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

Unusual reactivity of a flavin in a bifurcating electron-transferring flavoprotein leads to flavin modification and a charge-transfer complex

“…Upon release from the protein, the 8fF returned to the OX state, based on disappearance of both these features, indicating that the E° OX/ASQ is elevated by interactions in the ET site. This replicates the effect of the ET site on authentic FAD, which experiences greatly enhanced stabilization of the ASQ state when bound in the ET site ( 38 , 39 , 40 , 41 ). Similarly, the flavin optical spectrum when bound differs from that when free, confirming that 8fF experiences a distinct environment when bound in the ET site.…”

“…This suggests reduction of one of Rpa ETF's two flavins, as also seen in other enzymes where a flavin gets modified ( 47 , 48 ). The retained OX flavin spectrum in the anaerobic reaction resembled that of Bf flavin based on the λ max values and unresolved vibronic structure ( 38 ), indicating reduction of the ET flavin. In the same time interval, the aerobic reaction's signals changed shape, gaining new intensity between 500 and 550 nm, and near 320 nm, consistent with conversion of one flavin to 8fF (absorption maxima of free OX state near 352 and 463 vs .…”

“…These amino acid substitutions are all distant from the ET site, in a different domain and subunit of the protein ( Fig. 1 ) ( 38 ), so perhaps the presence of Bf FAD slows modification by favoring the closed conformation that seems less prone to ET flavin modification ( 25 ). Overall, the modification affects only one of the two FADs and so can be attributed to reactivity conferred on the ET flavin by its binding site.…”

“…These mechanisms imply that stabilization of an anionic state of the flavin should favor 8fF formation, as has been demonstrated in the FOX enzyme ( 43 , 46 ) and Hsa ETF ( 25 ). In Rpa ETF, the conserved Arg-273 that is adjacent to the ET flavin has been shown to stabilize the anionic ASQ of the flavin ( 38 ) as in canonical ETFs ( 39 , 41 ). Therefore, we tested the possibility that flavin modification occurs via a stabilized anionic intermediate, by replacing Arg-273.…”

“…Base residues in pink were mutated to prevent binding of the Bf FAD (Thr-94.B and Thr-97.B, in the numbering of Rhodopseudomonas palustris ETF, Rpa ETF ( 23 )). The head's Arg-273.A (in purple ) has been shown to modulate the reduction midpoint potentials (E°s) of the ET FAD ( 38 ). …”