307 Biochimica et Biophysica Acta, 501 (1978) 307--316 © Elsevier/North-Holland Biomedical Press BBA 47438 PROTON ELECTROCHEMICAL GRADIENT AND PHOSPHATE POTENTIAL IN MITOCHONDRIA G.F. AZZONE, T. POZZAN and S. MASSARI With the technical assistance of Mr. L. PREGNOLATO C.N.R. Unit for the Study of Physiology of Mitochondria and Institute of General Pathology, University of Padua, Padua (Italy) (Received April 1st, 1977) (Revised manuscript received August 31st, 1977) Summary The paper reports an analysis of the relationship between A~H the proton electrochemical potential difference, and AGp, the phosphate potential. Depression of A~H and AGp has been obtained by titration with: (a) carbonylcyanide trifluoromethoxyphenylhydrazone; (b)nigericin (+ valino- mycin); (c)KCI (+ valinomycin); and (d)rotenone. The uncoupler depresses A~H more than nigericin (+ valinomycin), KC1 (+ valinomycin) and rotenone at equivalent AGp. The AGp/A~H ratio is about 3 at high values of A~ H. When AGp and A~H are depressed by nigericin {+ valinomycin) the AGp/A/~ H ratio remains constant. When AGp and A/~ H are depressed by uncouplers, the AGp/A~ H ratio increases hyperbolically tending to infinity while A~H tends to zero. The absence of con- stant proportionality between AGp and A~H indicates that the proton gradients driving ATP synthesis presumably operate within microscopic environments. Introduction Lardy [1] and Chance and Williams [2,3] considered respiratory control to be of kinetic origin. Klingenberg [4,5] considered respiratory control as due to an increased rate of reversed electron transfer. It is now generally accepted that oxidation reduction reactions and ADP phosphorylation are near equilibrium i.e. mitochondrial respiration is dependent on AGp, the phosphate potential [6--10]. If A~H is the obligatory intermediate between electron transfer and ATP synthesis some sort of constant relationship should exist between A~H and AGp. Abbreviations: TPMP, triphenylmethylphosphonium; FCCP, carbonyl cyanide p-trifluoromethyl- phenylhydrazone.