INHIBITOR PROBES OF THE QUINONE BINDING-SITES OF MAMMALIAN COMPLEX-IIAND ESCHERICHIA-COLI FUMARATE REDUCTASE

The structural and catalytic properties of beef heart succinate dehydr ogenase (succinate-ubiquinone oxidoreductase, complex II) and Escheric hia coli fumarate reductase are remarkably similar, One exception is t hat whereas electron exchange between the mammalian enzyme and its qui none pool is inhibited by thenoyltrifluoroacetone. and carboxanilides, the enzyme from E. coli is not sensitive to these inhibitors, The lac k of good inhibitors has seriously hampered the elucidation of the mec hanism of quinone oxidation/reduction in the E. coli enzyme, We have p reviously reported (Tan, A. K., Ramsay, R. R., Singer, T. P., and Miyo shi, H. (1993) J. Biol. Chem. 268, 19328-19333) that 2-alkyl-4,6-dinit rophenols inhibit mammalian complexes I, II, and III, but with differe nt potencies and kinetic characteristics. Based on these studies we ha ve selected a series of 2-alkyl-4,6-dinitrophenols which proved to be very effective noncompetitive inhibitors of mammalian complex II, part icularly when acting in the direction of quinone reduction, the physio logical event. These compounds turned out to be even more potent inhib itors E. coli fumarate reductase, particularly when acting in the dire ction of quinol oxidation, again, the physiological event. Kinetic ana lysis revealed that with both enzymes 2 inhibitor binding sites seem t o be involved in the oxidation of succinate by quinone, but one seems to be functioning when fumarate is reduced by external quinol, Since t he E. coli enzyme can be modified by site-directed mutagenesis, these studies were extended to four mutants of fumarate reductase, impaired by single amino acid substitutions at either of the putative quinone b inding sites (Q(A) or Q(B)) of the enzyme. The results were analyzed i n terms of the model of these dual sites of quinone binding in fumarat e reductase, as well as the nature of the substituent in the 2-positio n of the dinitrophenol inhibitors.