Abstract
We have investigated the kinetic behavior of the electron-bifurcating crotonyl-CoA-dependent NADH: ferredoxin oxidoreductase EtfAB:bcd from Megasphaera elsdenii. The overall behavior of the complex in both the reductive and the oxidative half-reactions is consistent with that previously determined for the individual EtfAB and bcd components. This includes an uncrossing of the half-potentials of the bifurcating flavin of the EtfAB component in the course of ferredoxin-reducing catalysis, ionization of the bcd flavin semiquinone and the appearance of a charge transfer complex upon binding of the high potential acceptor crotonyl-CoA. The observed rapid-reaction rates of ferredoxin reduction are independent of [NADH], [crotonyl-CoA], and [ferredoxin], with an observed rate of ∼0.2 s-1, consistent with the observed steady-state kinetics. In enzyme-monitored turnover experiments, an approach to steady-state where the complex's flavins become reduced but no ferredoxin is generated is followed by a steady-state phase characterized by extensive ferredoxin reduction but little change in overall levels of flavin reduction. The approach to the steady-state phase can be eliminated by prior reduction of the complex, in which case there is no lag in the onset of ferredoxin reduction; this is consistent with the et FAD needing to be reduced to the level of the (anionic) semiquinone for bifurcation and concomitant ferredoxin reduction to occur. Single-turnover experiments support this conclusion, with the accumulation of the anionic semiquinone of the et FAD apparently required to prime the system for subsequent bifurcation and ferredoxin reduction.