The catalytic mechanism of electron-bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD+

Abstract

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...

Rapid kinetics reveal surprising flavin chemistry in bifurcating electron transfer flavoprotein from Acidaminococcus fermentans

“…Electrons from -FADH  bifurcated intramolecularly to α-FAD of the same EtfAB molecule and intermolecularly to α-FAD of another EtfAB molecule, whereby the stable anionic red semiquinone of α-FAD (α-FAD  ) at 377 nm got its maximum value (Figure 4). The intermolecular electron transfer agrees well with previous suggestions based on the enzyme structures (7,20). Addition of EtfaB was expected to inhibit the formation of α-FAD  , due to the dilution of the acceptor, but the opposite happened ( Figure 3C).…”

Rational Design of Fe2(μ‐PR2)2(L)6 Coordination Compounds Featuring Tailored Potential Inversion

“…As can be deduced from the values of ΔΔG, the introduction of one phosphine (20) favours PI (compared to 1 or 3). However, by increasing the number of phosphines, PI is progressively disadvantaged, with ΔΔG going from À 2.3 kcal/ mol in 20 to 2.4 kcal/mol in 21 and to 7.3 kcal/mol in 22.…”

“…[10] After the first observation of flavin-based electron bifurcation, [4] several other electron bifurcating enzymes containing flavins have been reported, showing that this mechanism of energy coupling is widespread in biological systems. [11][12][13][14][15][16][17][18][19][20][21] Even if a general consensus has not yet been reached, it has been proposed that one of the key functional thermodynamic features of electron bifurcation (EB) depends on the peculiar redox properties of the flavin cofactor. [11,13,18,22,23] Already in 1932 Michaelis noted that some heteronuclear aromatic compounds, such as quinones and flavins, are characterized by versatile 2electron redox properties.…”

Modulations of the reduction potentials of flavin‐based electron bifurcation complexes and semiquinone stabilities are key to control directional electron flow

“…According to the high reduction potential of α‐FAD (+134 mV, see section: Reduction potentials of EtfAB), we assume that in vivo , where NADH is present, it occurs in the semiquinone (Sq) form [12]. Schut et al made the same assumption for the Pyrobaculum aerophilum EtfABCX [21]. Hence, in electron bifurcation only the Sq/Red potential is of relevance (Fig.…”