Localization of the free radical on the flavin mononucleotide of the air-stable semiquinone state of NADPH-cytochrome P-450 reductase using 31P NMR spectroscopy.
Localization of the free radical on the flavin mononucleotide of the air-stable semiquinone state of NADPH-cytochrome P-450 reductase using 31P NMR spectroscopy.
复制标题
使用 31P NMR 光谱将自由基定位在 NADPH-细胞色素 P-450 还原酶的空气稳定半醌状态的黄素单核苷酸上。
DOI：
10.1021/bi00370a068
复制
发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Masters,BS
中科院分区：
文献类型：
--
作者：
Otvos,JD;Krum,DP;Masters,BS
生物化学系，威斯康星州医学院，密尔沃基，威斯康星州53226，和化学系，威斯康星-密尔沃基大学，密尔沃基，威斯康星州53201接收于1986年2月25日;修订的Mandarin pt接收于1986年6月24日摘要：微粒体NADPH-细胞色素P-450还原酶是已知的唯一含有FAD和FMN作为辅基的哺乳动物黄素蛋白。空气稳定的半醌的发现[Masters，B. S.美国，Kamin，H.，吉布森，Q. H、&威廉姆斯，C. H、Jr.（1965）J.Biol.Chem.240，921-931]及其作为单电子还原态的鉴定[Iyanagi，T.，& Mason，H. S.（1973）Biochemistry 12，2297-2308]已经产生了许多研究来阐明其独特的催化机制。在本文中，31 P NMR光谱被用来探测在这个空气中稳定的半醌形式的自由基的本地化，并确定由顺磁性离子Mn（II）的影响的FAD和FMN辅基的环境。与从利用无FMN还原酶的研究中得出的结论一致[朱红，J.L.，& Coon，M. J.（1978）J.Biol.Chem.253，8812-8819]，通过31 P NMR谱中FMN部分的特征共振的加宽，显示自由基存在于FMN部分上。此外，顺磁性离子Mn（II）的影响，确定了四个共振归因于FAD和FMN和额外的贡献NADP+导致的生理还原剂NADPH的氧化。Mn（II）的加入对FMN和FAD信号的线宽几乎没有影响，但由于Tx弛豫时间的减少而导致它们的强度增加。另一方面，结合NADP+的焦磷酸盐共振仅受顺磁性离子的最小影响，表明NADP+的焦磷酸盐部分比FAD的焦磷酸盐更多地与溶剂隔离。这些研究表明，31 P NMR作为直接探针的NADPH-细胞色素P-450还原酶的含磷辅因子在各种条件下，包括氧化还原状态的变化的环境的效用。
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and Department of Chemistry, University of Wisconsin—Milwaukee, Milwaukee, Wisconsin 53201 Received February 25, 1986; Revised Manuscript Received June 24, 1986 abstract: Microsomal NADPH-cytochrome P-450 reductase is the only mammalian flavoprotein known to contain both FAD and FMN as prosthetic groups. The discovery of the air-stable semiquinone [Masters, B. S. S., Kamin, H., Gibson, Q. H., & Williams, C. H., Jr.(1965) J. Biol. Chem. 240, 921-931] and its identification as a one-electron-reduced state [Iyanagi, T., & Mason, H. S.(1973) Biochemistry 12, 2297-2308] have engendered a number of studies to elucidate its unique catalytic mechanism. In this paper, 31P NMR spectroscopy is utilized to probe the localization of the free radical in this air-stable semiquinone form and to ascertain the environments of the FAD and FMN prosthetic groups as affected by the para-magnetic ion Mn (II). Consistent with conclusions drawnfrom studies utilizing FMN-free reductase [Vermilion, J. L., & Coon, M. J.(1978) J. Biol. Chem. 253, 8812-8819], the free radical was shown to reside on the FMN moiety by the broadening of its characteristic resonance in the 31P NMR spectrum. In addition, the effect of the paramagnetic ion Mn (II) was determined on the four resonances attributable to FAD and FMN and the additional ones contributed by NADP+ resulting from the oxidation of the physiological reductant NADPH. The addition of Mn (II) had little effect on the line widths of the FMN and FAD signals but resulted in an increase in their intensities due to a decrease in Tx relaxation times. On the other hand, the pyrophosphate resonances of bound NADP+ were only minimally affected by the paramagnetic ion, indicating that the pyrophosphate moiety of NADP+ is more sequestered from the solvent than the pyrophosphate of FAD. These studies demonstrate the utility of 31P NMR as a direct probe of the environments of the phosphorus-containing cofactors of NADPH-cytochrome P-450 reductase under various conditions, including changes in redox state.