Abstract
The structural and catalytic properties of beef heart succinate dehydrogenase (succinate-ubiquinone oxidoreductase, complex II) and Escherichia coli fumarate reductase are remarkably similar. One exception is that whereas electron exchange between the mammalian enzyme and its quinone pool is inhibited by thenoyltrifluoroacetone and carboxanilides, the enzyme from E. coli is not sensitive to these inhibitors. The lack of good inhibitors has seriously hampered the elucidation of the mechanism of quinone oxidation/reduction in the E. coli enzyme. We have previously reported (Tan, A. K., Ramsay, R. R., Singer, T. P., and Miyoshi, H. (1993) J. Biol. Chem. 268, 19328-19333) that 2-alkyl-4,6-dinitrophenols inhibit mammalian complexes I, II, and III, but with different potencies and kinetic characteristics. Based on these studies we have selected a series of 2-alkyl-4,6-dinitrophenols which proved to be very effective noncompetitive inhibitors of mammalian complex II, particularly when acting in the direction of quinone reduction, the physiological event. These compounds turned out to be even more potent inhibitors of E. coli fumarate reductase, particularly when acting in the direction of quinol oxidation, again, the physiological event. Kinetic analysis revealed that with both enzymes 2 inhibitor binding sites seem to be involved in the oxidation of succinate by quinone, but one seems to be functioning when fumarate is reduced by external quinol. Since the 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 binding sites (QA or QB) of the enzyme. The results were analyzed in terms of the model of these dual sites of quinone binding in fumarate reductase, as well as the nature of the substituent in the 2-position of the dinitrophenol inhibitors.
Highlights
The structural and catalytic properties of beef heart succinate dehydrogenase and Escherichia coli fumarate reductase are remarkably similar
Chem. 268, 19328 –19333) that 2-alkyl-4,6-dinitrophenols inhibit mammalian complexes I, II, and III, but with different potencies and kinetic characteristics. Based on these studies we have selected a series of 2-alkyl-4,6dinitrophenols which proved to be very effective noncompetitive inhibitors of mammalian complex II, when acting in the direction of quinone reduction, the physiological event
Since the E. coli enzyme can be modified by sitedirected mutagenesis, these studies were extended to four mutants of fumarate reductase, impaired by single amino acid substitutions at either of the putative quinone binding sites (QA or QB) of the enzyme
Summary
Materials—Beef heart electron transport particles (ETP, an inverted submitochondrial particle preparation) and complex II were isolated as in previous work [6]. Membrane preparations from wild-type and mutant strains of E. coli were isolated as described elsewhere [7, 8]. Construction and nomenclature of the mutant strains were described previously by Westenberg et al [7]. Assays—Succinate oxidation was determined at 38 °C with 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzo-quinone utilizing 2,6-dichlorophenolindophenol as a terminal electron acceptor (EmM ϭ 19.1), as in previous work [6]. Samples of enzyme were preactivated in assay buffer (50 mM Tris, 0.1 mM EDTA, pH 7.6) containing 20 mM succinate and 1 mM KCN (38 °C for 7 min). Fumarate reduction was measured under strictly anaerobic conditions with 2,3-dimethyl-1,4-naphthoquinol as electron
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
