Nisimoto Y, Edmondson D E
Department of Biochemistry, Aichi Medical University, Nagakute, Japan.
Eur J Biochem. 1992 Mar 15;204(3):1075-82. doi: 10.1111/j.1432-1033.1992.tb16731.x.
Significant dissociation of FMN from NADPH:cytochrome P-450 reductase resulted in loss of the activity for reduction of cytochrome b5 as well as cytochrome c and cytochrome P-450. However, the ability to reduce these electron acceptors was greatly restored upon incubation of FMN-depleted enzyme with added FMN. The reductions of cytochrome c and detergent-solubilized cytochrome b5 by NADPH:cytochrome P-450 reductase were greatly increased in the presence of high concentrations of KCl, although the stimulatory effect of the salt on cytochrome P-450 reduction was less significant. No apparent effect of superoxide dismutase could be seen on the rate or extent of cytochrome reduction in solutions containing high-salt concentrations. Complex formation of the flavoprotein with cytochrome c, which is known to be involved in the mechanism of non-physiological electron transfer, caused a perturbation in the absorption spectrum in the Soret-band region of cytochrome c, and its magnitude was enhanced by addition of KCl. Similarly, an appreciable increase in ellipticity in the Soret band of cytochrome c was observed upon binding with the flavoprotein. However, only small changes were found in absorption and circular dichroism spectra for the complex of NADPH:cytochrome P-450 reductase with either cytochrome b5 or cytochrome P-450. It is suggested that the high-salt concentration allows closer contact between the heme and flavin prosthetic groups through hydrophobic-hydrophobic interactions rather than electrostatic-charge pairing between the flavoprotein and the cytochrome which causes a faster rate of electron transfer. Neither alterations in the chemical shift nor in the line width of the bound FMN and FAD phosphate resonances were observed upon complex formation of NADPH:cytochrome P-450 reductase with the cytochrome.
FMN 与 NADPH：细胞色素 P - 450 还原酶的显著解离导致细胞色素 b5、细胞色素 c 以及细胞色素 P - 450 还原活性丧失。然而，用添加的 FMN 孵育耗尽 FMN 的酶后，还原这些电子受体的能力得到了极大恢复。在高浓度 KCl 存在下，NADPH：细胞色素 P - 450 还原酶对细胞色素 c 和去污剂溶解的细胞色素 b5 的还原作用显著增强，尽管盐对细胞色素 P - 450 还原的刺激作用较小。在含有高盐浓度的溶液中，超氧化物歧化酶对细胞色素还原的速率或程度未产生明显影响。已知黄素蛋白与细胞色素 c 的复合物形成参与非生理性电子传递机制，这会导致细胞色素 c 的 Soret 带区域吸收光谱发生扰动，并且添加 KCl 会增强其扰动幅度。同样，细胞色素 c 与黄素蛋白结合时，在 Soret 带观察到椭圆率有明显增加。然而，NADPH：细胞色素 P - 450 还原酶与细胞色素 b5 或细胞色素 P - 450 的复合物在吸收光谱和圆二色光谱中仅发现微小变化。这表明高盐浓度通过疏水 - 疏水相互作用使血红素和黄素辅基之间接触更紧密，而非黄素蛋白与细胞色素之间的静电 - 电荷配对，从而导致电子传递速率加快。NADPH：细胞色素 P - 450 还原酶与细胞色素形成复合物后，未观察到结合的 FMN 和 FAD 磷酸共振的化学位移或线宽发生改变。
