Spectroscopy and Chemistry of Microperoxidases

Author: Low, Donald W.

Year: 1997

Degree: Dissertation (Ph.D.)

Advisor: Gray, Harry B.

Committee Members: Bercaw, John E.; Gray, Harry B.; Chan, Sunney I.; Richards, John H.

Option: Chemistry

DOI: 10.7907/1zqe-ts23

Abstract

Microperoxidases (MP) fragments of horse cytochrome c (cyt c) were prepared and used as models for heme proteins. The spin state and electronic structure of the ferric heme octapeptide fragment (MP8) was investigated by NMR, EPR, UV-visible, resonance Raman and magnetic susceptibility methods. The heme iron is in equilibrium between S = 5/2 and S = 1/2 spin states. NMR studies on the ferric cyanide complex of MP8 indicates that residues 14-18 form a rigid structural motif in cyt c that is preserved in MP8. The imidazole of His 18 is oriented along the α-γ meso axis of the heme in both MP8 and cyt c. The reactivity of ferric MP8 towards single-electron transfer oxidations was investigated by nanosecond flash-photolysis methods. Spectroscopic and kinetic evidence indicate that the MP8 porphyrin π cation radical is readily generated by oxidation with photochemically generated Ru(bpy)33+, and is an intermediate in the formation of ferryl MP8. The analogous oxidation reaction was also investigated in manganese substituted MP8, and high-valent manganese-oxo MP8 was detected by nanosecond transient absorption. The single electron transfer oxidation of cytochrome c peroxidase (CcP) and thiol bound H93G sperm whale myoglobin were also explored. Reverse proteolysis reactions were used to modify the C-terminus of the heme nonapeptide (MP9). Histidine, methionine, tyrosine and tryptophan amides were readily added by trypsin catalyzed peptide synthesis reactions in aqueous DMF solutions. The intramolecular ligand binding properties of the X23MP10 (X = H, M, Y) series was examined by UV-visible and resonance Raman methods. H23MP10 readily forms a low-spin bis-histidine ligated heme center, Y23MP10 appears to bind phenolate at high pH, and M23MP10 does not appear to bind its methionine thioether. The ferrous carbonmonoxy form of MP8 (Fe(II)-CO MP8) was prepared and its photochemistry was explored by transient absorption and resonance Raman methods. Fe(II)-CO MP8 photolysis reactions were used for comparison with cyt c folding reactions initiated by CO photodissociation.

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