Open AccessKinetics of redox-linked proton pumping activity of native and subunit III-depleted cytochrome c oxidase: a stopped-flow investigation.
Author(s) -
Paolo Sarti,
Meriel G. Jones,
Giovanni Antonini,
Francesco Malatesta,
Alfredo Colosimo,
Michael T. Wilson,
Maurizio Brunori
Publication year - 1985
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.82.15.4876
Subject(s) - cytochrome c oxidase , chemistry , redox , kinetics , electron transport complex iv , cytochrome , cytochrome c , proton , electron transport chain , oxidase test , cytochrome c1 , stoichiometry , proton pump , respiratory chain , biophysics , photochemistry , coenzyme q – cytochrome c reductase , biochemistry , inorganic chemistry , mitochondrion , enzyme , biology , atpase , physics , quantum mechanics
The kinetics of oxidation of reduced cytochrome c by cytochrome c oxidase reconstituted into unilamellar vesicles (COV) has been followed by stopped-flow method in the time range 3 msec-1 sec. In the presence of valinomycin, the oxidation of cytochrome c is linked to proton ejection in the external medium, with an apparent stoichiometry (H+/e-) of 0.93 +/- 0.22, under conditions in which the enzyme is in the more active "pulsed" state (i.e., having undergone oxidation-reduction cycles). The time course of reaction indicates that the conformational change(s) involved in coupling the redox reaction to proton translocation is fast. Similar experiments carried out with cytochrome c oxidase depleted of subunit III show that proton-pumping is maintained, although with a lower efficiency (H+/e- = 0.5). The number of protons ejected per electron appears to be correlated to the value of the respiratory control ratio; although this result is partly due to an increase in the rate of diffusion back into the vesicles, a relationship between the respiratory control ratio and the efficiency of the proton pump may be inferred, suggesting a control of the H+/e-ratio.