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Cytochrome c oxidase : studies of electron input and intramolecular electron transfer

Citation

He, Qizhi (1995) Cytochrome c oxidase : studies of electron input and intramolecular electron transfer. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-10052007-085601

Abstract

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Cytochrome [...] oxidase is the terminal enzyme of the electron transport chain in mitochondria. This enzyme catalyzes the transfer of electrons from ferrocytochrome [...] to dioxygen and reduces it to water. Concomitant with electron transfer and dioxygen reduction, cytochrome [...] oxidase pumps protons from the matrix side to the cytosolic side of the inner membrane, contributing to the formation of a transmembrane electrochemical gradient. As such electron transfer plays a central role in the function of the enzyme. This thesis investigated the input of electrons into the enzyme, the flow of electrons among redox-active metal centers in the enzyme as well as conformational changes associated with the redox state changes of the protein.

During electron transport, cytochrome [...] diffuses to a binding site or sites on cytochrome [...] oxidase. Extensive chemical modification studies have indicated that seven highly conserved lysine amino acids surrounding the heme crevice of cytochrome [...] are involved in the formation of an electrostatic complex with cytochrome [...] oxidase. Lysine 86 in cytochrome [...] is believed to be crucial in the binding of cytochrome [...] with cytochrome [...] oxidase. In order to study the effect of binding orientation between cytochrome [...] and cytochrome [...] oxidase with respect to the intracomplex electron transfer, we modified cytochrome [...] with ruthenium bis(bipyridine) dicarboxybipyridine at lysine 86 (Ru-86-cytochrome [...]). Our results showed that upon laser excitation of the preformed complex of Ru-86-cytochrome [...] and cytochrome [...] oxidase, electron was transferred rapidly from the ruthenium group to the ferric heme of cytochrome [...]; subsequently to the cytochrome [...] oxidase. The observed intracomplex rate constants for the oxidation of cytochrome [...] are found to be biphasic with magnitudes of 560 [...] for one phase and 114 [...] for the other phase. The rate constant for the reduction of cytochrome [...] in cytochrome [...] oxidase is 2.3 x [...]. No reduction of [...] was observed at 830 nm. Although the observed rate constants for the oxidation of cytochrome [...] are slow, we believe there is a fast kinetic phase for this process beyond the resolution capability of the instrument. Apparently, the bulky ruthenated moiety on cytochrome [...] alters the binding orientation of cytochrome [...] with cytochrome [...] oxidase. As a result, cytochrome [...] preferentially transfers electron directly to cytochrome [...] rather than to [...]. The intracomplex electron transfer rate also exhibited ionic strength dependence as expected.

Internal electron transfer in cytochrome [...] oxidase was investigated by photolysis of CO-bound mixed-valence form of the enzyme. Upon CO photodissociation, ferrocytochrome [...] was generated in less than 0.1 [...]sec, and a subset of the reduced cytochrome [...] was reoxidized with biphasic rate constants of [...] = 1.0 x [...] and [...] = 7.8 x [...]. Reduction of cytochrome [...] was also observed with biphasic rate constants of [...] = 1.6 x [...] and [...] = 9 x [...]. The stoichiometry of oxidized cytochrome [...] to reduced cytochrome [...] was found to be 1:1. No apparent electron transfer to [...] was observed at 830 nm. These results indicate that there is a significant electron reequilibration only between cytochrome [...] and cytochrome [...] upon photodissociation of the CO-bound mixed-valence enzyme.

The nature of zinc in cytochrome [...] oxidase was investigated by depletion of zinc with mercuric chloride. The removal of zinc does not alter the steady-state and transient electron transfer activities of the enzyme. The study indicates that zinc plays a structural role in the enzyme by serving as a bridge between subunit VIa and VIb. Based on indications that there is allosteric interaction companying redox state changes of cytochrome [...] oxidase, we probed the protein matrix for conformational changes using a fluorescence label on the enzyme. As a result of our findings, we concluded that there is a redox-linked conformational change in cytochrome [...] oxidase.

Item Type:Thesis (Dissertation (Ph.D.))
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Restricted to Caltech community only
Research Advisor(s):
  • Chan, Sunney I.
Thesis Committee:
  • Chan, Sunney I. (chair)
  • Rees, Douglas C.
  • Gray, Harry B.
  • Okumura, Mitchio
Defense Date:9 November 1994
Record Number:CaltechETD:etd-10052007-085601
Persistent URL:http://resolver.caltech.edu/CaltechETD:etd-10052007-085601
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:3935
Collection:CaltechTHESIS
Deposited By: Imported from ETD-db
Deposited On:18 Oct 2007
Last Modified:26 Dec 2012 03:04

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