Contrasting roles for two conserved arginines: Stabilizing flavin semiquinone or quaternary structure, in bifurcating electron transfer flavoproteins

Bifurcating electron transfer flavoproteins (Bf ETFs) are important redox enzymes that contain two flavin adenine dinucleotide (FAD) cofactors, with contrasting reactivities and complementary roles in electron bifurcation. However, for both the "electron transfer" (ET) and the "bifurcating" (Bf) FADs, the only charged amino acid within 5 Å of the flavin is a conserved arginine (Arg) residue. To understand how the two sites produce different reactivities utilizing the same residue, we investigated the consequences of replacing each of the Arg residues with lysine, glutamine, histidine, or alanine. We show that absence of a positive charge in the ET site diminishes accumulation of the anionic semiquinone (ASQ) that enables the ET flavin to act as a single electron carrier, due to depression of the oxidized versus. ASQ reduction midpoint potential, E_ OX/ASQ. Perturbation of the ET site also affected the remote Bf site, whereas abrogation of Bf FAD binding accelerated chemical modification of the ET flavin. In the Bf site, removal of the positive charge impaired binding of FAD or AMP, resulting in unstable protein. Based on pH dependence, we propose that the Bf site Arg interacts with the phosphate(s) of Bf FAD or AMP, bridging the domain interface via a conserved peptide loop ("zipper") and favoring nucleotide binding. We further propose a model that rationalizes conservation of the Bf site Arg even in non-Bf ETFs, as well as AMP's stabilizing role in the latter, and provides a mechanism for coupling Bf flavin redox changes to domain-scale motion.