Effects of active site arginine residue on redox behavior of flavins in bifurcating electron transfer flavoprotein

Flavin based electron bifurcation (FBEB) is observed in bifurcating transfer flavoproteins (BfEtfs). Electron couples endergonic and exergonic reactions to exploit lower potential reaction step therefore this considered as a third mode of energy conservation mechanism biological systems. We study about the EtfAB system from Rhodoseudomonas palustris (Rpal). This heterodimer containing two flavin adenine dinucleotides (FADs), one each domain. One accepts electrons NADH bifurcates (Bf-FAD) higher FAD (ET-FAD) ferredoxin or flavodoxin. ET-FAD likely rests its anionic semiquinone (ASQ) state upon accepting an Bf-FAD it becomes hydroquinone (HQ). BF-FAD pair form HQ distributes separate acceptors without accumulating SQ intermediate. contrasting redox behavior flavins turn points spotlight on whether conserved active site arginine residues have different effects binding sites. R273 favors ASQ ET-FAD, whereas R165 near appears not to, possibly due neutralization positive charge by nearby C174. forms pi- pi stacking interaction with hydrogen bond interactions Bf-FAD. performed directed mutagenesis change size positively charged residue replacing lysine, neutralize inserting alanine make tunable via modulation pH introducing histidine. Amino acid substitutions were made flavin-binding sites separately. Based Spectro-electrochemical titrations, all variants retained WT pattern single reactivity at ET pairwise Reduction midpoint titrations showed 50% less accumulation R273H R273A, R273K produced similar amount that WT. confirms helps stabilization ASQ. Redox 7 8 suggest substituting H273 remains neutral these pHs. replacement site, R165K did affect formation but removal 165's R165H resulted diminished stability prevented Bf pH8. Therefore, plays vital role Bf-ETF stabilizing ET-FAD. On other hand, stabilizes essential for RpalEtf.