Iyanagi T, Makino R, Anan F K
Biochemistry. 1981 Mar 31;20(7):1722-30. doi: 10.1021/bi00510a004.
The mechanism of hepatic NADPH-cytochrome P-450 reductase has been investigated by using a stopped-flow technique. The reduction of the oxidized native enzyme (FAD-FMN) by NADPH proceeds by both one-electron equivalent and two-electron eqiuvalent mechanisms. The air-stable semiquinone form (FAD-FMNH.) of the native enzyme, which is characterized by an absorption shoulder at 635 nm, is also rapidly reduced to another semiquinone form (FADH-FMNH2) by NADPH with the disappearance of the shoulder at 635 nm, but the absorbance change at 585 nm is relatively constant. The FAD moiety in the FMN-depleted enzyme is rapidly reduced by NADPH, and reduced FAD is oxidized in successive one-electron steps by O2 or potassium ferricyanide. These results indicate the possibility of intra-molecular one-electron transfer between FAD and FMN. The rate of cytochrome P-450 reduction decreases in the presence of FMN-depleted enzyme but is nearly restored to the value of the original enzyme with FMN-reconstituted enzyme. These data suggest that FAD is the low-potential flavin, which serves as an electron acceptor from NADPH. On the other hand, FMN, which is the high-potential flavin, appears to participate as an electron carrier in the process of electron transfer from NADPH to cytochrome P-450 during the mixed-function catalytic cycle.
已通过使用停流技术研究了肝脏NADPH-细胞色素P-450还原酶的机制。NADPH对氧化型天然酶（FAD-FMN）的还原通过单电子当量和双电子当量机制进行。天然酶的空气稳定半醌形式（FAD-FMNH.）在635nm处有一个吸收肩，它也会被NADPH迅速还原为另一种半醌形式（FADH-FMNH2），635nm处的肩消失，但585nm处的吸光度变化相对恒定。FMN缺失的酶中的FAD部分会被NADPH迅速还原，还原型FAD会被O2或铁氰化钾以连续的单电子步骤氧化。这些结果表明FAD和FMN之间可能存在分子内单电子转移。在FMN缺失的酶存在下，细胞色素P-450的还原速率降低，但用FMN重组的酶几乎可恢复到原始酶的值。这些数据表明FAD是低电位黄素，作为NADPH的电子受体。另一方面，作为高电位黄素的FMN似乎在混合功能催化循环中作为电子载体参与从NADPH到细胞色素P-450的电子转移过程。
