CHARACTERIZATION OF THE INHIBITORY MECHANISM OF 1-METHYL-4-PHENYLPYRIDINIUM AND 4-PHENYLPYRIDINE ANALOGS IN INNER MEMBRANE PREPARATIONS

We have investigated the mechanism of the inhibition of membrane-bound NADH dehydrogenase by 1-methyl-4-phenylpyridinium (MPP+) and a series of its 4'-alkyl-substituted analogs of increasing hydrophobicity, as well as their neutral, desmethyl congeners. Comparison of hydrophobici ty, as measured by partition coefficients, with the IC50 for the inhib ition of NADH oxidase activity in mitochondrial inner membrane prepara tions shows a negative correlation, but the cationic inhibitors are mo re effective than the neutral analogs with similar hydrophobicity. The presence of 10 mum tetraphenylboron (TPB-) potentiates the inhibitory power of positively charged analogs up to 4'-pentyl-MPP', while the n eutral inhibitors are unaffected by TPB-. Without TPB-, the more hydro philic analogs give incomplete inhibition, but the inclusion of TPB- p ermits the attainment of complete inhibition, accompanied by the appea rance of sigmoidal titration curves. These data support the hypothesis that MPP+ analogs, like rotenone, are bound at two sites on the enzym e and occupancy of both is required for complete inhibition. TPB-, by forming ion pairs with the cationic analogs, facilitates their equilib ration to both sites in membrane preparations. When present in molar e xcess over the MPP+ analog, TPB- partially reverses the inhibition by decreasing its concentration in the more hydrophilic binding site. The effect of temperature and of pH on the IC50 values for inhibition sup port the concept of dual binding sites, and the pH dependence of the i nhibition reveals the participation of two ionized protein groups in t he binding, one of which may be a thiol group.