The H+/site ratio of mitochondrial electron transport

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. Measurement of the uptake of weak acids during loading of mitochondria with Ca²⁺ demonstrated that 2.0 weak acid anions were accumulated per Ca²⁺ ion. Since 1.7 to 2.0 Ca²⁺ ions were taken up per site, these data correspond to an H⁺/site ratio of 3.5 to 4.0. 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. 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; the results show that the H⁺/site ratio is at least 3.0 and may be as high as 4.0.