Stoichiometry of proton translocation coupled to substrate oxidation in plant mitochondria

The proton translocation coupled to the electron flux from succinate, exogenous NADH, and NAD(+)-linked substrates (malate and isocitrate) to cytochrome c and to oxygen was studied in purified potato (Solanum tuberosum) mitochondria using oxygen and ferricyanide pulse techniques. In the presence of valinomycin plus K(+) (used as a charge compensating cation), optimum values of H(+)/2 e(-) were obtained when low amounts of electron acceptors (oxygen or ferricyanide) were added to the mitochondria (1-2 nanogram [2 e(-)] equivalents per milligram protein). The stoichiometry of proton translocation to electron flux was unaffected in the presence of N-ethylmaleimide, an inhibitor of the Pi/H(+) symport. With succinate as substrate, H(+)/2 e(-) ratios were 4.0 +/- 0.2 and 3.7 +/- 0.3 with oxygen and ferricyanide as electron acceptors, respectively. With exogenous NADH, H(+)/2e(-) ratios were 4.1 +/- 0.9 and 3.4 +/- 0.2, respectively. The proton translocation coupled to the oxidation of NAD(+)-linked substrates (malate, isocitrate) was dependent upon the presence of adenylates (ADP, AMP, or ATP). For malate (+ glutamate) oxidation the observed H(+)/2 e(-) ratios were increased from 3.6 +/- 2.2 to 6.5 +/- 0.5 in the presence of 20 micromolar ADP.