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Electron bifurcation produces high-energy products based on less energetic reagents. This feat enables biological systems to exploit abundant mediocre fuel to drive vital but demanding reactions, including nitrogen fixation and CO2 capture. Thus there is great interest in understanding principles that can be portable to man-made devices. Bifurcating electron transfer flavoproteins (Bf ETFs) employ two flavins with contrasting reactivities to acquire pairs of electrons from the modest reductant, NADH. The flavins then disperse the electrons individually to high- and a low reduction midpoint potential (E°) acceptors, the latter of which captures most of the energy. Maximum efficiency requires that only one electron access the exergonic path that will 'pay for' production of the low-E° product. It is therefore critical that one of the flavins, the 'electron transfer' (ET) flavin, is tuned to execute single-electron (1e—) chemistry only. To learn how, and extract fundamental principles, we systematically altered interactions with the flavin O2 position. Removal of a single hydrogen bond (H-bond) disfavored formation of the flavin anionic semiquinone (ASQ) relative to the oxidized (OX) state, lowering E°ASQ/OX by 150 mV and retuning the flavin's tendency to 1e— vs. 2e— reactivity. This was achieved by replacing conserved His 290 with Phe while also replacing the supporting Tyr 279 with Ile. Although this variant binds oxidized FADs at 90% the WT level, the ASQ state of the ET flavin is not stable in the absence of H290's H-bond, and dissociates, contrary to WT. Removal of this H-bond also altered the ET flavin's covalent chemistry. Whereas the WT ETF accumulates modified flavins whose formation is believed to rely on an anionic paraquinone methide intermediate, the FADs of the H-bond lacking variant remain unchanged over weeks. Hence the variant that destabilizes the anionic semiquinone also suppresses the anionic intermediate in flavin modification, testifying to electronic similarities between these two species. These correlations suggest that the H-bond that stabilizes the crucial flavin ASQ also promotes flavin modification. The two effects may indeed be inseparable, as a Jekyll and Hydrogen bond. 

The flavodoxin of Rhodopseudomonas palustris CGA009 (Rp9Fld) supplies highly reducing equivalents to crucial enzymes such as hydrogenase, especially when the organism is iron-restricted. By acquiring those electrons from photodriven electron flow via the bifurcating electron transfer flavoprotein (ETF), Rp9Fld provides solar power to vital metabolic processes. To understand Rp9Fld's ability to work with diverse partners, we solved its crystal structure. We observe the canonical flavodoxin (Fld) fold and features common to other long-chain Flds, but not all the surface loops thought to recognize partner proteins. Moreover some of the loops display alternative structures and dynamics. To advance studies of protein-protein associations and conformational consequences, we assigned the 19F NMR signals of all 5 tyrosines (Tyrs). Our electrochemical measurements show that incorporation of 3-19F-Tyr in place of Tyr has only a modest effect on Rp9Fld's redox properties even though Tyrs flank the flavin on both sides. Meanwhile, the 19F probes demonstrate the expected paramagnetic effect, with signals from nearby Tyrs becoming broadened beyond detection when the flavin semiquinone is formed. However the temperature dependencies of chemical shifts and linewidths reveal dynamics affecting loops close to the flavin, and regions that bind to partners in a variety of systems. These coincide with patterns of amino acid type conservation but not retention of specific residues, arguing against detailed specificity with respect to partners. We propose that the loops surrounding the flavin adopt altered conformations upon binding to partners and may even participate actively in electron transfer.