Department of Molecular Biology, University of California, Irvine, California 92697-3900, USA.
Biochemistry. 2010 Jan 12;49(1):58-67. doi: 10.1021/bi901872s.
In the cytochrome P450cam-dependent monooxygenase system from Pseudomonas putida, putidaredoxin (Pdx) shuttles electrons between putidaredoxin reductase (Pdr) and P450cam and, thus, must form transient complexes with both partners. 1-Ethyl 3-[3-(dimethylamino)propyl]carbodiimide (EDC) was found to promote formation of stoichiometric Pdr-Pdx complexes only when carboxyl groups on Pdx were activated. The yield of the EDC-mediated cross-link depended on the Pdx variant used and the redox state of both partners, decreasing in the following order: Pdr(ox)-Pdx(ox) > Pdr(ox)-Pdx(red) > or = Pdr(red)-Pdx(red). The Pdr-Pdx C73S/C85S conjugate was purified and characterized. Compared to the equimolar mixture of intact Pdr and Pdx, the fusion protein was more efficient in electron transfer to cytochrome c and, in the presence of saturating levels of P450cam, more effectively supported camphor hydroxylation. On the basis of our results, we conclude that (i) the cross-linked complex is physiologically relevant and represents a suitable model for mechanistic studies, (ii) molecular recognition between Pdr and Pdx is redox-controlled and assisted by the Glu72(Pdx)-Lys409(Pdr) charge-charge interactions, and (iii) the high specificity of the Pdr-Pdx couple may be due to finely tuned interactions at the protein-protein interface resulting in only one strongly preferred docking orientation leading to efficient FAD-to-[2Fe-2S] electron transfer.
在恶臭假单胞菌依赖细胞色素 P450cam 的单加氧酶系统中，假单胞菌还原酶（Pdr）和 P450cam 之间的电子穿梭由假单胞菌黄素蛋白（Pdx）完成，因此，Pdx 必须与这两种蛋白形成瞬时复合物。发现 1-乙基-3-[3-（二甲基氨基）丙基]碳二亚胺（EDC）仅在 Pdx 的羧基基团被激活时才能促进形成化学计量的 Pdr-Pdx 复合物。EDC 介导的交联产物的产率取决于所使用的 Pdx 变体以及两种蛋白的氧化还原状态，按以下顺序降低：Pdr（ox）-Pdx（ox）> Pdr（ox）-Pdx（red）≥ Pdr（red）-Pdx（red）。纯化并表征了 Pdr-Pdx C73S/C85S 融合蛋白。与等摩尔比的完整 Pdr 和 Pdx 的混合物相比，融合蛋白在向细胞色素 c 传递电子方面更有效，并且在存在饱和水平的 P450cam 时，更有效地支持樟脑羟化。根据我们的结果，我们得出结论：（i）交联复合物在生理上是相关的，并且是用于机制研究的合适模型；（ii）Pdr 和 Pdx 之间的分子识别受氧化还原控制，并受 Glu72（Pdx）-Lys409（Pdr）电荷-电荷相互作用的辅助；（iii）Pdr-Pdx 对的高特异性可能是由于在蛋白质-蛋白质界面上精细调节的相互作用，导致只有一个强烈优选的对接取向，从而导致有效的 FAD-至-[2Fe-2S]电子转移。
