Jenkins C M, Waterman M R
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA.
Biochemistry. 1998 Apr 28;37(17):6106-13. doi: 10.1021/bi973076p.
In addition to their endogenous roles as an activation system for various Escherichia coli metabolic pathways, the soluble flavoproteins flavodoxin (Fld) and NADPH-flavodoxin (ferredoxin) reductase (Fpr) can serve as an electron-transfer system for microsomal cytochrome P450s. Furthermore, since Fld and Fpr are structurally similar to the functional domains (FMN binding and NADPH/FAD binding domains, respectively) of NADPH-cytochrome P450 reductases (P450 reductases), these bacterial proteins represent a potentially useful model system for eukaryotic P450 reductases. Here we delineate similarities and differences between the E. coli Fpr-Fld system and rat P450 reductase as electron donors to bovine 17alpha-hydroxylase/17,20-lyase P450 (P450c17). Importantly, recombinant Fpr, in combination with recombinant Fld, supports both the hydroxylase and lyase activities of P450c17 to the same proportional extent (hydroxylase-to-lyase ratio) as does P450 reductase. Maximum P450c17 turnover [5-6 mol of 17alpha-OH-progesterone (mol of P450c17)-1 min-1] was achieved using a large molar excess (50-100-fold over P450c17) of a 1:1 ratio of Fpr-Fld, although this rate was an order of magnitude less than the maximal P450 reductase-supported activity. Using these conditions, we have examined the effects of increasing ionic strength and the presence of cytochrome b5 (b5) on these two systems. Critical Fld-P450c17 electrostatic interactions are disrupted at moderate ionic strength (>100 mM NaCl) as evidenced by significant inhibition (>50%) of Fpr-Fld-supported P450c17 activity while much higher ionic strength (300 mM NaCl) is required to disrupt P450 reductase-P450c17 interactions to the same extent. Interestingly, cytochrome b5 was found to dramatically inhibit both P450 reductase- and Fpr-Fld-supported P450c17 progesterone 17alpha-hydroxylase activity while in contrast 17alpha-OH-pregnenolone lyase activity was stimulated by b5. Investigation of the fate of reducing equivalents from NADPH added to Fpr under aerobic conditions revealed that the majority of the protein-bound FAD of Fpr is converted to the hydroquinone form. In constrast, the FMN of Fld is reduced by Fpr to a stable blue, neutral semiquinone which serves as the predominant electron donor to P450c17 in reconstitution assays. Thus, while the Fpr-Fld system and P450 reductase are fundamentally different with respect to their electrostatic interactions with P450c17, their ability to support maximal P450c17 turnover, and the FMN redox states (one-electron-reduced for Fld and two-electron-reduced for P450 reductase) capable of transferring electrons to microsomal cytochrome P450s, these differences do not appear to influence the relative catalytic efficiency of the P450c17 hydroxylase and lyase reactions.
除了作为各种大肠杆菌代谢途径的激活系统发挥内源性作用外，可溶性黄素蛋白黄素氧还蛋白（Fld）和NADPH - 黄素氧还蛋白（铁氧化还原蛋白）还原酶（Fpr）还可作为微粒体细胞色素P450的电子传递系统。此外，由于Fld和Fpr在结构上分别与NADPH - 细胞色素P450还原酶（P450还原酶）的功能域（FMN结合域和NADPH / FAD结合域）相似，这些细菌蛋白代表了一种潜在有用的真核P450还原酶模型系统。在此，我们描述了大肠杆菌Fpr - Fld系统与大鼠P450还原酶作为牛17α - 羟化酶/17,20 - 裂解酶P450（P450c17）电子供体之间的异同。重要的是，重组Fpr与重组Fld结合，支持P450c17的羟化酶和裂解酶活性达到与P450还原酶相同的比例程度（羟化酶与裂解酶比率）。使用大摩尔过量（比P450c17高50 - 100倍）的1:1比例的Fpr - Fld可实现最大P450c17周转率[5 - 6摩尔17α - 羟基孕酮/（摩尔P450c17）-1分钟-1]，尽管该速率比最大P450还原酶支持的活性低一个数量级。在这些条件下，我们研究了增加离子强度和细胞色素b5（b5）的存在对这两个系统的影响。在中等离子强度（>100 mM NaCl）下，关键的Fld - P450c17静电相互作用被破坏，这表现为Fpr - Fld支持的P450c17活性受到显著抑制（>50%），而需要更高的离子强度（300 mM NaCl）才能在相同程度上破坏P450还原酶 - P450c17相互作用。有趣的是，发现细胞色素b可以显著抑制P450还原酶和Fpr - Fld支持的P450c17孕酮17α - 羟化酶活性，而相反，17α - 羟基孕烯醇酮裂解酶活性受到b5的刺激。对在有氧条件下添加到Fpr的NADPH中还原当量的命运进行研究表明，Fpr中大部分与蛋白质结合的FAD转化为对苯二酚形式。相比之下，Fld的FMN被Fpr还原为稳定的蓝色中性半醌，在重组测定中它作为向P450c17的主要电子供体。因此，虽然Fpr - Fld系统和P450还原酶在与P450c17的静电相互作用、支持最大P450c17周转率的能力以及能够将电子转移到微粒体细胞色素P450的FMN氧化还原状态（Fld为单电子还原，P450还原酶为双电子还原）方面存在根本差异，但这些差异似乎并不影响P450c17羟化酶和裂解酶反应的相对催化效率。
