Kinetics of redox-linked proton pumping activity of native and subunit III-depleted cytochrome c oxidase: a stopped-flow investigation.
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.
Sarti
P
P
Jones
M G
MG
Antonini
G
G
Malatesta
F
F
Colosimo
A
A
Wilson
M T
MT
Brunori
M
M
D000818:Animals; D001188:Artiodactyla; D001693:Biological Transport, Active; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000378:metabolism; D046911:Macromolecular Substances; D008564:Membrane Potentials; D008929:Mitochondria, Heart / Q000201:enzymology*; D009206:Myocardium / Q000201:enzymology; D011522:Protons
D007473:Ion Channels; D046911:Macromolecular Substances; D011522:Protons; D003576:Electron Transport Complex IV