80- Figure 21. Plot of In(kf) versus protein(ZnP) experiments. distance for four -81- 24----------------------------------------- 18 c: ,..... 12 4 8 16 12 R (~) 20 24 -82- determined by From revised the transient absorption rate-distance spectroscopy, expression is ± 10 s-1 .8 2 the rate at 100 (Equation 9), 20 A is predicted to be 24 s- 1 , a value that is considerably smaller than observed similar experimentally. to This discrepancy, though not very great, is that encountered in the a 5Ru(His-12)Mb(ZnP) rate-distance possible explanation for this minor is that the conformation of the hemoglobin subunit chains, perhaps because of waters of hydration that comparisons. A crystal are structure rigidly solution, represents incorporated however, an into relaxation of to freely move waters recover from its in tersi te distance. This relaxation Though specific protein regions others, the molecular-scale appear to be expanded structure, thereby allows in is expected in may exhibit greater and The myoglobins structure. rigidity resulting compression zinc/ruthenium-modified protein crystalline and isotropic. 7 0 largely crystalline the the the expanded the enables discrepancy a requirements the protein reduction myoglobin extension of to the as well. flexibility than of rate-distance In the protein relationship in be valid, because the structure and solution con- discontinuity in the should same protein structure is involved in every experiment. Such formation a difference should be detectable the solution transfer rates solution temperature, rate than as the between the highest the crystal as is frozen compressed glass a into electron- a glass. At conformation would have temperature. Electron-transfer the a lowest faster rates in the solution (250 - 310 K) and solid (77 - 175 K) phase regions have been observed in [Zn, Fe]Hb, but in the critical temperature range, -83near the freezing point of the glass, no data have been reported. 8 2 examination close of the temperature effects in both A [Zn, Fe]Hb and Zn/Ru-Mb is needed. More recent utilize systems talloproteins of work the that has involved protein-protein complexes. These crysta llogra phica ll y characterized single-site me- form stable electron-transfer and reaction specific is complexes. evaluated by The using distance computer- generated models of the docked protein-protein complex. The complex formed between cytochrome b 5 derivative of ([cx~n, hemoglobin and the zinc-substituted ~;e{III)CN]Hb) has studied. 2 2 been The edge-to-edge distance between the coplanar porphyrins, 7 - 8 A (16 A metal-to-metal), -+ evaluated by computer A rate of 8x I o3 s- 1 the complex. 8 3 Fe 3+ was ZnP+-Fe 2 + electron-transfer graphics simulation of has been reported for the 3ZnP *reaction, where the driving force is comparable to that reported in the hybrid hemoglobin system. In complex the cytochrome separation the b5 , between model A metal (17 to zinc-su bsti tu ted structure metal) cytochrome predicts between the an A 8 porphyrin edge-to-edge rings. 8 4 is similar to the other Zn/Fe-hybrid systems (.6.E 0 driving force and c The 0.85 - V), and the observed rate is 5xl05 s- 1 . 2 3 One final physiologically and Zn/Fe system that has relevant complex c. has cytochrome Zinc From single-crystal X-ray diffraction found nearly parallel to be modelling between been peroxidase. been investigated consists of the studies with yeast substituted cytochrome c into cytochrome the with the data structures, the porphyrin an edge-to-edge peroxidase from distance c individual rings were of - I7 I8 -84- A. 8 5 The electron-transfer rate is 138 12 s- 1 :!: at a driving force of - 0.90 v. 2 4 The results from summarized in 8. pared with the distance-dependence general, the distances In Table protein-protein distance estimates pack as at The intervening mechanism for A possible that electron the two These rate-distance are the short that interface as could distinguishing feature transfer occur are studies are graphically com- in 9) Figure rates m respect the zinc/ru- possible to explanation complexes in the computer bear on the issue, rather than of the across donor-acceptor aqueous electronic the for the do not modelling. although enhancement protein-protein an 21. observed protein-protein also decoupling The with assumed electronic may data the A the complex (Equation with results. is medium proteins. results too myoglobin low well protein-protein associ a ted complexes thenium-modified these a is needed. complexes interface coupling is between could be complexes are adversely affected by such an intervening polar environment. Currently, investigating medium to cytochrome mediate c is glycine The and (Gly). electron (CcP). protein-protein these in studies, yeast a phylogenetically cytochrome transfer between Through site-directed (Cc) is c and mutagenesis, the c cytochrome tyrosine (Tyr), serine (Ser), and Tyr contain aromatic side chains (tolyl respectively), serine with replaced p-hydroxytolyl, In (Phe-87) peroxidase phenylalanine involving effects. 8 0 phenylalanine conserved believed experiments Phe and glycine contains are studied (hydroxymethyl), and electron-transfer kinetics a contains a heteroatom hydrogen. The long-range within the protein-protein -85- Table 8. Observed and calculated rate data for Zn/Fe-hybrid protein- protein complexes. Protein-Protein R Complex (A) [3 ZnP *, Fe 3+]Hb/Cyt b 5 (Fe 3+) 8x10 3 8.99 7 <3 znP *)Cyt c/Cyt b 5 (Fe 3+) 5x10 5 13.12 8 ( 3 ZnP *)CcP /Cyt c (Fe 3 +) 1.4x10 2 4.93 17 a. Calculated from Equation model studies (refs. 79-81 ). 9 with R values taken from computer- -86complex between the zinc-substituted CcP and the various Cc mutants. The results of these studies show that the CcP( 3 ZnP *) electron transfer proceeds containing cytochromes c. slower. More CcP(ZnP+), is at similar The Gly significantly, four orders rates reverse magnitude modifications than in the Ser and Gly. m CcP( 3 ZnP*) the attributed residue. the to an improper Conformational These Phe, rate in the reverse reaction. transfer of the associated changes diminish the Alternatively, intersite may electron-transfer direction of rates electron the distance. Phe may transfer not can and -+ Tyr with bring the known, the the reduction have a -+ of residues CcP(ZnP+) changes may explanation of observation profound be aromatic aromatic detailed the easily intervening conformational Though is Ser considerably Cc(Fe( 2 +P) favorable orientation for the very rapid Cc(Fe( 2 +P) into a and The absence of a medium effect electron are Tyr, reaction, faster orientation changes c. ferricytochrome these Cc(Fe 3+P) -+ the mutant exhibits a the of in Cc(Fe3+p) -+ effect on that the the rate suggests further lines of research. Rigid Organic Linkers: In addition biologically to synthetic models that by organic spacers rigid · intramolecular is a incorporate electron relevant aromatic have been transfer. In electrons. acceptor is one The of a spacer series of is discussed above, and acceptors linked in the investigation system, the electron donors employed one biphenyl radical anion generated from solvated systems a a rigid, organic of donor bimolecular reaction with steroidal The molecules with skeleton. a 7r-electron -87network (for tron-transfer radical example, naphthyl rate measured anion was and quinonyl) by time-resolved absorption. 8 6 •8 7 transient (Figure With edge distance of 10 A. The elec- of the monitoring a from 0 to 2.4 V, the observed rates are 10 6 to 22a). driving-force 10 9 s- 1 range at an edge to The maximum rate was found at a driving force of -1.2 V. In a similar system, separates the electron mophore, from a the spacer, a donor, cyanoethylene a rigid, aliphatic photoexcitable , naphthalene acceptor 22b). 8 8 (Figure added or removed to vary the range from 7 to 15 A. A are of 9 (to -14 greater results A) than s-1 . an electron-transfer in bridge, An chro- Spacers were Rates at distances increase in rate least two orders phenyl (or biphenyl) at the spacer length of magnitude slower. A bridge third system incorporates to separate a anthracene acceptor (Figure as 109 s- 1 tance by were aromatic dimethylaniline observed A with 4 an 22c). 8 9 at a another donor Electron-transfer distance phenyl from of 7.5 A. spacer caused a photoexcited rates as fast Increasing the dis- fall by the rate to only 20 - 30 times. A final attached to (Figure 22d). 9 0 system a utilizes quinone At at a acceptor photoexcited by a metalloporphyrin donor bicyclo[2.2.2]octane spacer of 10 A, electron transfer takes place With the addition another spacer, the a distance 1.0 V). of rate falls by two orders of magnitude. From s- 1 at a Equation distance of 09, the predicted 10 A. This value electron-transfer is similar to rate the is 5xl0 6 low-driving -88- Figure 22. Structures of donor-spacer-acceptor steriod-naphthyl (R cyanoethylene (R anthracene = 10 cated. A). (R 10 9 7.5 A), Edge-to-edge A), A), and d) systems, b) c) a) biphenyl- naphthalene-bridge- dimethylaniline-phenyl- porphyrin-linker-quinone donor-acceptor distances are (R indi- -89- b) c) d) .........- CN === --CN -90- rate, 10 6 s- 1 , Furthermore, the rate linker off in force falls reported in the biphenyl-steroid-naphthyl dependence on distance fashion similar a to through the system. a saturated intraprotein rate-dis- tance dependence determined in this work (-10 2 s- 1 for .6.R = 4 A). the rate falls less dra- available to the aro- presence matically, of an aromatic suggesting that spacer, however, the ~-interactions the matic system may enhance the electronic coupling. 9 1 In The importance of the intervening medium, in particular an aromatic-group enhancement of the as donor-acceptor tron transfer mental rates by in ex hi bit dependence in the a 5 Ru(His-12)Mb(ZnP), is indicated The myoglobins the rna y the structures suggest that mechanism, of these the electron the -+ by systems a 5 Ru 3+ electhese experi- and the rapid transfers take place similarity with the distance through-space intraprotein work indicates that the through-space long-range coupling effects are the reason for the slower rates may be an artifact of the activated electron-transfer of the primary charge-separation it is through-bond same. for through-bond a 3 znP* proposed While results. they coupling and observed in the zinc/ruthenium processes that occur in them. Photosynthetic Reaction Center: step The electron-transfer will be considered kinetics here, as analogous to the reactions measured in the zinc/ruthenium-modified myoglobins. The spatial tionships of the prosthetic constitute the photosynthetic reaction center cen tl y been in the el ucida ted. 9 2 groups that bacterium Rhodopseudomonas The involved groups in viridis the have initial rela- reelec- -91- tron-transf er events two consist monomeric bacteriopheophytin of a bacteriochlorophyll b molecules, BPh. The in close proximity to each other as little as BChl is associated The 3 A. A porphyrin-ring b, bacteriochlorophylls separation with appears BChl, and two of dimer lie pyrrole rings separated by components their dimer, b the with each BChl in the dimer. be 3.5 A. to about BPh A is then positioned about 4 A (closest approach) to each of the monomeric BChl's. The rate of formation of BChC from (BChl) * 2 has been reported to The subsequent rate of reduction of BPh is 2xl01 1 s 1 7 2 The activation small. For electron-transfer A, the rates predicted by Equation 9 are 1x10 10 s- 1 free energies of reactions these across reactions distances are of negligibly 3.5 and 4 and 8xi0 9 s -1 ' re- spectively. The center arrangement does not of favor components a in through-bond the photosynthetic electron-transfer reaction mechanism. Within the framework of a through-space mechanism, the rapid electrontransfer rates would need to the fixed-site Zn/Ru-hybrids. are less in the reorganization center acceptor than appears to distance the be act-i.vationless, The driving must be finely and forces zinc/ruthenium-modified energy a be tuned system energy the for to results these proteins, correspondingly reorganization electron transfers to take place. given from reactions indicating lower. that The reaction that minimizes donor- allow extremely rapid -92Directions of Future Work: This study on zinc/ruthenium-modified long-range intraprotein Promising areas extension of for the of continuing range thenium-modified properties electron-transfer of and myoglobins rate future electron-transfer proteins, b) intraprotein dependence revealed a on distance. investigation include a) distances the zinc/ru- determination electron has transfer, for of the and c) an low-temperature measurement of the dependence of the rate on driving force in the ruthenium-modified metalloprotein systems. The temperature dependence of the electron transfer rates in the a 5 Ru(His)Mb(ZnP) system should be examined at low temperature in order to assess the importance tunneling motions. protein conformational detected magnetic that electron-transfer the of resonance in well as Preliminary the rates as in optically a 5Ru(His)Mb(ZnP) systems indicates the four derivatives not a 5Ru(His-48)Mb(ZnP) measurably faster counterparts. the sites. glass, it By His-48 edge-to-edge other If at 4 may K exist in in an extended 17 A, is nearly this the conformation is explain the information are low about the temperature is expected to with longer-ranged its conformation show in which is of the protein a frozen similarity of the low-temperature rates. temperature of the glass concerning the proposed change driving comparable a which the melting was comparison distance would approaching below, rate of with That the rates are slow at though freezing work same (2 s- 1 ) at 4 K. surprising, the in from to above conformational the and changes should be obtained. One way to the force is to vary the me- -93talloporphyrin that Ideally, metalloporphyrins in the solution ms. at is room Triplet inserted into the ruthenium-modified selected temperature and should have triplet lifetimes less than ms quenching at long distances. palladium-mesoporphyrin IX (PdP) (Pd 2 + tron-transf er Rh 3+, Sn 4 + and are other exhibit are phophorescence lifetimes of at least short allow elec- work with to Preliminary as possibilities) too the myoglobin. central metal: demonstrates the feasibility of this line of attack. Palladium-mesoporphyrin myoglobin Mb(PdP) derivatives was in IX the successfully was inserted same purified into fashion as on CM-52 a ruthenium-labelled zinc porphyrin column; was. however, a 5Ru(His-48)Mb(PdP) apparently denatured on the column. Transient absorption (A. nm) measurements yielded a quenching on triplet was 391 = Mb(PdP), decay rate observed in nm) and performed on I.Ox10 3 of triplet emission (A. the s- 1 = 671 pulsed laser system, 25°C. No triplet biphasic triplet at a 5 Ru(His-81 )Mb(PdP). A emission was seen in a 5 Ru(His-48)Mb(PdP) with component rates of 8x10 3 s-1 . The a 5 Ru(His-48)Mb( 3 PdP*) was and incomplete transfer purification rate in complicated attributed procedure to (no a 5 Ru(His-48)Mb(PdP) behavior in impurities remaining CM-52 the column). appears to be magnitude slower than in the corresponding zinc system. emission from The an by the electronorder of Though 3 PdP * is of higher energy than 3ZnP *, -0.1 V, the cation radical is expected to result, be higher still, -0.2 V. As a the driving force is ex- pected to be -0.1 V lower in the Pd/Ru-Mb than in the Zn/Ru-Mb, and may explain the slower electron-transfer rate observed. Slightly -94longer lifetimes reactions in are the needed in order longer-ranged the phosphorescence of the flexibility conducting of the to detect deri va ti ves. The triplet by the ability time-resolved experiments with electron-transfer to monitor methods enhances these electron-transfer systems. In addition quenching to studying a 5 Ru 3+, by several should allow investigations excited state and in processes in the reverse esting, because especially those recent driving so force effects metalloporphyrins of reductive doing enable direction. theoretical involving aromatic the oxidative example, Sn 4 +P) m (for quenching of the observations of electron-transfer This porphyrin could be particularly inter- indicates that medium effects, groups, should not work oxidative and reductive quenching processes. 7 9 be the same for -95- APPENDIX A Calculation of Model System Coordinates -96- The procedure employed using the Brookhaven myoglobin is presented coordinates whale calculations for coordinates and to calculate are also included. results pentaammineruthenium crystallographic in a 5 Ru(His-48) final the for this data base The details section. derivative are shown. the 116, and His-81, for sperm of the Pertinent 12 derivatives The assumptions made about the position of the ru- thenium relative to the imidazole ring are (Figure #): 1) Ruthenium is attached to the N €; 2) Ruthenium lies in the plane of the imidazole the reflex the ruthenium ring defined by c 5 - N € - C€; The 3) N€ - Ru vector bisects angle, c 5 -N€-C€; The 4) coordination sphere of is octahedral. The general procedure used to determine the ruthenium coordinates involves the definition of of a set of linear of coordinates, equations in - N € - C€ three plane and then solution variables. the distance formula, d 2 = are produced from equation the C5 the plane. (x,y,z), and The distances c5 the and The equations x 2 + y 2 + z 2 , and between the atoms of Cf: linear sought the the ruthenium imidazole ring are calculated based on a Nf: - Ru distance of 2.12 A and criterion 2) mentioned above. direction vectors (the ammine Having from ligand is taken as 2.12 A. the determined ruthenium positions) are the to the calculated. ruthenium coordinates, vertices of The - Ru an the octahedron NH 3 distance -97N€(x,y,z) = (30.874, 28.896,-1.164) C€(x,y,z) = (29.747, 29.559, -1.315) C 0 (x,y ,z) = (31.311, 28.457, -2.380) Ru(x,y,z) = (x,y,z) R2 R N€Ce = -1.127i + 0.663j- 0.151k 1.732499 1.316244 NeCs = 0.437i + 0.439i- I.216k 1.862346 1.364678 NeRu = (x- 30.874)i + (y- 28.896)j + (z + 1.216)k 4.494400 2.120000 2.866585 1.693099 Generate the normal vector of the ring plane, C 0 - N € - C€ Nece x NeCs = -0.872497i- 1.436419i+ 0.205022k Determine the angular position with respect to c 6 - N € - C€ IINeCe x NeCsii/(IINeCell IINeCsll) =sin!= 0.942575 ! = 70.488542 ! ' = 180-! = 109.511458 e = 360 - ' ' = 250.488542 9/2 = 125.244271 cos 9/2 = -0.577064 Calculate the equation of the plane, requiring the Ru to be coplanar NeRu • NeCe x NeCl> = -0.872497x - 1.436419y + 0.205022z + 68.682881 = 0 -4.255626x - 7.006170y + z + 335.002492 = 0 Evaluate the distances to the Ru IINeRull 2 = 4.494400 Apply the Law of Cosines to determine the remaining two distances a 2 = b 2 + c 2 -2bc cos(9/2) (A-1) -982 = 9.447421 whereb= IINf:Rull andc= IINf:Cf:ll IIC Rull 2 = 9.695774 5 where b = IINf:Rull and c = liNf:c 5 11 11Cf:Rull From the distance formula, generate three equations 11Nf:Rull 2 = (x- 30.874) 2 + (y- 28.896) 2 + (z + 1.164) 2 = 4.494400 x 2 + y 2 + z 2 -61.748x- 57.792y + 2.328z + 1785.043188 = 0 11Cf:Rull 2 = (x- 29.747) (A-2) 2 + (y- 29.559) 2 + (z + 1.315) 2 = 9.447421 x 2 + y 2 + z 2 -59.494x - 59.118y + 2.630z + 1750.900294 = 0 IIC Rull 2 = (x - 31.311) 2 + (y- 28.457) 5 (A-3) 2 + (z + 2.380) 2 = 9.695774 x 2 + y2 + z 2 -62.622x - 56.914y + 4.760z + 1786.148196 = 0 (A-4) Reduce quadratic equations to linear form (A-4) - (A-2) = -0.874x + 0.878y + 2.432z + 1.105008 = 0 -0.359375x + 0.361020y + z + 0.454362 = 0 (A-5) (A-3) - (A-2) = 2.254x - 1.326y + 0.302z - 34.142894 = 0 7.463576x - 4.390728y + z - 113.055940 = 0 (A-6) (A-4) - (A-3) = -3.128x + 2.204y + 2.130z + 35.247902 = 0 -1.468545x + 1.034742y + z + 16.548311 = 0 {(A-4) - (A-2)} independent - {(A-3) equations - (A-2)} = remain and an {(A-4) - (A-7) (A-3)}, additional one hence is onl y two needed. The equation of the plane is used. -4.255626x - 7.006170y + z + 335.002492 = 0 (A-1) -99Now solve the system of three linear equations (A-6)- (A-1) = 11.719202x + 2.615442y- 448.058432 = 0 4.480773x + y- 171.312701 = 0 (A-8) (A-7) - (A-1) = 2.787081x + 8.040912y - 318.454181 = 0 0.346613x + y- 39.604237 = 0 (A-9) (A-8)- (A-9) = 4.134160x - 131.708464 = 0 Final values are X = 31.858579 y = 28.561639 z = 0.683455 Ru(x,y,z) = (31.859, 28.562, 0.683) The vector N£Ru now provides the direction vector for RuN1 N£Ru = (x- 30.874)i + (y- 28.896)j + (z + 1.216)k N £Ru = 0.985i- 0.334j + 1.847k = RuN1 Normal vector to the plane, C6 - N € - C€. N = -0.872497i- 1.436419j + 0.205022k Con vert normal vector to a normal vector with length NH 3 distance a.N = -0.872497a.i- 1.436419a.j + 0.205022a.k lla.NII 2 = (-0.872497a.) 2 + (1.436419a.) 2 + (0.205022a.) 2 = (2.12) 2 2.866585a. 2 = (2.12) 2 (X = 2.12/(2.866585) 0·5 = 1.252142 2.12, the Ru - -100N 2 _12 = -1.092490i- 1.798600j + 0.256717k = RuN2 N_ 2 _12 = 1.092490i + 1.798600j- 0.256717k = RuN4 Generate normal vector to plane containing N 1 - Ru - N 2 RuN1 = 0.985i- 0.334j + 1.847k RuN2 = -1.092i- 1.799j + 0.257k RuN1 x RuN2 = 3.236915i- 2.270069j- 2.136743k = NJ. This vector provides the direction from Ru ot N 3 and N 5 Converting to vector length 2.12 aNJ. = 3.236915ai- 2.270069aj- 2.136743ak llaNJ.II 2 = (3.236915a) 2 + (2.270069a) 2 + (2.136743a) 2 = (2.12) 2 20.196503a 2 = (2.12) 2 CJ. = 2.12/(20.196503) 0·5 = 0.471735 NJ. 2 _1 2 = 1.526965i- 1.070870j- 1.007976k = RuN3 NJ._ 2 _1 2 = -1.526965i + 1.070870j + 1.007976k = RuN5 Using the direction vectors generated ates of the five ammines follow are provided to confirm the from octahedral with vector length addition. structure The distance between cis-ammines should be 2.9981. 2.12, of the coordin- Some distances the ligand shell. -101His-48 N E (x,y,z) = (30.874, 28.896,-1.164) CE(x,y,z) = (29.747, 29.559, -1.315) C 0 (x,y,z) = (31.311, 28.457, -2.380) IIRuNII IINENII IIN;Ni+lll N 1(x,y,z) = (32.844, 28.228, 2.530) 2.120 4.240 2.999 N 2 (x ,y,z) = (30.766, 26.763, 0.940) 2.120 2.998 2.999 N 3 (x,y,z) = (33.386, 27.491, -0.325) 2.120 2.998 2.998 N 4 (x,y,z) = (32.951, 30.360, 0.427) 2.120 2.998 2.998 N 5(x,y ,z) = (30.332, 29.633, 1.691) 2.120 2.998 IIRuNII IINENII IIN;Ni+lll N 1(x,y,z) = (-7.498, 23.314, 21.938) 2.120 4.240 2.998 N 2 (x,y,z) = (-5.065, 24.238, 23.426) 2.120 2.998 2.998 N 3 (x,y,z) = (-5.843, 25.565, 20.853) 2.120 2.998 2.998 N 4 (x,y,z) = (-5.837, 22.901, 19.477) 2.120 2.998 2.998 N 5 (x,y,z) = (-5.059, 21.574, 22.050) 2.120 2.998 Ru(x,y,z) = (31.859, 28.562, 0.683) His-81 NE(x,y,z) = (-3.404, 23.826, 20.964) CE(x,y,z) = (-2.473, 22.904, 21.094) C 0 (x,y,z) = (-2.853, 24.970, 20.469) Ru(x,y,z) = (-5.451, 23.570, 21.451) -102His-116 NE(x,y,z) = (18.265, 3.269, 10.484) CE(x,y,z) = (17.492, 3.255, 11.543) C 0 (x,y,z) = (18.462, 4.552, 10.080) IIRuNII IINENII IIN;Ni+lll N 1(x,y ,z) = (19.90 1, -0.163, 8.606) 2.120 4.240 2.999 N 2 (x,y,z) = (17.374, 1.422, 8.297) 2.120 2.998 2.999 N 3(x,y,z) = (20.036, 2.791, 8.112) 2.120 2.998 2.998 N 4 (x,y,z) = (20.791, 1.684, 10.794) 2.120 2.998 2.998 N 5 (x,y,z) = (18.130, 0.315, 10.978) 2.120 2.998 IIRuNII liNE Nil liN-N - 1 11 l l+ N 1(x,y,z) = (17.084, 5.951, 24.805) 2.120 4.240 2.999 N 2 (x,y,z) = (14.147, 6.269, 25.319) 2.120 2.998 2.998 N 3 (x,y,z) = (15.355, 7.364, 22.803) 2.120 2.998 2.998 N 4 (x,y,z) = (17.338, 8.915, 24.430) 2.120 2.998 2.997 N 5 (x,y,z) = (16.131, 7.821, 26.946) 2.120 2.998 Ru(x,y,z) = (19.083, 1.553, 9.545) His-12 N E(x,y,z) = (14.402, 9.2 33, 24.945) CE(x,y,z) = (14.120, 9.934, 26.019) C 0 (x,y,z) = (13.704, 9.719, 23.886) Ru(x,y,z) = (15.743, 7.592, 24.875) -103- APPENDIX B Kinetic Analysis -104- The bleaching derivation of the of the kinetic a 5 Ru(His)Mb(ZnP) expression systems for is the presented. ground-state Given the proposed reaction mechanism where k1 , k2 , and k3 rate constant, and the A is the radical, sion for triplet the time the forward intrinsic 3znP*-Ru 3+, C is the ground dependence of C and and reverse triplet-decay state, excited ZnP+-Ru 2 +, are B state, following electron-transfer constant, is the porphyrin ZnP-Ru 3 +. the respectively, An excitation cation expres- pulse sought. d[A]/dt = -k 1 [A] - k 3 [A] (B-1) d[B]/dt = k 1 [A] - k 2 [B] (B-2) d[C]/dt = k 3 [A] + k 2 [B] (B-3) d[A]/[A] = -(k 1 + k 3 )dt (B-4) A = A 0 exp{ -(k 1 + k 3 )t} (B-5) (B-6) multiplying both sides by exp{k 2 t} B = [k 1 A 0 /(k 2 - k 1 - k 3 )][exp{-(k 1 + k 3 )t}- exp{-k 2 t}] (B-7) is -105- d[C] = kg A 0 exp{ -(k 1 + kg )t} + [k 2 k 1 A 0 /(k 2 - k kg)] (B-8) C = A 0 [l +[(kg - k 2 )/(k 2 - k 1 - kg )]exp{-(k 1 + k 3 )t} + (B-9) 1 - [exp{-(k 1 + kg )t} - exp{-k 2 t}] [k 1 /(k 2 - k 1 - k 3 )]exp{-k 2 t}] From ground Equation state is a B-9, the biphasic time dependence first-order of the return to the exponential. In the event that (k 1 + k 3 ) is much greater than k 2 , or vice versa, the equation reduces to a single first-order rate constant, k 1 , can expression. The be extracted from (k 1 the intrinsic decay rate, kg. forward electron-transfer + kg) by difference with -106- APPENDIX C Evaluation of Nonbonded Repulsions -107The principal bond of rotation is Ca.-C/}. This bond attaches the imidazole ring via a methylene (C/}) to the protein backbone. Rotation about side-chain this bond causes residue. A secondary bond "flips" the the bond maximum of imidazole displacement rotation ring. is A of Cy-C/}. 180° the Rotation rotation about this essentially dis- places the ruthenium pentaammine label to the c£. Nonbonded surface were repulsions evaluated Interaction distances determined by radii. These between a 5 Ru(His) and by van der Waals for the various atoms contact the appropriate covalent interaction radii were to used the radii chemical combining at protein (Table C-1). substituents were and van der Waals calculate the nonbonded repulsion distances in Table C-2. Rotations at sites of exhaustive were performed potential search to-metal and allowable ranges of while repulsion. interaction edge-to-edge for the monitoring Upon sites interatomic reaching was closest distances approach, an made. The resulting metal- presented in Table The distances are modified residues are included 7. to indicate the local restrictions on each of the histidines. The amount histidines covered 5.5 A His-48 24.1 A, 48)Mb a in crystal dynamic allowed to each of the ruthena ted considerable range, from only 0.6 A in His-81 (metal-to-metal). The well with the estimated structure ( -24 A).T 7 a molecule conformation, aqueous extension compares of the so I utwn. 9 3 0 and that histidines The maximum crystal can be minimum distance Bearing in the extension .in mind structure expected accessible in the that a to His-48, a 5Ru(Hisprotein is represents only one rotate freely m were selected as to distances -108Table C-1. Selected covalent and van der Waals radii of atoms. covalent v.d.Waals radius a radiusb (A) (A) H 0.37 1.2 c 0.77 1.6 0 0.74 1.4 N 0.74 1.5 half -thickness of aromatic ring Interaction 1.7 radii of relevant groups are calculated from the appropriate covalent and van der Waals radii. (A) 2.0 (0 COY + H COY ) + H Yd W 2.3 2.3 a. Wells, A. F. Structural Inorganic Chemistry Oxford Uni- versity Press: New York 1962. b. Pauling, L. The Nature of the Chemical Bond, University Press: Ithaca, New York 1960, p. 260. 3d ed ., Cornell -109- Table C-2. Nonbonded repulsion distances calculated from covalent and crys- van der Waals radii Other Interaction Distance Groups A CH 2 -0H 3.4 NH 3 -0H 3.7 CH 2 -CH2 4.0 NH 3 - CH 2 4.3 NH 3 - NH 2 4.6 interaction tallographic distances interatomic are based on the initial distances prior to any rotations mod if ica tions. amide - amide 3.2 amide- CH 2 3.6 or -110the ranges to-metal for minimum the rate-distance distances are from the native structure {Table 6). correlation quite similar (Figure 18). to values the The metal- estimated -111- REFERENCES and NOTES -1121. Hatefi, Y. Annu. Rev. Biochem. 1985, 54, 1015-1069. 2. Dixit, P. B. S. N.; Vanderkooi, M. E., J. M. Curr. Top. Bioenerg. and Reaction 1984, 13, 159-202. 3. Michel-Beyerle, ed. Antennas Centers of Plants and Photosynthetic Bacteria Springer-Verlag: Berlin 1985. 4. Govindjee, Photosynthesis: ed . Energy Conversion by Bacteria, Vol. I, Academic Press: New York 1982. 5. Deisenhof er, J.; Epp, Miki, 0.; K.; Huber, R.; Michel, H. Middendorf, D.; Parson, w. w. J. Mol. Bioi. 1984, 180, 385-398. 6. Woodbury, N. Biochemistry photosynthetic W.; Becker, 1985, 24, M.; 7516-7521. reaction center in In this the related work, the bacterium Rhodopseudomonas sphaeroides was studied. 7. Rehm, D.; Weller, A. Isr. J. Chern. 1970, 8, 259-271. 8. Miller, J . R .; Beitz, V.; J. Huddelston, R. K. Am. Chern. J. Soc. 1984, 106, 5057-5068. 9. Miller, J. R.; Beitz, J. V. J. Chern. Phys. 1981, 74, 6746-6756. 10. Mauk, A. G.; Scott, R. A.; Gray, H. B. J. Am. Chern. Soc. 1980, 102, 4360-4363. 11. English, A. M.; Lum, V. R.; DeLaive, P. J.; Gray, H. J. Am. 1979, 3, B. Chern. Soc. 1982, 104, 870-871. 12. I.; Koga, S. S.; Vassilian, A. S. S.; Magnuson, R. Tabushi, N.; Yanagita, M. Tetrahedron Lett. 257-260. 13. Isied, J. Am. Chern. Soc. 1984, 106, 1732- Am. Chern. Soc. 1736. 14. Isied, H. Schwarz, H. A. J. -1131985, 107, 7432-7438. 15. Bolton, J. R.; Ho, K.; Weedon, A. C. T.-F.; J. Liauw, S.; Siemiarczuk, Chern. Soc. Chern. A.; Wan, C. S. 1985, 1985, 559- Commun. 560. 16. Hush, N. S.; Paddon-Row, M. N.; Verhoeven, J. W.; M. Chern. Heppener, Cotsaris, Phys. E.; Oevering, H.; Lett. 1985, 117, 8- A.; Pewitt, E. B. Hopfield, J. J.; 11. 17. Wasielewski, M. R.; Niemczyk, M. P.; Svec, W. J. Am. Chern. Soc. 1985, 107, 1080-1082. I 8. Franco, C.; McLendon, G. lnorg. Chern. 1984, 23, 2370-2372. 19. Leland, A.; B. Joran, A. D.; Felker, P. M.; Zewail, A. H.; Dervan, P. B. J. Phys. Chern. 1985, 89, 5571-5573. 20. Ohta, K.; Closs, L.; G. Morokuma, K.; Green, N. J. Am. Chern. Soc. 1986, 108, 1319-1320. 21. McGourty, J. L.; Blough, N. V.; Hoffman, B. M J. Am. Chern. Soc. 1983, 105, 4470-4472. 22. Simolo, K. P.; McLendon, G. L.; Mauk, M. R.; Mauk, A. G. J. Am. M. R.; B. M. Chern. Soc. 1984, 106, 5012-5013. 23. McLendon, L.; G. Winkler, J. R .; Nocera, D. N.; Mauk, Mauk, A. G.; Gray, H. B. J. Am. Chern. Soc. 1985, 107, 739-740. 24. Ho, P. S.; Sutoris, C.; Liang, N.; Margoliash, E.; Hoffman, J. Am. Chern. Soc. 1985, 107, 1070-1071. 25. Mathews, C. R.; Erickson, P. M.; Van Vliet, D. L.; Petersheim, M. J. Am. Chern. Soc. 1978, 100, 2260-2262. 26. Mathews, C. R.; Erickson, Acta 1980, 624, 499-510. P. M.; Froebe, C. L. Biochim. Biophys. -11427. Yocom, K. Worosila, M.; G.; Shelton, Isied, S. J. B.; Shelton, J. R.; S.; Bordignon, E.; Schroeder, Gray, W. B. H. A.; Proc. Nat!. Acad. Sci. USA 1982, 79, 7052-7055. 28. Kostic, N. M.; Margalit, R.; Che, C.-M.; Gray, H. B. Am . J. Chern. Soc. 1983, 105, 7765-7767. 29. Winkler, J. R.; Nocera, D. G.; Yocom, K. M; Bordignon, E.; Gray, H. B. J. Am. Chern. Soc. 1982, 104, 5798-5800. 30. Nocera, D. G.; Winkler, J. R.; Yocom, K . M.; Bordignon, E.; Gray, H. B. J. Am. Chern . Soc. 1984, 106, 5145-5150. 31. Crutchley, R. J.; Shelton, J. B.; Shelton, J. R.; Schroeder, W. B. Frontiers in A.; Gray, H. B., unpublished results. 32. Crutchley, R. Bioinorganic J.; Ellis, Chemistry, W. R., Xavier, Jr.; Gray, A. V., H. ed. VCH Verlagsges- H. B. J. Am. Chern. Chemistry, 2nd ed., Chern. 1950, 263, H. J. Am. Chern . ellschaft: Weinheim, FRG, 1986, pp. 679-693. 33. Crutchley, R. J.; Ellis, W. R., Jr.; Gray, Soc. 1985, 107, 5002-5004. 34. Barry's Methods Bible: in Bioinorganic Pasadena, California 1981, p. 94. 35. Schilt, A. A.; Taylor, R . C. J. lnorg. Chern. 1959, 9, 211-221. 36. Pfeiffer, P.; Werdelmann, B. Z. Anorg. Allg. 31-38. 37. Ford, P.; Rudd, De F. P.; Gaunder, R.; Taube, Soc.1968, 90,1187-1194. 38. Winter, A.; Electrojocusing Perlmutter, in a H.; Granulated Application Nate 198, revised 1980. Davies, H. Preparative Flat-BEd Gel the LKB Multiphor, with 211 7 -11539. Adler, A. D.; Longo, F. R.; Kampas, F.; Kim, J. Inorg. J. Nucl. Chern. 1970, 32, 2443-2445. 40. Doss, M. Z. Klin. Chern. u. Klin. Biochem. 1970, 8, 208-211. 41. Teale, F. W. J. Biochim. Biophys. Acta 1959, 35, 543. 42. Yonetani, T. J. Bioi. Chern. 1967, 242, 5008-5013. 43. Schichman, Barry's S. Bible: Methods in Bioinorganic J. B. Chemistry, 2nd ed., Pasadena, California 1981, pp. 84-85. 44. Stryer, L. J. J. Mol. Bioi. 1965, 13, 482-495. 45. Leonard, J. J.; Yonetani, T.; Callis, Biochemistry 1974, 13, 1460-1464. 46. Rice, S. F.; Gray, H. B. J. Am. Chern. Soc. 1983, 105, 4571-4575. 47. Henkel, J. G.; Clarke, F. H. Molecular Graphics on the IBM PC Microcomputer, Academic Press: New York 1985. 48 . Takano, T. J. Mol. Bioi. 1977, 110, 537-568. 49. Takano, T. J. Mol. Bioi. 1977, 110, 569-584. 50. Stynes, H. C.; lbers, J. A. J. Inorg. Chern. 1971, 10, 2304-2308. 51. LKB Ampholine polyacrylamide gel plates (PAG plates) were used as per instructions of the supplier. A pH range of 3.5 - 9.5 (LKB # 1804-101) was employed. 52. = 0.355, After heme extraction, 17.2 mg (A 2 8 0 17200 m.w} 8 45 tion. The zinc centrifugation 0.403, 40 protein is and mL) mL) of protein mesoporphyrin a Sephadex were actually in were available for reconstitu- was inserted, and following G-25-80 recovered. = 15.8 mM- 1 em - 1 , t The apomyoglobin porphyrin is not included in the calculation. column, 17.5 amount equivalents mg of (A 2 8 0 reconstituted as the zinc -11653. Edmundson, A. B.; Hirs, C. H. W. J. Mol. Bioi. 1962, 5, 663-682. 54. Hanania, I. G. H.; Yeghiayan, A.; B. Cameron, Biochem. F. J. 1966, 98, 189-192. 55. Bowen, W. J. J. Bioi. Chern. 1949, 179, 235-245. 56. Sundberg, R. J.; Gupta, G. Bioinorg. Chern. 1973, 3, 39-48. 57. Emission experiments were conducted by J. L. Marshall; many thanks. 58. Hoffman, B. M. J. Am. Chern. Soc. 1975, 97, 1688-1694. 59. Kalyanasundaram, K.; J. Phys. Chern. results. These experiments Neumann-Spallart, M. 1982, 86, 5163-5169. 60. Zang, L.-H.; Maki, utilize optically A. H., detected unpublished magnetic resonance (ODMR) and involve = 698 nm) J. Biochem. the direct measurement of the Mb( 3 ZnP*) lifetime (i\ max in frozen rna trices. 61. Vanderkooi, J. M.; F.; Adar, M Erecinska, Eur. 1976, 64, 381-387. 62. Conrad, the R. H.; dipole-dipole (mor 1 ), n is Biochemistry 1978, orientation factor, NA is refractive index of the Brand, the L. natural lifetime of the donor, and donor and acceptor (em). (cm 6 mor 1 ) is the fF 0 (i\)€ A (i\)i\ 4 di\ is Avogadro's number medium, is the distance between the is the T overlap integral emission as a function of wavelength and fF 0 (i\)di\ = 1. the molar spectrum of the (cm 2 mor 1 ). The overlap calculated by normalizing the by the extinction extinction integral spectrum and was describes K probability decadic F 0 (i\) 777-787. the emission where R 7, multiplying F0 of acceptor acceptor donor € A (i\) is donor -117coefficient and i\ 4 at each wavelength. The product was plotted vs. i\ and the area is the value of the overlap integral. 63. G.; Navon, Sutin, !nor g. N. lowest-lying excited energy to Chern. 4 state, 13, 1974, T 1 g' in 16700 cm- 1 2159-2164. the Ru 3+N 600 nm). The complex 6 a 6 Ru(CI0 4 ) 3 is at 435 nm (23000 cm- 1 ). The a Ru(His) 3+ spectrum 5 is red-shifted with respect to the a Ru 3+ lowering the 4T 1g 6 ' level as little as ( Beyond 650 nm, the extinction coefficients are negligible. 5 5 64. Dexter, D. L. J. Chern. Phys. 1953, 21, 836-850. 65. Ideally, direct observation of would provide species, however, either R u(II) or ZnP+ this cone! us ion. These ' an electron-transfer unequivocable have product, evidence for not been spectro- Work with zinc-sub- scopically observed in the a 5 Ru(His)Mb(ZnP) systems. 66. Winkler, J. sti tu ted cytochrome sees, feature not b) The amount a action. R., personal communication. a). reveals that the slow expected m the reverse c of impurity appears component to phosphore- electron-transfer re- correspond the to number of fractions pooled together to make-up the sample .. 67. A more stringent selection of elution with sodium fractions lowers the minor in an inert four cycles of component contribution. 68. Samples atmosphere were box. treated Excess dithionite was dithionite removed by concentration and dilution with an Amicon ultrafiltration unit. 69. Ringe, D.; Petsko, G. A.; Kerr, D. E.; Ortiz de Montellano, P. R . Biochemistry 1984, 23, 2-4. 70. Hartmann, H.; Parak, F.; Steigemann, W.; Petsko, G. A.; Ringe -118- Ponzi, D.; Frauenfe1der, Proc. H. Nat/. Acad. Sci. U.S.A . 1982, 79, 4967-4971. 71. Ringe, G. A. Prog. Biophys. Molec. Bioi. 1985, 45 Sutin, N. Biochirn. Biophys. Acta 1985, 811, 265- J. J. Chern. Phys. Institute of D.; Petsko, R. A .; 197-235. 72. Marcus, 322. 73. Hush, N. S. Coord. Chern. Rev. 1985, 64, 135-157. 74. Beratan, D. N.; Onuchic, J. N.; Hopfield, J. 1985, 83, 5325-5329. 75. Scheidt, W. R. Ace. Chern. Res. 1977, 10, 339-345. 76. Ayrovainen, M Candidacy report, California Technology 1984, p. 5. 77. Ringe, D.; Petsko, G., unpublished results. 78. Hopfield, J. J. Proc. Nat!. Acad. Sci. USA 1974, 71, 3640-3644. 79. Beratan, D. N., personal communication. 80. Liang, N.; Pielak, G. J.; Mauk, A. G.; Smith, M.; Hoffman, B. M. J.; H. B. M. J. Am. Am. Chern. Proc. Nat!. Acad. Sci. U.S.A., in press. 81. Mayo, S. L.; Ellis, W. R., Jr.; Crutchley, R. Gray, Science 1986, 233, 948-952. 82. Peterson-Kennedy, S. E.; McGourty, J. L.; Hoffman, B. Chern. Soc. 1984, 106, 5010-5012. 83. Poulos, T. L.; Mauk, A. G. J. Bioi. Chern. 1983, 258, 7369-7373 . 84. Salemme, F. R. J. Mol. Bioi. 1976, 102, 563-568. 85. Poulos, T. L.; Kraut, J. J. Bioi. Chern. 1980, 255, 10322-10330. 86. Calcaterra, L. T.; Soc.1983, 105, 670-671. Closs, G. L.; Miller, J. R. J. -11987. Miller, J. R.; Calcaterra, L. T.; Closs, G. L. Hush, N. Am. J. Chern. Soc.1984, 106, 3047-3049. 88. Verhoeven, J. W.; Paddon-Row, M N.; S.; Oevering, H.; 1985, 107, Heppener, M. Pure & Appl. Chern. 1986, 58, 1285-1290. 89. Heitele, H.; Michel-Beyerle, M D.; A.; E. Am. J. Chern. Soc. 8286-8288. 90. Joran, A. Leland, B. Geller, G. G.; Hopfield, J. J.; Michel, H. J. Dervan, P. B. J. Am. Chern. Soc. 1984, 106, 6090-6092. 91. Larsson, S. J. Am. Chern. Soc. 1981, 103, 4034-4040. 92. Deisenhofer, J.; Epp, 0.; Miki, K.; Huber, R.; Mol. Bioi. 1984, 180, 385-398. 93 . Karplus, M.; McCammon, J. A. Sci. Amer. 1986, 254( 4 ), 42-51.