FERREDOXINS IN HYDROXYLASE ENZYME ELECTRON TRANSFER PATHWAYS

在过去的几年里，我们小组一直在调查 一类Cys4Fe2S2铁氧还蛋白的详细结构和功能 它们作为细胞色素P450的还原剂和效应剂 单加氧酶，将氧气中的一个氧原子还原为水的酶 同时将第二氧插入未活化的C-H键中 底物。P450超家族的蛋白质广泛分布于 从好氧细菌到哺乳动物。在许多这样的系统中， 细胞色素P450接受铁氧还蛋白的电子，而铁氧还蛋白 被NADH或NAD(P)H依赖的黄素蛋白还原。这个 在这些系统中氧化还原蛋白之间的相互作用往往是相当的 特异性的，同源系统之间的交叉反应性低。为 例如，有高度的结构和序列同源性 在腐达还蛋白(PDX)和肾上腺素(ADX)之间，两种铁氧还蛋白 为细菌和哺乳动物细胞色素P450提供电子， 分别进行了分析。然而，ADX没有能力转移第二个 转换为细胞色素P450所需的电子，生理的 PDX的氧化还原合作伙伴。即使是与之密切相关的特普来辛(TDX) 是一种细菌蛋白质，具有高度的序列、功能和 与PDX的结构同源性。只显示了10%的交叉反应 P450，，.系统。为了进一步探索这些结构-功能 关系，我们正在确定特来多辛(TDX)的结构。 我们已经获得了750 MHz的高分辨率‘H，’H-NOESY，1H， “N-TOCSY-HSQC和‘H，”N-NOESY-HSQC数据，我们已经分析到 产生几乎完全的1H和“N共振的指认 TDX的抗磁区。

For the past several years, our group has been investigating the detailed structure and function of a class of Cys4Fe2S2 ferredoxins which act as reductants and effectors of cytochrome P450 monooxygenases, enzymes which reduce one oxygen atomof 02 to water while inserting the second oxygen into an unactivated C-H bond in a substrate. Proteins of the P450 superfamily are widely distributed in nature from aerobic bacteria to mammals. In many of these systems, the cytochrome P450 accepts electrons from ferredoxins, which in turn are reduced by NADH or NAD(P)H-dependent flavoproteins. The interactions between redox proteins in these systems tend to be quite specific, with low cross-reactivity between homologous systems. For example, there is a high degree of structural and sequence homology between putidaredoxin (Pdx) and adrenodoxin (Adx), two ferredoxins which donate electrons to bacterial and mammalian cytochromes P450, respectively. However, Adx is not competent to transfer the second electron required for turnover to cytochrome P450, the physiological redox partner of Pdx. Even the closely related terpredoxin (Tdx) which is a bacterial protein with a high degree of sequence, functional and structural homology to Pdx. shows only 10% cross-reactivity with the P450,,. system. To further explore these structure-function relationships, we are determining the structure of terpredoxin (Tdx). We have obtained 750 MHz high resolution 'H,'H-NOESY, 1H, "N-TOCSY-HSQC and 'H, "N-NOESY-HSQC data, which we have analyzed to yield nearly complete assignment of 1H and "N resonances for the diamagnetic region of Tdx.