Noble Michael A, Girvan Hazel M, Smith Susan J, Smith W Ewen, Murataliev Marat, Guzov Victor M, Feyereisen René, Munro Andrew W
Anadys Pharmaceuticals, Inc., San Diego, California, USA.
Drug Metab Rev. 2007;39(2-3):599-617. doi: 10.1080/03602530701468458.
Interactions between a soluble form of microsomal cytochrome b(5) (b(5)) from Musca domestica (housefly) and Bacillus megaterium flavocytochrome P450 BM3 and its component reductase (CPR), heme (P450) and FAD/NADPH-binding (FAD) domains were analyzed by a combination of steady-state and stopped-flow kinetics methods, and optical spectroscopy techniques. The high affinity binding of b(5) to P450 BM3 induced a low-spin to high-spin transition in the P450 heme iron (K(d) for b(5) binding = 0.44 microM and 0.72 microM for the heme domain and intact flavocytochrome, respectively). The b(5) had modest inhibitory effects on steady-state turnover of P450 BM3 with fatty acids, and the ferrous-carbon monoxy P450 complex was substantially stabilized on binding b(5). Single turnover reduction of b(5) by BM3 using stopped-flow absorption spectroscopy (k(lim) = 116 s(-1)) was substantially faster than steady-state reduction of b(5) by P450 BM3 (or its CPR and FAD domains), indicating rate-limiting step(s) other than BM3 flavin-to-b(5) heme electron transfer in the steady-state reaction. Steady-state b(5) reduction by P450 BM3 was considerably accelerated at high ionic strength. Pre-reduction of P450 BM3 by NADPH decreased the k(lim) for b(5) reduction approximately 10-fold, and also resulted in a lag phase in steady-state b(5) reduction that was likely due to BM3 conformational perturbations sensitive to the reduction state of the flavocytochrome. Ferrous b(5) could not reduce the ferric P450 BM3 heme domain under anaerobic conditions, consistent with heme iron reduction potentials of the two proteins. However, rapid oxidation of both hemoproteins occurred on aeration of the ferrous protein mixture (and despite the much slower autoxidation rate of b(5) in isolation), consistent with electron transfer occurring from b(5) to the oxyferrous P450 BM3 in the complex. The results demonstrate that strong interactions occur between a eukaryotic b(5) and a model prokaryotic P450. Binding of b(5) perturbs BM3 heme iron spin-state equilibrium, as is seen in many physiologically relevant b(5) interactions with eukaryotic P450s. These results are consistent with the conservation of structure of P450s (particularly at the heme proximal face) between prokaryotes and eukaryotes, and may point to as yet undiscovered roles for b(5)-like proteins in the control of activities of certain prokaryotic P450s.
通过稳态和停流动力学方法以及光谱技术相结合，分析了家蝇可溶性微粒体细胞色素b(5)（b(5)）与巨大芽孢杆菌黄素细胞色素P450 BM3及其组成还原酶（CPR）、血红素（P450）结构域和FAD/NADPH结合（FAD）结构域之间的相互作用。b(5)与P450 BM3的高亲和力结合诱导了P450血红素铁从低自旋到高自旋的转变（b(5)结合的解离常数K(d)，血红素结构域为0.44 microM，完整黄素细胞色素为0.72 microM）。b(5)对P450 BM3利用脂肪酸进行的稳态周转有适度的抑制作用，并且亚铁-一氧化碳单氧P450复合物在结合b(5)后得到显著稳定。利用停流吸收光谱法（k(lim)=116 s(-1)）由BM3对b(5)进行的单周转还原比P450 BM3（或其CPR和FAD结构域）对b(5)的稳态还原快得多，这表明在稳态反应中除了BM3黄素到b(5)血红素的电子转移之外还存在限速步骤。在高离子强度下，P450 BM3对b(5)的稳态还原显著加速。NADPH对P450 BM3的预还原使b(5)还原的k(lim)降低约10倍，并且还导致稳态b(5)还原出现滞后相，这可能是由于黄素细胞色素的还原状态引起的BM3构象扰动所致。在厌氧条件下，亚铁b(5)不能还原三价铁P450 BM3血红素结构域，这与两种蛋白质的血红素铁还原电位一致。然而，在亚铁蛋白混合物曝气时两种血红素蛋白都迅速氧化（尽管单独的b(5)自氧化速率要慢得多），这与复合物中从b(5)到亚铁-氧合P450 BM3发生电子转移一致。结果表明真核生物的b(5)与模型原核生物P450之间发生了强烈相互作用。b(5)的结合扰乱了BM3血红素铁的自旋态平衡，这在许多与真核生物P450的生理相关b(5)相互作用中都能看到。这些结果与原核生物和真核生物之间P450结构（特别是在血红素近表面）的保守性一致，并且可能表明类b( like proteins)-5在控制某些原核生物P450活性方面尚未被发现的作用。
