Kinetic Studies on the Electron-Transfer Reaction between Cytochrome c3 and Flavodoxin from Desulfovibrio vulgaris Strain Hildenborough

Abstract

The kinetic properties of the electron-transfer process between reduced Desulfovibrio vulgaris cytochrome c3 and D. vulgaris flavodoxin have been studied by anaerobic stopped-flow techniques. Anaerobic titrations of reduced cytochrome c3 with oxidized flavodoxin show a stoichiometry of 4 mol of flavodoxin required to oxidize the tetraheme cytochrome. Flavodoxin neutral semiquinone and oxidized cytochrome c3 are the only observable products of the reaction. At pH 7.5, the four-electron-transfer reaction is biphasic. Both the rapid and the slow phases exhibit limiting rates as the flavodoxin concentration is increased with respective ratesof 73.4 and 18.5 s-I and respective &values of 65.9 f 9.4 pM and 54.5 f 13 pM. A biphasic electron-transfer rate is observed when the ionic strength is increased to 100 mM KCl; however, the observed rate is no longer saturable, and relative second-order rate constants of 5.3 X lo5 and 8.5 X lo4 M-l s-1 are calculated. The magnitude of the rapid phase of electron transfer diminishes with the level of heme reduction when varying reduced levels of the cytochrome are mixed with oxidized flavodoxin. No rapid phase is observed when 0.66e--reduced cytochrome c3 reacts with an -25-fold molar excess of flavodoxin. At pH 6.0, the electron-transfer reaction is monophasic with a limiting rate of 42 f 1.4 s-1 and a Kd value of -8 pM. Increasing the ionic strength of the pH 6.0 solution to 100 pM KC1 results in a biphasic reaction with relative second-order rate constants of 5.3 X lo5 and 1.1 X lo4 M-l s-l. Azotobacter vinelandii flavodoxin reacts with reduced D. vulgaris cytochrome c3 in a slow, monophasic manner with limiting rate of electron transfer of 1.2 f 0.06 s-l and a Kd value of 80.9 f 10.7 pM. These results are discussed in terms of two equilibrium conformational states for the cytochrome which are dependent on the pH of the medium and the level of heme reduction [Catarino et al. (1991) Eur. J. Biochem. 207, 1107-1 1131.