Lamb David C, Kim Youngchang, Yermalitskaya Liudmila V, Yermalitsky Valery N, Lepesheva Galina I, Kelly Steven L, Waterman Michael R, Podust Larissa M
Wolfson Laboratory of P450 Biodiversity, Swansea Medical School University of Wales Swansea, Swansea, Wales SA2 8PP, UK.
Structure. 2006 Jan;14(1):51-61. doi: 10.1016/j.str.2005.09.015.
NADPH-cytochrome P450 reductase transfers two reducing equivalents derived from a hydride ion of NADPH via FAD and FMN to the large family of microsomal cytochrome P450 monooxygenases in one-electron transfer steps. The mechanism of electron transfer by diflavin reductases remains elusive and controversial. Here, we determined the crystal structure of truncated yeast NADPH-cytochrome P450 reductase, which is functionally active toward its physiological substrate cytochrome P450, and discovered a second FMN binding site at the interface of the connecting and FMN binding domains. The two FMN binding sites have different accessibilities to the bulk solvent and different amino acid environments, suggesting stabilization of different electronic structures of the reduced flavin. Since only one FMN cofactor is required for function, a hypothetical mechanism of electron transfer is discussed that proposes shuttling of a single FMN between these two sites coupled with the transition between two semiquinone forms, neutral (blue) and anionic (red).
NADPH-细胞色素P450还原酶通过黄素腺嘌呤二核苷酸（FAD）和黄素单核苷酸（FMN），以单电子转移步骤将源自NADPH氢化物离子的两个还原当量转移至微粒体细胞色素P450单加氧酶大家族。双黄素还原酶的电子转移机制仍然难以捉摸且存在争议。在此，我们确定了截短的酵母NADPH-细胞色素P450还原酶的晶体结构，该酶对其生理底物细胞色素P450具有功能活性，并在连接结构域和FMN结合结构域的界面处发现了第二个FMN结合位点。这两个FMN结合位点对大量溶剂具有不同的可及性以及不同的氨基酸环境，这表明还原黄素的不同电子结构得到了稳定。由于功能仅需要一个FMN辅因子，因此讨论了一种假设的电子转移机制，该机制提出单个FMN在这两个位点之间穿梭，并伴随着中性（蓝色）和阴离子（红色）两种半醌形式之间的转变。
