Cytochrome c oxidase is not a proton pump

Measurements of mitochondrial ←H+/O quotients: Effects of phosphate and N‐ethylmaleimide

“…In this system, previous work on cytochrome c oxldase [6], and measurements of +e-10 quotients by means of -+K'/O quotients in the present paper, show that the only significant process producing ApH, apart from a very slow leak through the antimycin block, is as given in eq. (1):…”

“…1 A, and the equivalent slight shortfall of net alkalinisation in fig.lB, are accounted for by the normally protomnotive antimycin-insensitive respira-tion, which proceeds at about 10% of the rate of ferrocytochrome c oxidation in this type of experiment (and see [6] has not been corrected for the antimycin-insensitive respiration, but the pK, time-course has been corrected for a baseline shift caused by injecting the ah-saturated (K+-free) sucrose solution. The pK, was 3.1-3.2 when the 0, pulses were injected after the 20 min preincubation period.…”

“…The general experimental procedures for the 02-pulse experiments with the antimycin-and rotenonetreated mitochondria were as before [6]. To allow for the small protonmotive and electrogenic effect of the antimycin-insensitive respiration during the O2 pulses with ferrocytochrome c or DADHa as reductant, we estimated the rate of oxygen consumption in the absence of added reductant and expressed it as a fraction of that in the presence of each reductant.…”

Cytochrome c Oxidase is a proton pump

“…Since ferrocyanide specifically reduces cytochrome c in mitochondria [S ] initiating respiration, we interpreted the proton ejection as being linked to cytochrome c oxidase activity. This interpretation was, however, rejected in [7], where H' ejection was suggested to be an artefact due to oxidation of an unidentified hydrogenated mitochondri~ reductant by the ferricyanide formed during respiration with ferrocyanide. Moyle and Mitchell [7] proposed further that the reduction of formed ferricyanide by the endogenous reductant is not only linked to mere release of H', but is in addition linked to the activity of a protonmotive system catalysing proton translocation across the mitochondrial membrane.…”

“…Moyle and Mitchell [7] proposed further that the reduction of formed ferricyanide by the endogenous reductant is not only linked to mere release of H', but is in addition linked to the activity of a protonmotive system catalysing proton translocation across the mitochondrial membrane. We may interject here that such a process would have to be insensitive to both rotenone (which inhibits NADH dehydrogenase [9]) and antimycin (which inhibits electron transfer in the cytochrome b-cl complex [lo]), since the proton ejection observed during ferrocyanide oxidation is not sensitive to these inhibitors [I ,2] In support of the proposal in [7], the consumption of H' during uncoupled respiration with ferrocyanide, in many conditions, was reported not to match the rate of oxygen consumption. In cases uncomplicated by side reactions, eq.…”