Proton and calcium circuits across the mitochondrial inner membrane.

polation of the H + back-flow rate to the half-time for 0, reduction obtained from the independently measured state 3 0, uptake rate. However, the oxygen-pulse method, even when improved by elimination of very fast H + back-flow on the H +/H,PO,symporter (Reynafarje et al., 1976), always underestimates the mechanistic H + /O ratio for 3 reasons. ( I ) The half-time for 0, reduction under oxygen-pulsed conditions is much shorter than calculated from state 3 0, consumption rates. (2) H+/O flow ratios obtained from the amount of H + at the half-time for O2 reduction, by which time much H + back-flow has already taken place, are necessarily much lower than those obtained at level flow. (3) There is in fact no kinetically valid way of obtaining the H + /O ratio at close to level flow by extrapolation of the total amount of H + ejected to some point at which a given fraction of the added O2 has been consumed, because H + back-flow begins at time zero and increases in rate during the course of 0, uptake as ApH increases. Only by measurement of the rates of both 0, uptake and H + ejection at conditions approaching level flow, or by measurement of both flows and forces in conditions between level flow and static head, can the mechanistic H + / O ratio be closely approached. H +/O or q+/O ratios of 8 for succinate and approaching 12 for NADH oxidation, as we and some others have observed, raise questions as to which of the approx. 20 redox centres of the chain are directly involved in energy transduction, and by what mechanism (whether by ligand conduction, conformational transitions, or other processes) does this occur.