Department of Molecular Biology, 10550 North Torrey Pines Road, The Scripps Research Institute, La Jolla, California 92037, USA.
Biochemistry. 2010 Apr 27;49(16):3412-9. doi: 10.1021/bi100183g.
P450cam from Pseudomonas putida is the best characterized member of the vast family of cytochrome P450s, and it has long been believed to have a more rigid and closed active site relative to other P450s. Here we report X-ray structures of P450cam crystallized in the absence of substrate and at high and low [K(+)]. The camphor-free structures are observed in a distinct open conformation characterized by a water-filled channel created by the retraction of the F and G helices, disorder of the B' helix, and loss of the K(+) binding site. Crystallization in the presence of K(+) alone does not alter the open conformation, while crystallization with camphor alone is sufficient for closure of the channel. Soaking crystals of the open conformation in excess camphor does not promote camphor binding or closure, suggesting resistance to conformational change by the crystal lattice. This open conformation is remarkably similar to that seen upon binding large tethered substrates, showing that it is not the result of a perturbation by the ligand. Redissolved crystals of the open conformation are observed as a mixture of P420 and P450 forms, which is converted to the P450 form upon addition of camphor and K(+). These data reveal that P450cam can dynamically visit an open conformation that allows access to the deeply buried active site without being induced by substrate or ligand.
假单胞菌 P450cam 是细胞色素 P450 大家族中研究最为透彻的成员，长期以来被认为其活性位点相对于其他 P450 更为僵化和封闭。在这里，我们报告了无底物和高、低 [K(+)] 条件下 P450cam 的晶体结构。在没有樟脑的情况下，观察到了明显的开放构象，其特征是 F 和 G 螺旋回缩、B' 螺旋无序以及 K(+)结合位点缺失，从而形成了一个充满水的通道。单独存在 K(+)的情况下，结晶不会改变开放构象，而单独存在樟脑就足以关闭通道。将开放构象的晶体浸泡在过量的樟脑中不会促进樟脑结合或关闭，表明晶体晶格对构象变化有抵抗力。这种开放构象与结合大的连接底物时观察到的构象非常相似，表明这不是配体引起的扰动的结果。重新溶解的开放构象晶体观察到 P420 和 P450 两种形式的混合物，在加入樟脑和 K(+)后，该混合物转化为 P450 形式。这些数据表明，P450cam 可以动态地访问开放构象，从而无需底物或配体诱导即可进入深埋的活性位点。
