The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer

Electron-transfer flavoprotein (ETF) serves as an intermediate electron car rier between primary flavoprotein dehydrogenases and terminal respiratory c hains in mitochondria and prokaryotic cells. The three-dimensional structur es of human and Paracoccus denitrificans ETFs determined by X-ray crystallo graphy indicate that the 4'-hydroxyl of the ribityl side chain of FAD is hy drogen bonded to N(1) of the flavin ring. We have substituted 4'-deoxy-FAD for the native FAD and investigated the analog-containing ETF to determine the role of this rare intra-cofactor hydrogen bond. The binding constants f or 4'-deoxy-FAD and FAD with the apoprotein are very similar, and the energ y of binding differs by only 2 kJ/mol. The overall two-electron oxidation-r eduction potential of 4'-deoxy-FAD in solution is identical to that of FAD. However, the potential of the oxidized/semiquinone couple of the ETF conta ining 4'-deoxy-FAD is 0.116 V less than the oxidized/semiquinone couple of the native protein. These data suggest that the 4'-hydoxyl-N(1) hydrogen bo nd stabilizes the anionic semiquinone in which negative charge is delocaliz ed over the N(1)-C(2)O region. Transfer of the second electron to 4'-deoxy- FAD reconstituted ETF is extremely slow, and it was very difficult to achie ve complete reduction of the flavin semiquinone to the hydroquinone. The tu rnover of medium chain acyl-CoA dehydrogenase with native ETF and ETF conta ining the 4'-deoxy analogue was essentially identical when the reduced ETF was recycled by reduction of 2,6-dichlorophenolindophenol. However, the ste ady-state turnover of the dehydrogenase with 4'-deoxy-FAD was only 23% of t he turnover with native ETF when ETF semiquinone formation was assayed dire ctly under anaerobic conditions. This is consistent with the decreased pote ntial of the oxidized semiquinone couple of the analog-containing ETF. ETF containing 4'-deoxy-FAD neither donates to nor accepts electrons from elect ron-transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) at significant rates (less than or equal to 0.5% the wild-type rates). These results indi cate that the 4'-hydroxyl-N(1) hydrogen bond plays a major role in the stab ilization of the anionic semiquinone and anionic hydroquinone oxidation sta tes of ETF and that this hydrogen bond may provide a pathway for electron t ransfer between the ETF flavin and the flavin of ETF-QO.