KINETIC-STUDIES ON THE ELECTRON-TRANSFER REACTION BETWEEN CYTOCHROME-C(3) AND FLAVODOXIN FROM DESULFOVIBRIO-VULGARIS STRAIN HILDENBOROUGH

The kinetic properties of the electron-transfer process between reduce d Desulfovibrio vulgaris cytochrome c(3) and D. vulgaris flavodoxin ha ve been studied by anaerobic stopped-flow techniques. Anaerobic titrat ions of reduced cytochrome c(3) with oxidized flavodoxin show a stoich iometry of 4 mol of flavodoxin required to oxidize the tetraheme cytoc hrome. Flavodoxin neutral semiquinone and oxidized cytochrome c(3) are the only observable products of the reaction. At pH 7.5, the four-ele ctron-transfer reaction is biphasic. Both the rapid and the slow phase s exhibit limiting rates as the flavodoxin concentration is increased with respective rates of 73.4 and 18.5 s(-1) and respective K-d values of 65.9 +/- 9.4 mu M and 54.5 +/- 13 CIM. A biphasic electron-transfe r rate is observed when the ionic strength is increased to 100 mM KCl; however, the observed rate is no longer saturable, and relative secon d-order rate constants of 5.3 X 10(5) and 8.5 x 10(4) M(-1) s(-1) are calculated. The magnitude of the rapid phase of electron transfer dimi nishes 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 c(3) reacts with an similar to 25-fold molar excess of flavodoxin. At pH 6.0, the electron-transfe r reaction is monophasic with a limiting rate of 42 +/- 1.4 s(-1) and a Kd value of similar to 8 mu M. Increasing the ionic strength of the pH 6.0 solution to 100 mu M KCl results in a biphasic reaction with re lative second-order rate constants of 5.3 x 10(5) and 1.1 x 10(4) M(-1 ) s(-1) Azotobacter vinelandii flavodoxin reacts with reduced D. vulga ris cytochrome cs in a slow, monophasic manner with limiting rate of e lectron transfer of 1.2 +/- 0.06 s(-1) and a K-d value of 80.9 +/- 10. 7 mu M. These results are discussed in terms of two equilibrium confor mational states for the cytochrome which are dependent on the pH of th e medium and the level of heme reduction [Catarino et al. (1991) Eur. J. Biochem. 207, 1107-1113].