UC Riverside

Flavin-based electron bifurcation (FBEB) is an evolutionarily ancient mode of energy conservation found in a multitude of archaea and bacteria. FBEB allows these organisms to conserve energy by coupling the generation of low-potential reducing equivalents to the reduction of a high-potential electron acceptor/pathway. The thermodynamics of these separate pathways and the bifurcating cofactor (flavin adenine dinucleotide or FAD) have all been previously well-studied. However, in the context of FBEB the kinetics and nature of electron transfer (ET) in the system remain unknown.The present work serves to elucidate the discrete steps of ET involved in bifurcation of the crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase complex from the bacterium Megasphaera elsdenii. The complex consists of two flavoenzymes, an electron transferring flavoprotein (EtfAB) and a butyryl-CoA dehydrogenase (bcd). The kinetics of the reductive and oxidative half-reactions of the individual proteins, the intact-complex, and the reduction of ferredoxin have been investigated. The various techniques that have been employed include: spectral deconvolution of the UV-vis spectra, kinetic steady-state assays, rapid-reaction kinetics, enzyme monitored turnover and electron paramagnetic resonance (EPR) spectroscopy. The work has revealed, despite the favorability of transfer of the low-potential electron into the high-potential pathway, this does not occur in an enzymatic time-frame. There is no “leakage” into the high-potential pathway that would “short-circuit” bifurcation. Spectral deconvolution and EPR analysis have confirmed not only the presence, but also the importance of, the flavin semiquinone in ET. Rapid-reaction kinetic studies of both reductive and oxidative half-reactions have revealed that after the initial reduction by NADH there are a series of slower ETs in the high-potential pathway that impede the immediate transfer of the low-potential electrons, thus preserving the fidelity of ferredoxin reduction. Enzyme-monitored turnover and steady-state studies have shown that the rate of ferredoxin reduction is significantly slower than the initial reduction of the complex. The experimental findings provide evidence supporting the rate of bifurcation being preserved through controlled, slow ET into the high-potential pathway, with the FAD constituting the first site in the high-potential pathway operating exclusively between the semiquinone and hydroquinone oxidation states.