Determination of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the human oxidoreductase NR1
Determination of the redox potentials and electron transfer properties of the FAD- and FMN-binding domains of the human oxidoreductase NR1
复制标题
DOI：
10.1046/j.1432-1033.2003.03474.x
复制
发表时间：
2003-03-01
期刊：
影响因子：
--
通讯作者：
Scrutton, NS
中科院分区：
文献类型：
--
作者：
Finn, RD;Basran, J;Scrutton, NS
人类新型还原酶 1 (NR1) 是一种与细胞色素 P450 还原酶 (CPR) 相关的 NADPH 依赖性双黄素氧化还原酶。 NR1 的 FAD/NADPH 和 FMN 结合结构域已得到表达和纯化，并通过停流和稳态动力学方法以及电位测定法研究了它们的氧化还原特性。 FAD/NADPH 结构域的氧化/半醌 (-315 +/- 5 mV) 和半醌/二氢醌 (-365 +/- 15 mV) 对的中点还原电位与人类 CPR 的 FAD/NADPH 结构域相似，但氢化物从 NADPH 转移到 NR1 的 FAD/NADPH 结构域的速率大约慢 200 倍。在 FAD/NADPH 结构域与人工氧化还原受体的稳态反应中，氢化物转移是限速的。停流研究表明，从 NR1 的 FAD/NADPH 结构域到 NADP(+) 的氢化物转移比生理方向上的氢化物转移（NADPH 到 FAD）更快，与测量的 FAD 对还原电位一致 [FAD 氧化还原对的中点电位为 -340 mV，对比 NAD(P)H 为 -320 mV]。 FMN 结构域中黄素对的中点还原电位为 -146 +/- 5 mV（氧化/半醌）和 -305 +/- 5 mV（半醌/二氢醌）。 FMN 氧化/半醌对表明 FMN 半醌稳定，符合 (a) 将电子从 FAD/NADPH 结构域转移到 FMN 结构域的需要，以及 (b) CPR 和一氧化氮合酶中 FMN 结构域的热力学性质。尽管 NR1 和 CPR 总体结构相似，但我们的研究揭示了热力学相似性，但黄素结合域催化的电子转移反应存在主要动力学差异。
Human novel reductase 1 (NR1) is an NADPH dependent diflavin oxidoreductase related to cytochrome P450 reductase (CPR). The FAD/NADPH- and FMN-binding domains of NR1 have been expressed and purified and their redox properties studied by stopped-flow and steady-state kinetic methods, and by potentiometry. The midpoint reduction potentials of the oxidized/semiquinone (-315 +/- 5 mV) and semiquinone/dihydroquinone (-365 +/- 15 mV) couples of the FAD/NADPH domain are similar to those for the FAD/NADPH domain of human CPR, but the rate of hydride transfer from NADPH to the FAD/NADPH domain of NR1 is approximate to 200-fold slower. Hydride transfer is rate-limiting in steady-state reactions of the FAD/NADPH domain with artificial redox acceptors. Stopped-flow studies indicate that hydride transfer from the FAD/NADPH domain of NR1 to NADP(+) is faster than hydride transfer in the physiological direction (NADPH to FAD), consistent with the measured reduction potentials of the FAD couples [midpoint potential for FAD redox couples is -340 mV, cf -320 mV for NAD(P)H]. The midpoint reduction potentials for the flavin couples in the FMN domain are -146 +/- 5 mV (oxidized/semiquinone) and -305 +/- 5 mV (semiquinone/dihydroquinone). The FMN oxidized/semiquinone couple indicates stabilization of the FMN semiquinone, consistent with (a) a need to transfer electrons from the FAD/NADPH domain to the FMN domain, and (b) the thermodynamic properties of the FMN domain in CPR and nitric oxide synthase. Despite overall structural resemblance of NR1 and CPR, our studies reveal thermodynamic similarities but major kinetic differences in the electron transfer reactions catalysed by the flavin-binding domains.