Electron bifurcation plays a key role in anaerobic energy metabolism, but it is a relatively new discovery, and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum. The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using nondenaturing MS, cross-linking, and homology modeling in which EtfA, -B, and -C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR, and optical titrations with NADH or inorganic reductants with and without NAD ؉ , we propose a catalytic cycle involving formation of an intermediary NAD ؉ -bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low-and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically unrelated bifurcating NADH-dependent ferredoxin NADP ؉ oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD ؉ , the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and the two twoelectron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETFs and can be applied to the large bifurcating ETF family.Electron-bifurcating enzymes couple exergonic and endergonic reactions, thus maximizing conservation of free energy available from exergonic reactions (1). In this way, electrochemical energy can be captured for cellular metabolism, lowering the demands on transmembrane gradients or substrate-level phosphorylation. Thus, electron bifurcation provides a unifying explanation for many peculiar fermentative pathways found in anaerobic microorganisms, with important implications for understanding anaerobic microbial physiology in general (2-9).So far, the bifurcating enzymes that have been characterized fall into one of four phylogenetically unrelated groups: electron transfer flavoproteins (EtfAB-containing), [FeFe]-hydrogenase/formate dehydrogenases (HydABC-containing), heterodisulfide reductases (HdrA-containing), and transhydrogenases (NfnAB-containing) (8, 10). These enzymes catalyze more than a dozen different reactions, most involving the oxidation or reduction of ferredoxin, and are found mainly in anaerobic organisms (reviewed in Refs. 3,8,11,and 12). However, some of the EtfAB-containing complexes, such as that described below, can also be found in microaerophiles and aerobes.Bifurcating ETFs 2 are the best-studied bifurcating enzymes, and they form a subset of the large and well-known family of ETFs, which ar...

Experiments and computation establish the locations of the two flavins whose contrasting reactivities produce electron bifurcation in ETFs. They confirm the accepted model and support homologies & distinctions between bifurcating and canonical ETFs.

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A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris (RpaETF). RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-E° carrier and the other to the higher-E° electron transfer FAD (ET-FAD). Previous work noted that the CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that RpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this may be the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact.