The Cuᴀ Site in Cytochrome c Oxidase: Its Role in Coupling Electron Transport to Proton Pumping

Citation

Gelles, Jeff Daniel (1986) The Cuᴀ Site in Cytochrome c Oxidase: Its Role in Coupling Electron Transport to Proton Pumping. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/vr49-7048. https://resolver.caltech.edu/CaltechTHESIS:10112019-095757311

Abstract

Cytochrome c oxidase contains a copper-ion electron transfer site, Cu_A, which has previously been found to be unreactive with externally added reagents under conditions in which the protein remains structurally intact. We have studied the reaction of cytochrome oxidase with sodium p-hydroxymercuribenzoate (pHMB) and found that the reaction proceeds, under appropriate conditions, to give an excellent yield of a particular derivative of the Cu_A center which has electron paramagnetic resonance and near-infrared absorption spectroscopic properties which are distinctly different from those of the unmodified center. Spectroscopic and chemical characterization of the other metal-ion sites of the enzyme reveals little or no effect of the pHMB modification on the structures of and reactions at those sites. Of particular interest is the observation that the modified enzyme still displays a substantial fraction of the native steady-state activity of electron transfer from ferrocytochrome c to O₂. Although the modified copper center retains the ability to receive electrons from the powerful reductant Na₂S₂O₄ and to transfer electrons to O₂, it is not significantly reduced when the enzyme is treated with milder (higher potential) reductants such as NADH/phenazine methosulfate or the physiological substrate ferrocytochrome c.

Cu_A exhibits many spectroscopic and chemical properties which make it highly atypical of cuproprotein active sites; the singular nature of this site has prompted speculation about the importance of the structural peculiarities of this metal-ion center in the catalytic cycle of the enzyme. In this work, we demonstrate that the unusual features of this site are not prerequisites for competent catalysis of electron transfer and O₂ reduction by the enzyme. These observations suggest that a secondary electron transfer pathway not involving Cu_A exists between the cytochrome c and oxygen binding sites which can function at a rate at least 20% of the total turnover rate of the native enzyme.

Cytochrome c oxidase converts free energy released in respiratory electron transport into a metabolically useful form by contributing to the potential gradient across the mitochondrial inner membrane. Both a process involving electron transfer linked proton pumping and a process involving electron transfer from ferrocytochrome c to O₂ contribute to the potential gradient. Taken together with those from other recent studies, the results of the experiments support a model for electron transfer in cytochrome oxidase in which Cu_A and Fe_a are parts of separate, parallel electron transport pathways between cytochrome c and the cytochrome oxidase O₂ reduction site. This model has important implications for the role of Cu_A in respiratory energy transduction by cytochrome oxidase. It suggests that Cu_A is the best candidate among the four metal center sites in cytochrome oxidase to be the site of redox-linked proton pumping.

In order to explore more fully the mechanistic aspects of energy transduction in cytochrome oxidase, we propose a complete chemical mechanism for the enzyme's proton pump. The mechanism achieves pumping with chemical reaction steps localized at a single redox site within the enzyme; no indirect coupling through protein conformational changes is required. The proposed mechanism is based on a novel redox-linked transition metal ligand substitution reaction. The use of this reaction leads in a straightforward manner to explicit mechanisms for achieving all of the processes determined by Blair, et al. (D.F. Blair, J. Gelles, and S.I. Chan, Biophys. J., in press) to be needed to accomplish redox-linked proton pumping. These processes include: 1) modulation of the energetics of protonation/deprotonation reactions and modulation of the energetics of redox reactions by the structural state of the pumping site; 2) control of the rates of the pump's redox reactions with its electron transfer partners during the turnover cycle (gating of electrons); and 3) regulation of the rates of the protonation/deprotonation reactions between the pumping site and the aqueous phases on the two sides of the membrane during the reaction cycle (gating of protons). The model is the first proposed for the cytochrome oxidase proton pump which is mechanistically complete and specific enough that a realistic assessment can be made of how well the model pump would function as a redox-linked energy transducer. This assessment is accomplished via analyses of the thermodynamic properties and steady-state kinetics expected of the model. These analyses demonstrate that the behavior of a pump based on the model would be very similar to that observed of cytochrome oxidase both in the mitochondrion and purified preparations. Specifically, calculation of the properties of the model pump at equilibrium demonstrates that the behavior expected of the model pump in an electrochemical titration is the same as that observed for the Cu_A center: a nearly pH-independent midpoint potential of approximately 290 mV. An analysis of the performance of the pump during steady-state turnover demonstrates that the model pump is expected to function efficiently and with good power output under physiological conditions, and that its properties under conditions of varying load are similar to those observed in experiments on respiring mitochondria and on purified cytochrome oxidase reconstituted into artificial lipid vesicles.

Although the analysis presented here concerns only a single model of a redox-linked proton pump, it leads to some important general conclusions regarding the mechanistic features of such pumps. The first is that a workable proton pump mechanism does not require large protein conformational changes. Another conclusion is that a redox-linked proton pump need not display a pH-dependent midpoint potential as has frequently been assumed. A final conclusion is that mechanisms for redox-linked proton pumps that involve transition metal ligand exchange reactions are quite attractive because such reactions readily lend themselves to the linked gating processes necessary for proton pumping.

Several of the results of this research form the basis of a new approach to studying the role of Cu_A in energy transduction by cytochrome oxidase. I describe several continuing experimental programs based on this method, and summarize the goals, experimental design, and progress of these studies. I conclude by considering the physiological significance of the new conceptions about the role of Cu_A in energy transduction by cytochrome oxidase arising from this research.

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