Progress Toward Elucidating the Mechanisms of Energy Transduction in Cytochrome c Oxidase

Citation

Blair, David Francis (1986) Progress Toward Elucidating the Mechanisms of Energy Transduction in Cytochrome c Oxidase. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/g33b-bw96. https://resolver.caltech.edu/CaltechTHESIS:08162019-111201815

Abstract

The exothermic transfer of four electrons from cytochrome c to dioxygen is catalyzed in eucaryotes by mitochondrial cytochrome c oxidase. A large fraction of the energy made available from these electron transfers is conserved in the form of a proton electrochemical gradient across the mitochondrial inner membrane. Two energy-conserving mechanisms operate in cytochrome c oxidase: First, the electron transfers to dioxygen are themselves electrogenic, since the electrons originate from the outer side of the membrane while the protons required to produce water originate from the inner side. Second, the electron transfers are coupled to the active transport across the membrane of additional protons, perhaps as many as one for every electron transferred. In this work, investigations which are intended to contribute to a detailed understanding of the mechanisms of energy transduction in cytochrome c oxidase are described.

Cytochrome c oxidase performs its catalytic functions with the assistance of four metal ion sites. The redox thermodynamic properties of three of these sites have been extensively characterized by spectroelectrochemical methods. A fairly complex interactive scheme involving all four metal centers is required to account for the observed thermodynamic behavior. Via measurements of reduction potential temperature dependences, it is found that the standard entropies of reduction of the two sites which have been suggested to play a role in proton pumping (cytochrome a and Cu_A) are unusually large and negative, suggesting that a substantial protein-ordering conformational change accompanies their reduction. The reduction potential of cytochrome a is only moderately pH-dependent, indicating that its reduction is not stoichiometrically coupled to proton uptake.

The energy made available for transduction by the oxidase comes primarily from the exothermic transfer of electrons from the metal centers to dioxygen. The mechanism of these electron transfers has been examined in a detailed kinetic study of the reaction of the enzyme with dioxygen at low temperatures. Two different reaction intermediates have been found in which dioxygen is reduced to the same, namely, the three-electron level. The conversion between these two intermediates is rather highly activated but is promoted by entropic factors. It is suggested that this step corresponds to the breaking of the oxygen-oxygen bond. Earlier suggestions that electrons may be transferred via multiple pathways through the enzyme have been confirmed and extended. An examination of the reaction of partially reduced samples of the oxidase with dioxygen indicates that the rates of internal electron transfer within the oxidase are different at different steps in dioxygen reduction. Multiple pathways and step-dependent rates are expected to have important implications for the mechanisms of energy conservation by the oxidase.

The steady-state kinetics of a simple model for redox-linked proton pumping has been examined in a final chapter. In this analysis, special emphasis was given to the importance of controlling electron flows through the transducing site. The consideration of electron flows results in some novel conclusions which assist in rationalizing the results of the redox thermodynamic study. It is expected that the analysis should also contribute to the design and interpretation of further experiments intended to reveal the mechanisms of proton pumping.

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