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