¹⁹F NMR probes of conformational change in a bifurcating electron transfer flavoprotein

Bifurcating electron transfer flavoproteins (bETFs) accept a pair of modestly reducing electrons and produce a more potent electron carrier based on energy derived from favorable transfer of the other electron. A domain-scale conformational change is believed to gate electron transfer within the bETF, allowing only one electron to use the favorable path. 80° rotation of the so-called head domain carries the electron transfer flavin adenine dinucleotide (ET-FAD) from near the bifurcating FAD in bETF's closed conformation to a position >35 Å away in bETF's open conformation. In this, the ET-FAD is positioned to transfer electrons to partner proteins. Because conformation change is critical to bETF activity, understanding the motions and drivers of the conformational change is crucial to harnessing bifurcating enzymes for practical applications. After placing 5-¹⁹F-tryptophans at strategic locations, we used ¹⁹F NMR to identify Trp side chains that report on conformation. Surface exposure of Trp residues was assessed on the basis of paramagnetic relaxation enhancement (PRE) from Gd³⁺ in bulk solvent. Trp side chains responding to the conformation change were identified based on chemical shift or PRE changes upon ligand binding and/or flavin reduction, or substitution of a residue known to affect the conformational equilibrium. In both wild-type and mutant Acidaminococcus fermentans bETF reduced by excess NADH, we observed selective broadening of the NMR signals of Trps 144 and 149 near the bifurcating FAD to which NADH binds. Thus, reduction of the flavin or formation of its charge-transfer complex with NAD⁺ could produce structural change. Trp 149 and 239 were responsive to mutation of a residue reported to affect the equilibrium between the open and closed conformations and displayed temperature dependence suggestive of dynamics. Thus, we establish ¹⁹F NMR of fluorinated Trps as a tool for elucidating conformational changes involved in electron transfer bifurcation by bETF.