Talfournier F, Munro A W, Basran J, Sutcliffe M J, Daff S, Chapman S K, Scrutton N S
Departments of Biochemistry and Chemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom.
J Biol Chem. 2001 Jun 8;276(23):20190-6. doi: 10.1074/jbc.M010853200. Epub 2001 Apr 2.
The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the alphaR237A 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 alphaArg-237 for Ala in M. methylotrophus ETF is to engineer a remarkable approximately 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 alphaR237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the alphaR237A 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 alphaR237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.
通过厌氧氧化还原滴定法测定了野生型嗜甲基甲基ophilus（菌株W3A1）电子传递黄素蛋白（ETF）和αR237A突变体中FAD辅因子的中点还原电位。野生型ETF中氧化型 - 半醌对的FAD还原电位（E'(1)）为 +153 ± 2 mV，表明黄素阴离子半醌物种具有异常的稳定性。由于FAD完全还原存在动力学和热力学障碍，转化为二氢醌是不完全的（E'(2) < -250 mV）。ETF的结构模型（Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231 - 236）表明，位于黄素异咯嗪环si面上方的精氨酸 - 237的胍基在野生型ETF中阴离子半醌的异常稳定中起关键作用。在嗜甲基甲基ophilus ETF中用丙氨酸替换α精氨酸 - 237的主要作用是使黄素阴离子半醌显著不稳定约200 mV（E'(2) = -31 ± 2 mV，E'(1) = -43 ± 2 mV）。此外，αR237A ETF中还原为FAD二氢醌相对容易，表明在αR237A ETF中野生型ETF中看到的动力学障碍基本消除。因此，动力学（以及热力学）因素在填充蛋白质结合黄素的氧化还原形式中很重要。此外，我们表明，由于电子传递复合物组装受损，三甲胺脱氢酶向αR237A ETF的电子传递严重受损。
