Abstract
The number of H^+^ ejected during passage of 2e^−^ through each energy‐conserving site of the mitochondrial respiratory chain (the H^+^/site ratio) was measured in three ways. In each case transmembrane movements of endogenous phosphate were minimized. (1) Measurement of the uptake of weak acids during loading of mitochondria with Ca^2+^ demonstrated that 2.0 weak acid anions were accumulated per Ca^2+^ ion. Since 1.7 to 2.0 Ca^2+^ ions were taken up per site, these data correspond to an H^+^/site ratio of 3.5 to 4.0. (2) More direct measurement of H^+^ ejection using the oxygen pulse technique demonstrated that the H^+^/site ratio was 3.0. In these experiments phosphate movements were prevented by addition of N‐ethylmaleimide to inhibit phosphate‐hydroxide antiport, by washing the mitochondria to remove endogenous phosphate, or by working at 5°C to reduce the rate of phosphate transport. When phosphate movements were allowed, H^+^/site ratios of 2.0 were observed. (3) Measurement of the initial steady rates of oxygen consumption and H^+^ ejection following addition of substrate to aerobic, substrate‐limited mitochondria yielded H^+^/site ratios of 2.0, which were elevated to 4.0 when phosphate transport was prevented as described above.
Previous determinations of the H^+^/site ratio were thus underestimates due to the unrecognized movements of endogenous phosphate; our results show that the H^+^/site ratio is at least 3.0 and may be as high as 4.0.