Dwyer T M, Mortl S, Kemter K, Bacher A, Fauq A, Frerman F E
Department of Pediatrics, Cell and Developmental Biology Program, University of Colorado School of Medicine, Denver 80262, USA.
Biochemistry. 1999 Jul 27;38(30):9735-45. doi: 10.1021/bi9903906.
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.
电子传递黄素蛋白（ETF）作为线粒体和原核细胞中初级黄素蛋白脱氢酶与末端呼吸链之间的中间电子载体。通过X射线晶体学确定的人类和反硝化副球菌ETF的三维结构表明，FAD核糖醇侧链的4'-羟基与黄素环的N(1)形成氢键。我们用4'-脱氧-FAD替代天然FAD，并研究了含该类似物的ETF，以确定这种罕见的辅因子内氢键的作用。4'-脱氧-FAD和FAD与脱辅基蛋白的结合常数非常相似，结合能仅相差2 kJ/mol。溶液中4'-脱氧-FAD的整体双电子氧化还原电位与FAD相同。然而，含4'-脱氧-FAD的ETF的氧化/半醌对的电位比天然蛋白的氧化/半醌对低0.116 V。这些数据表明，4'-羟基-N(1)氢键稳定了阴离子半醌，其中负电荷在N(1)-C(2)O区域离域。第二个电子转移到4'-脱氧-FAD重构的ETF极其缓慢，并且很难将黄素半醌完全还原为对苯二酚。当中链酰基辅酶A脱氢酶与天然ETF以及含4'-脱氧类似物的ETF一起作用时，当还原的ETF通过2,6-二氯酚靛酚的还原进行循环时，其周转基本相同。然而，当在厌氧条件下直接测定ETF半醌形成时，脱氢酶与4'-脱氧-FAD的稳态周转仅为与天然ETF周转的23%。这与含类似物的ETF的氧化半醌对电位降低一致。含4'-脱氧-FAD的ETF既不以显著速率（≤野生型速率的0.5%）向电子传递黄素蛋白泛醌氧化还原酶（ETF-QO）供电子，也不接受其电子。这些结果表明，4'-羟基-N(1)氢键在稳定ETF的阴离子半醌和阴离子对苯二酚氧化态中起主要作用，并且该氢键可能为ETF黄素与ETF-QO的黄素之间的电子转移提供一条途径。
