UC Riverside

Electron bifurcation is an ancient form of energy conservation that occurs in anaerobic microbes and some aerobes. It involves the coupling of a high potential pathway with a low potential pathway to generate the low-potential reducing equivalents needed to drive a variety of intracellular processes such as CO2 and N2 fixation even when the intracellular redox poise is modest. Many electron-bifurcating proteins utilize electron-transferring flavoprotein (ETF) component to bifurcate. Unlike the long-investigated non-bifurcating ETFs found in many vertebrates and microorganisms, which contain only an electron-transferring flavin (ET-FAD), bifurcating ETFs possess a second equivalent of FAD instead of AMP. This FAD is unique in bifurcating systems and is the site of electron bifurcation. A key aspect of flavin-based electron bifurcation is that the quinone/semiquinone and semiquinone/hydroquinone half-potentials are highly crossed, meaning that the semiquinone oxidation state is highly unstable thermodynamically. Although the thermodynamics of bifurcation are well understood, the kinetics of this process are not. We focus here on the menaquinone-dependent NADH:ferredoxin oxidoreductase EtfABCX from the hyperthermophilic archaeon Pyrobacculum aerophilum and the hyperthermophilic bacterium Thermotoga maritima and have investigated the reductive half-reaction of both the EtfAB subcomplex and the intact EtfABCX both in the absence and presence of the low-potential acceptor ferredoxin. With EtfAB depleted of its ET-FAD and containing only the bifurcating (BF)-FAD, monophasic reduction with a hyperbolic dependence on NADH concentration is observed as the flavin becomes directly reduced to the FADH-. However, in the course of reductive titrations with sodium dithionite, the anionic semiquinone (FAD•-) accumulates, indicating that the half-potentials of the BF-FAD have become uncrossed, with the semiquinone oxidation state now thermodynamically stabilized, negating bifurcation. With replete EtfAB containing both FADs, and with the intact EtfABCX complex, reduction with NADH is multiphasic and necessarily more complex, particularly in the latter case due to the presence of two [4Fe-4S] clusters of EtfX and a quinone-reducing (QR) FAD of EtfC. We find clear evidence indicating that in both the replete EtfAB and EtfABCX the half-potentials of the BF-FAD again become uncrossed in at least a subpopulation of protein, presumably due to a change in the conformation of the domain containing the ET-FAD that has been documented previously via crystallography and cryoelectron microscopy. The work presented also includes a spectroscopic and kinetic deconvolution of the reductive half-reaction of the intact EtfABCX complex.