αArg-237 in Methylophilus methylotrophus (sp W3A1) electron-transferring flavoprotein affords ∼200-millivolt stabilization of the FAD anionic semiquinone and a kinetic block on full reduction to the dihydroquinone
αArg-237 in Methylophilus methylotrophus (sp W3A1) electron-transferring flavoprotein affords ∼200-millivolt stabilization of the FAD anionic semiquinone and a kinetic block on full reduction to the dihydroquinone
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DOI：
10.1074/jbc.m010853200
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发表时间：
2001-06-08
影响因子：
4.8
通讯作者：
Scrutton, NS
中科院分区：
文献类型：
--
作者：
Talfournier, F;Munro, AW;Scrutton, NS
通过厌氧氧化还原滴定法测定了FAD辅因子野生型嗜甲基菌（W3 A1）电子转移黄素蛋白（ETF）和α R237 A突变体的中点还原电位。野生型ETF（E ′（1））中氧化的半醌对的FAD还原电位为+153 +/-2 mV，表明黄素阴离子半醌物质的异常稳定。由于在FAD完全还原时存在动力学和热力学阻断，因此转化为二氢醌是不完全的（E ′（2）< -250 mV）。K.，Scrutton，N.美国，和Sutcliffe，M. J.（1998）Protein Pept. Lett. 5，231-236）表明Arg-237的胍基（位于黄素异咯嗪环的si面上）在野生型ETF中阴离子半醌的异常稳定中发挥着关键作用。M中Arg-237换成Ala的主要影响，甲基营养菌ETF的目的是设计一种显著类似于黄素阴离子半醌的200-mV去稳定化（E ′（2）= -31 +/-2 mV，和E '（1）= -43 +/-2 mV）。此外，在aR 237 A ETF中还原成FAD二氢醌相对容易，表明在野生型ETF中看到的动力学阻断在aR 237 A ETF中基本上被去除。（以及热力学）考虑在填充蛋白质结合的黄素的氧化还原形式中是重要的。另外，我们表明，由于电子转移复合物的组装受损，从三甲胺脱氢酶到α R237 A ETF的电子转移严重受损。
The midpoint reduction potentials of the FAD cofactor wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the alpha R237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidized-semiquinone couple in wild-type ETF (E'(1)) is + 153 +/- 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E'(2) < -250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD, A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF, The major effect of exchanging Arg-237 for Ala in M, methylotrophus ETF is to engineer a remarkable similar to 200-mV destabilization of the flavin anionic semiquinone (E'(2) = -31 +/- 2 mV, and E'(1) = -43 +/- 2 mV). In addition, reduction to the FAD dihydroquinone in alpha R237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the alpha R237A ETF, Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin, Additionally, we show that electron transfer from trimethylamine dehydrogenase to alpha R237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.