Tryptophan 697 Modulates Hydride and Interflavin Electron Transfer in Human Methionine Synthase Reductase
Tryptophan 697 Modulates Hydride and Interflavin Electron Transfer in Human Methionine Synthase Reductase
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DOI：
10.1021/bi2012228
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发表时间：
2011-12-27
期刊：
影响因子：
2.9
通讯作者：
Woltherst, Kirsten R.
中科院分区：
文献类型：
--
作者：
Meints, Carla E.;Gustafsson, Frida S.;Woltherst, Kirsten R.
人甲硫氨酸合成酶还原酶（MSR）是一种二黄素氧化还原酶，通过维持甲硫氨酸合成酶（MS）的活性在甲硫氨酸和叶酸代谢中起着重要作用。MSR催化NADPH的氧化，并通过其FAD和FMN辅因子将电子穿梭为无活性的MS-cob（II）丙氨酸。位于FAD异咯嗪环旁边的保守芳香族残基（Trp 697）控制烟酰胺结合和相关黄素蛋白的催化作用。我们创建了四个MSR突变体（W 697 S，W 697 H，S698 Delta和S698 A），并研究了它们相关的动力学行为。多波长停流分析表明，NADPH还原的C-末端Ser 698突变体发生在三个可分辨的动力学步骤，包括转移的氢化物离子到FAD，半醌的形成（表明FAD FMN电子转移），和缓慢的黄素还原的第二个分子的NADPH。相应的实验与W 697突变体显示两步黄素还原没有可观察到的半醌中间体，表明W 697支持FAD FMN电子转移。S698 Delta和W 697 H突变体中FAD还原、稳态细胞色素c（3+）周转和非偶联NADPH氧化的加速速率可能归因于NADPH置换W 697的能垒降低。NADP（+）的结合，而不是2 '5'-ADP，对于所有突变体比对于天然MSR更紧密。综合研究表明，虽然W 697减弱氢化物转移，但它确保了辅酶选择性并加速了FAD到FMN的电子转移。此外，类似的细胞色素P450还原酶（CPR）变体的分析指出了黄素还原驱动力的关键差异，并表明CPR中保守的FAD堆积色氨酸残基也促进了黄素间电子转移。
Human methionine synthase reductase (MSR), a diflavin oxidoreductase, plays a vital role in methionine and folate metabolism by sustaining methionine synthase (MS) activity. MSR catalyzes the oxidation of NADPH and shuttles electrons via its FAD and FMN cofactors to inactive MS-cob(II)alamin. A conserved aromatic residue (Trp697) positioned next to the FAD isoalloxazine ring controls nicotinamide binding and catalysis in related flavoproteins. We created four MSR mutants (W697S, W697H, S698 Delta, and S698A) and studied their associated kinetic behavior. Multiwavelength stopped-flow analysis reveals that NADPH reduction of the C-terminal Ser698 mutants occurs in three resolvable kinetic steps encompassing transfer of a hydride ion to FAD, semiquinone formation (indicating FAD to FMN electron transfer), and slow flavin reduction by a second molecule of NADPH. Corresponding experiments with the W697 mutants show a two-step flavin reduction without an observable semiquinone intermediate, indicating that W697 supports FAD to FMN electron transfer. Accelerated rates of FAD reduction, steady-state cytochrome c(3+) turnover, and uncoupled NADPH oxidation in the S698 Delta, and W697H mutants may be attributed to a decrease in the energy barrier for displacement of W697 by NADPH. Binding of NADP(+), but not 2'5'-ADP, is tighter for all mutants than for native MSR. The combined studies demonstrate that while W697 attenuates hydride transfer, it ensures coenzyme selectivity and accelerates FAD to FMN electron transfer. Moreover, analysis of analogous cytochrome P450 reductase (CPR) variants points to key differences in the driving force for flavin reduction and suggests that the conserved FAD stacking tryptophan residue in CPR also promotes interflavin electron transfer.