Distinct properties underlie flavin-based electron bifurcation in a novel electron transfer flavoprotein FixAB from Rhodopseudomonas palustris

Abstract

A newly recognized third fundamental mechanism of energy conservation in biology, electron bifurcation, uses free energy from exergonic redox reactions to drive endergonic redox reactions. Flavin-based electron bifurcation furnishes low-potential electrons to demanding chemical reactions, such as reduction of dinitrogen to ammonia. We employed the heterodimeric flavoenzyme FixAB from the diazotrophic bacterium to elucidate unique properties that underpin flavin-based electron bifurcation. FixAB is distinguished from canonical electron transfer flavoproteins (ETFs) by a second FAD that replaces the AMP of canonical ETF. We exploited near-UV-visible CD spectroscopy to resolve signals from the different flavin sites in FixAB and to interrogate the putative bifurcating FAD. CD aided in assigning the measured reduction midpoint potentials (° values) to individual flavins, and the ° values tested the accepted model regarding the redox properties required for bifurcation. We found that the higher-° flavin displays sequential one-electron (1-e) reductions to anionic semiquinone and then to hydroquinone, consistent with the reactivity seen in canonical ETFs. In contrast, the lower-° flavin displayed a single two-electron (2-e) reduction without detectable accumulation of semiquinone, consistent with unstable semiquinone states, as required for bifurcation. This is the first demonstration that a FixAB protein possesses the thermodynamic prerequisites for bifurcating activity, and the separation of distinct optical signatures for the two flavins lays a foundation for mechanistic studies to learn how electron flow can be directed in a protein environment. We propose that a novel optical signal observed at long wavelength may reflect electron delocalization between the two flavins.

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

“…3B). The values measured for P. aerophilum EtfAB of E m(OX/ASQ) ϭ ϩ33 mV and E m(ASQ/HQ) ϭ Ϫ94 mV fall well within values reported for other EtfABs (21,27), and the high E m(OX/ASQ) of ϩ33 mV for P. aerophilum EtfAB indicates that For the data in blue the "oxidized" flavin is OX, "reduced" flavin is the ASQ, and the dye is thionine. For the data in black the oxidized flavin is ASQ, reduced flavin is the HQ, and the dye is Nile blue.…”

“…However, herein we have identified key catalytic intermediates of P. aerophilum EtfABCX, including a novel NAD ϩ -dependent charge transfer complex, and describe a catalytic cycle that is very distinct from that established for NfnI. Moreover, the P. aerophilum EtfABCX catalytic cycle is consistent with previous structural observations and mechanisms of related EtfAB-containing complexes (8,9,13,24,27). We propose that the unifying principles proposed here can be applied to all bifurcating members of the large ETF family.…”

“…S5, phase 1). We assigned this to the ET-FAD, as it is expected to be more readily reduced than the BF-FAD (17,27). Further addition of reductant resulted in only a slight intensity loss at 374 nm, suggesting little change in the amount of ASQ ( Fig.…”

“…Thus, we can calculate a lower limit for the E m of the BF-FAD ASQ/HQ couple. Assuming that the ASQ/HQ couple of the ET-FAD is operative in turnover (27,30), electron transfer to ASQ-ET-FAD at Ϫ94 mV is exergonic by 191 mV. It follows that electron transfer to Fd can be endergonic at a similar level, placing a lower boundary of Ϫ476 mV on the E m of the BF-FAD OX/ASQ, which is presumably sufficient to reduce Fd (3,47).…”

“…Like the Fix system (24), P. aerophilum EtfABCX is shown to oxidize NADH and bifurcate electrons to both highpotential (quinone) and low-potential (Fd) acceptors. For the P. aerophilum EtfABCX complex, we refer to each of the flavins in the enzyme in terms of the role that each has been proposed to play in ETF proteins in general (13,16,24,27). Thus, the FAD unique to bifurcating ETFs is denoted the "bifurcating FAD," or BF-FAD, and this is present in the A-subunit of P. aerophilum EtfABCX (Fig.…”