Redox potentials and kinetic properties of fumarate reductase complex from Vibrio succinogenes

The isolated menaquinol: fumarate oxidoreductase (fumarate reductase complex) from Vibrio succinogenes was investigated with respect to the redox potentials and the kinetic response of the prosthetic groups. The following results were obtained. (1) The redox state of the components was measured as a function of the redox potential established by the fumarate/succinate couple, after freezing of the samples (173 K). From these measurements, the midpoint potential of the 2Fe-2S] cluster (?59 mV), the 4Fe-4S] cluster (?24 mV) and the flavin/flavosemiquinone couple (about ?20 mV) was obtained. (2) Potentiometric titration of the enzyme in the presence of electron-mediating chemicals gave, after freezing, apparent midpoint potentials that were 30–100 mV more negative than those found with the fumarate/succinate couple. (3) The rate constants of reduction of the components on the addition of succinate or 2,3-dimethyl-1,4-naphthoquinol were as great as or greater than the corresponding turnover numbers of the enzyme in quinone reduction by succinate or fumarate reduction by the quinol. In the oxidation of the reduced enzyme by fumarate, cytochrome b oxidation was about as fast as the corresponding turnover number of quinol oxidation by fumarate, while the 2Fe-2S] and half of the 4Fe-4S] cluster responded more than 2-times slower. The rate constant of the other half of the 4-Fe cluster was one order of magnitude smaller than the turnover number.