FMN Binding Site of Yeast NADPH-Cytochrome P450 Reductase Exposed at the Surface Is Highly Specific

被引:5
|
作者
Ivanov, Alexis S. [2 ]
Gnedenko, Oksana V. [2 ]
Molnar, Andrey A. [2 ]
Archakov, Alexander I. [2 ]
Podust, Larissa M. [1 ]
机构
[1] Univ Calif San Francisco, Dept Pharmaceut Chem, San Francisco, CA 94143 USA
[2] Russian Acad Med Sci, Inst Biomed Chem, Moscow 119121, Russia
基金
美国国家卫生研究院; 俄罗斯基础研究基金会;
关键词
NITRIC-OXIDE SYNTHASE; P-450; REDUCTASE; ELECTRON-TRANSFER; NADPH-CYTOCHROME-P-450; BACILLUS-MEGATERIUM; DIFLAVIN REDUCTASES; ESCHERICHIA-COLI; LIVER-MICROSOMES; STRUCTURAL BASIS; OXIDOREDUCTASE;
D O I
10.1021/cb100055v
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
NADPH-cytochrome P450 reductase (CPR) transfers two reducing equivalents derived from NADPH via FAD and FMN to microsomal P450 monooxygenases in one-electron transfer steps. The crystal structure of yeast CPR (yCPR) contains a surface-exposed FMN binding site (FMN2 site) at the interface of the FMN binding and connecting domains, in addition to the single buried site that has been observed in rat CPR. This finding provides a testable hypothesis of how intramolecular (between FAD and FMN) and intermolecular (between FMN and P450) electron transfer may occur in CPR. To verify that occupancy of the FMN2 site is not an artifact of crystallization, a surface plasmon resonance (SPR) biosensor technique has been applied to probe the selectivity of this site under functional conditions. A series of kinetic and equilibrium binding experiments involving yCPR immobilized on different sensor chip surfaces was performed using FMN and FAD, as well as FMN-derived compounds, including riboflavin, dimethylalloxazine, and alloxazine, and other molecules that resemble the planar isoalloxazine ring structure. Only FMN and FAD showed stoichiometric binding responses. Binding affinity for FMN was in the submicromolar range, 30 times higher than that for FAD. Association kinetic rates for the yCPR/FMN complex were up to 60-fold higher than for the yCPR/FAD complex. Taken together, these data indicate that (i) the surface-exposed site in yCPR is highly selective toward binding flavins, (ii) binding of FMN in this site is notably favored, and finally, (iii) both the phosphate group and the isoalloxazine ring of FMN are essential for binding.
引用
收藏
页码:767 / 776
页数:10
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