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Figure 1: Canonical Etf function as a universal serial bus (USB) passing electrons from dehydrogenases to the quinone pool of the respiratory electron transfer chain. From: Watmough & Frerman (2010).
 

Figure 2: Overlay of ribbon structures of a canonical Etf (Teals, human, 1EFV) and a bifurcating Etf (Blues, A. fermentans, 4L2I). The FADs of the bifurcating Etf are in yellow and green, the FAD and AMP of the canonical Etf are in amber and teal, respectively.

Figure 3: Reductive titration of R. pal. EtfAB from oxidized (red) through partially reduced (green) in the presence of thionine (600 nm).

Figure 4: TAS traces showing formation and decay of flavin anionic semiquinone and associated depletion and recovery of oxidized flavin. Controls with exogenous electron donors distinguished between non-productive charge recombination vs. productive electron transfer [90].

Figure 5: Model for the bifurcating FixABCX complex in which the Etf component (FixAB) is in grey and blue, as in the image of the Etf alone, second panel from the left in the set of images across the top of this page. The two electron transfer paths that bifurcate from the presumed bifurcating flavin are in blue (presumed high potential path via the presumed ET-FAD) and red (presumed low-potential path via FixX) [92].

Etfs provide a USB for 1-electron transfer to respiratory electron transfer OR versatile energy redistribution among electrons in a pair.
 
 

A Novel Reaction by a Well-Known Fold.

Electron Transfer Flavoproteins (Etfs) have been recognized for decades for their ability to convey electrons to the respiratory electron transport chain from a variety of very different reactions. They do this by accepting electrons one at a time (1-electron transfer) from a variety of dehydrogenases such as fatty acid dehydrogenases, and then donating them to quinone reductase which feeds the reduced quinone pool (Figure 1). These 'canonical' Etfs employ a flavin adenine dinucleotide (FAD) as their electron carrier, as it cycles between the flavin's oxidized and anionic semiquinone states. This FAD is bound in one domain of the heterodimeric protein (amber stick model in Figure 2). The other domain contains an AMP residue described as purely structural in the literature, and is made up of both protein monomers. Recently however, members of this venerable family have been shown to contain two FADs rather than one, and to have the 'new' second FAD bound in the site that had contained AMP in the canonical Etfs (FAD = green stick model, AMP = teal). Indeed the AMP portion of the new FAD binds in place of the structural AMP of canonical Etfs (Figure 2).
            The newly-recognized Etfs appear able to mediate flavin-based electron bifurcation.

Electron Transfer Bifurcation.

Electron transfer bifurcation ('electron bifurcation' or 'bifurcation', for short) represents a third fundamental mechanism of energy conservation, along with substrate-level phosphorylation and transmembrane electrochemical potentials. It is operative in the Q-cycle of respiratory electron transfer but has often been mistaken for confusing and obscure. New discoveries now indicate that bifurcation is pervasive and important in anaerobic metabolism and may be critical to their very high energy-efficiency and capacity for very difficult reactions [93]. Among these, production of H2 and NH3 from abundant substrates, have very high potential utility. We seek to understand flavin-based bifurcation at a fundamental level of individual electron transfer events and states with the objective that such insights may permit designers of electronic materials and components to incorporate the principles of bifurcation to increase the efficiency and versatility of deployable devices.

Mechanism and Requirements for Bifurcation.

In flavin-based bifurcation a flavin acquires a pair of electrons from NAD(P)H at an Eo near -320 mV. Such reducing equivalents are relatively abundant but not sufficiently energetic to drive reduction of H+ to H2 or N2 to NH3. The reduced flavin effectively reallocates the total energy of the pair of electrons, to release one electron at very low Eo (highly reducing, lower than that of NADH) and the other at a higher Eo. Thus energy is conserved but a highly energetic electron is produced. The enzyme effectively trades quantity for quality [83].
            Flavin-based bifurcation requires the following properties/capabilities. Therefore, we are testing a putative bifurcating Etf with respect to these fundamental requirements. 1- one flavin should have a highly reactive (thermodynamically unstable) semiquinone state: the bifurcating flavin, -2- the other should have a stable semiquinone state in order to enable it to accept electrons one at a time from the first flavin. Because transfer of single electrons is the activity of canonical Etfs we propose that this capability resides in the FAD that bifurcating Etfs share with canonical Etfs, and refer to this flavin as the putative electron transfer FAD (ET-FAD, yellow/amber in Figures 2 and 5). Because canonical Etfs do not have bifurcating activity and do not accept electrons from NADH we propose that this latter activity resides in the FAD unique to bifurcating Etfs, and refer to this flavin as the putative bifurcating FAD (Bf-FAD, green in Figures)

FixAB is a Bifurcating Etf.

Sequence homology studies have identified Etfs associated with nitrogen fixation, which therefore have the name FixAB. (The A and B indicate the two proteins that make up the heterodimer, and regrettably correspond to the B and A monomers of Etfs. Therefore this page minimizes the use of the A/B nomenclature.) A complex of FixABCX containing the Etf as well as FixC (homologous to Etf-ubiquinone oxidoreductase, ETF:QO in Figure 1) and FixX (homologous to a ferredoxin) has recently been shown to display bifurcating activity and therefore may represent the missing piece of the puzzle of how NADH can drive reduction of N2 [92]. Since neither ETF:QO nor ferredoxin are likely sites of the bifurcating activity, which moreover has been demonstrated in another Etf in a completely different metabolic context, the bifurcating activity is presumed to reside in the Etf component of FixABCX. We have overexpressed and purified the first Fix Etf to contain 2 FAD per heterodimer, and are performing redox titrations and mechanistic studies to learn whether it possesses the properties proposed to be required for bifurcating activity. If so, then this system will serve as an ideal molecular laboratory to understand flavin-based electron bifurcation.

Under the Hood: How does Flavin-Based Bifuctating Work?

A crucial hypothesis regarding flavin-based bifurcation is the participation of a thermodynamically unstable flavin semiquinone [83]. The proposed mechanism calls for doubly-reduced flavin to lose one electron to a high-potential electron acceptor nearby, thereby adopting its semiquinone state. If the flavin semiquinone state is sufficiently unstable, it will possess sufficient driving force to reduce a very low potential acceptor nearby, accomplishing bifurcation. In order for the latter to occur instead of the second electron following the first to the high-potential acceptor, that acceptor must only be able to acquire one electron (not a second) hence the requirement in Etfs that the ET-flavin have a very stable semiquinone state. In the bifurcating Nfn (NADH-dependent ferredoxin-NADP+ oxidoreductase I) the high potential acceptor appears to be an Fe2S2 cluster and therefore has a distinct optical signature from that of the presumed bifurcating flavin. As in the FixABCX system, the low-potential acceptor is a Fe4S4 cluster. Transient absorption spectroscopy (TAS) was be used to look for short-lived species resulting from photoexcitation of the presumed bifurcating flavin. Indeed, a very short-lived species consistent with anionic flavin semiquinone was observed! [87] Similar signals have also been observed in FixABCX and the Etf component of it. We have used TAS to test another premise of flavin-based bifurcation: that there should be pathways for highly efficient electron transfer from the flavin to the other electron carriers implied (the high- and low-potential acceptors at minimum, but extended pathways in more complex/complete systems). Figure 4 shows the immediate formation of anionic flavin semiquinone and its subsequent rapid decay in the flavin-based bifurcating Nfn[90] (red trace). Analysis of the lifetimes in conjuction with known Eos indicate that the energy spanned by the flavin's two bifurcated electrons rivals the energy of charge separation in photosynthesis [87]. Thus there is fascinating biophysics inherent in flavin-based electron bifurcation. Experiments are underway to explore this.

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Partial update: Oct 2017                                                                                               

Copyright 2017 A.-F. Miller     

Comments: A.-F. Miller