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 carrier between primary flavoprotein dehydrogenases and terminal respiratory chains in mitochondria and prokaryotic cells. The three-dimensional structures of human and Paracoccus denitrificans ETFs determined by X-ray crystallography indicate that the 4'-hydroxyl of the ribityl side chain of FAD is hydrogen 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 for 4'-deoxy-FAD and FAD with the apoprotein are very similar, and the energy of binding differs by only 2 kJ/mol. The overall two-electron oxidation-reduction potential of 4'-deoxy-FAD in solution is identical to that of FAD. However, the potential of the oxidized/semiquinone couple of the ETF containing 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 bond stabilizes the anionic semiquinone in which negative charge is delocalized 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 achieve complete reduction of the flavin semiquinone to the hydroquinone. The turnover of medium chain acyl-CoA dehydrogenase with native ETF and ETF containing the 4'-deoxy analogue was essentially identical when the reduced ETF was recycled by reduction of 2,6-dichlorophenolindophenol. However, the steady-state turnover of the dehydrogenase with 4'-deoxy-FAD was only 23% of the turnover with native ETF when ETF semiquinone formation was assayed directly under anaerobic conditions. This is consistent with the decreased potential of the oxidized semiquinone couple of the analog-containing ETF. ETF containing 4'-deoxy-FAD neither donates to nor accepts electrons from electron-transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) at significant rates (</=0.5% the wild-type rates). These results indicate that the 4'-hydroxyl-N(1) hydrogen bond plays a major role in the stabilization of the anionic semiquinone and anionic hydroquinone oxidation states of ETF and that this hydrogen bond may provide a pathway for electron transfer between the ETF flavin and the flavin of ETF-QO.
Dwyer
T M
TM
Department of Pediatrics, Cell and Developmental Biology Program, University of Colorado School of Medicine, Denver 80262, USA.
Mortl
S
S
Kemter
K
K
Bacher
A
A
Fauq
A
A
Frerman
F E
FE
D000818:Animals; D004579:Electron Transport; D044927:Electron-Transferring Flavoproteins; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives / Q000737:chemistry; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013552:Swine
D044927:Electron-Transferring Flavoproteins; D005415:Flavins; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; C023406:1-deaza-FAD