Inhibitor Probes of the Quinone Binding Sites of Mammalian Complex II and Escherichia coli Fumarate Reductase

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.

Identification of Quinone-binding and Heme-ligating Residues of the Smallest Membrane-anchoring Subunit (QPs3) of Bovine Heart Mitochondrial Succinate:Ubiquinone Reductase

“…The Q-binding domain in the proposed model of QPs3 is located at the end of transmembrane helix I toward the C side of the mitochondrial inner membrane (9). Location of Q-binding domains of QPs1 and QPs3 on opposite sides of the membrane is in line with a two-Q-binding site hypothesis formulated from inhibitor binding studies of this enzyme complex (10).…”

The Quinone-binding Sites of the Saccharomyces cerevisiae Succinate-ubiquinone Oxidoreductase

“…Sixth, the kinetics of s-BDNP inhibition (Table IV) show that only the low affinity inhibitor-binding site, K i (2) , is affected. This inhibitor-binding site likely corresponds to a quinone-binding site (38,44). Finally, close proximity of the residues Phe-69 and Ser-71 strongly argues for their involvement in a common function, which we propose is the formation of a quinone-binding site in the vicinity of the loop connecting the Sdh4p transmembrane segments I and II.…”

“…The stability of ubiquinone in such a hydrophobic milieu is indicative of interaction with protein. Photoaffinity-labeling experiments (31)(32)(33)(34)(35), mutagenesis (36,37), and inhibitor (1,4,38) studies have shown that SDH and FRD contain at least two putative quinone-binding pockets, which are located on the opposite sides of the membrane. The crystal structure of the Escherichia coli FRD (27) revealed two bound quinones.…”

“…Sensitivities of SDH4 Mutants to a Quinone Analog Inhibitor-The 4,6-dinitrophenol derivatives are quinone analogs (43) that inhibit electron transfer in many respiratory proteins Quinone-binding Sites in the Yeast SDH (44), including the bovine heart SDH and the E. coli FRD (38). We have previously shown that s-BDNP inhibits the yeast SDH with hyperbolic, noncompetitive kinetics (22,24,25).…”

Interaction of a menaquinol binding site with the [3Fe‐4S] cluster of Escherichia coli fumarate reductase

“…Given that HOQNO is a closer analog of MKH 2 than of been used to modify the His residues present in the succinatebinding active site of bovine heart succinate dehydrogenase [45], Q, it is possible that it binds only at the MKH 2 site. Using a range of alkylated dinitrophenols, Yankovskaya et al [21] dem-so it is reasonable to assume that the ethoxyformic anhydride treatment used herein would also modify such residues in the onstrated apparent inhibitor binding at two sites when FrdABCD catalyzes reduction of a Q analog, but only at one site when fumarate-binding active site of FrdABCD [9,10].…”