Structure-Function Studies on Cytochrome c Oxidase: Part I. An Investigation into the Nature of the Metal Sites in Cytochrome c Oxidase using X-Ray Absorption Spectroscopy. Part II. An Investigation into the Lipid Factors Affecting Protein Activity and Respiratory Control in Reconstituted Cytochrome c Oxidase Membranes

Author: Hu, Valerie Wailin

Year: 1978

Degree: Dissertation (Ph.D.)

Advisor: Chan, Sunney I.

Committee Member: Unknown, Unknown

Option: Chemistry; Biology

DOI: 10.7907/qxtt-0d68

Abstract

Cytochrome c oxidase is a membrane-bound enzyme of the mitochondrial respiratory chain which is ultimately responsible for almost all of the oxygen consumption in aerobic organisms. In a rapid four-electron process, it reduces molecular oxygen to water and, at the same time, couples the energy derived from this exothermic reaction with energy conservation by driving ATP synthesis. The proper functioning of cytochrome oxidase in oxygen reduction and energy coupling requires not only phospholipids but also an intact, vectorially-oriented membrane. In the study described briefly below, two different aspects of the cytochrome oxidase problem have been examined: 1) the nature of the metal sites in the isolated molecule with regard to their participation in the oxidation/reduction process, and 2) the functional role of lipids in the activation of the protein.

Part I deals with an investigation into the nature of the metal sites in cytochrome c oxidase. For this purpose, X-ray absorption edge spectroscopy was employed to examine the oxidation states of the copper and iron centers in the protein under oxidized, fully-reduced, and partially-reduced conditions. In addition, the edge spectra of oxidized and reduced plastocyanin (a "blue copper" protein containing a single Cu ion) as well as of a number of model Cu compounds in various oxidation states were obtained to establish energy ranges for the several bound to bound transitions of Cu in the different oxidation states. A comparison of the fine structure detail in the absorption edge spectra of cytochrome oxidase with those of the models indicates that one of the two copper ions in the oxidized protein is in the +1 oxidation state. Upon reduction of the protein with dithionite, the second copper becomes Cu(I). The Fe K-edge spectra of cytochrome c oxidase reveals a small shift(~ 2 eV) towards lower energies upon reduction. This is comparable with the shift observed for the reduction of the heme iron in cytochrome c [l]. Studies on the cyanide complexes of cytochrome oxidase indicate that 1) cyanide has no effect on the Cu K-edge spectrum of oxidized cytochrome oxidase, 2) the 1s → 3d transition of the Fe edge of oxidized oxidase is enhanced by the presence of cyanide, and 3) both of the Cu ions are reduced in the partially-reduced cyanide complex.

Part II of this thesis is concerned with an investigation into the lipid factors affecting protein activity and respiratory control in reconstituted cytochrome oxidase membranes. Delipidated cytochrome c oxidase was reconstituted with various natural and synthetic phospholipids and several aspects of protein-lipid interactions were studied. In a well-defined series of model membranes, it was shown that cytochrome oxidase activity, monitored at a constant temperature, was sensitive to the physical state of the lipids, being higher when the lipids were in the liquid-crystalline phase. In addition, the temperature at which the reconstitution was allowed to take place was found to affect the manifestation of phase-dependent oxidase activity. A passive charge diffusion model has been proposed to explain the observed phenomena.

The effect of protein on the lipid phase transition has also been investigated using light scattering techniques. A heating/cooling hysteresis in the absorbance versus temperature curves has been attributed to a reversible aggregation and dispersal of the proteins in the bilayer which is dependent on the physical state of the lipids.

Several lipid factors have been investigated with regard to their influence on the reconstitution of cytochrome oxidase membranes exhibiting respiratory control. It was found that high lipid/protein ratios and the presence of acidic polar head groups are conducive to the establishment of respiratory control as evidenced by increased rates of oxygen consumption in the presence of chemical uncouplers. In addition, the mode of vesicle preparation affected the responsiveness of the samples towards uncoupling agents.

Files