Gable Jessica A, Tripathi Sarvind, Poulos Thomas L
Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and Chemistry, University of California, Irvine, California 92697-3900, United States.
ACS Omega. 2022 May 27;7(22):18481-18485. doi: 10.1021/acsomega.2c00960. eCollection 2022 Jun 7.
A characteristic feature of cytochromes P450* is that the complex formed between the ferrous heme iron and carbon monoxide generates an intense absorption band at 450 nm. This unique feature of P450s is due to the proximal thiolate Cys ligand coordinated to the heme iron. Various harsh treatments shift this band to 420 nm, thereby giving P420 which is most often associated with an inactive form of the enzyme. Various explanations have been put forward to explain the P450-to-P420 change ranging from protonation of the Cys heme ligand, displacement of the Cys ligand, or replacement of the Cys ligand with His. There are two crystal structures of the well-studied cytochrome P450cam that have a high fraction of P420. In one, P450cam is cross-linked to its redox partner, putidaredoxin (Pdx), and the second is P450cam crystallized in the absence of substrate. In both of these structures, a significant part of the substrate pocket is disordered and the poor quality of the electron density for the substrate indicates substantial disorder. However, in both structures there is no detectable change in the Cys-iron ligation or surrounding structure. These results indicate that the P450-to-P420 switch is due primarily to an opening and disordering around the substrate binding pocket and not ligand displacement or ligand swapping. Since it remains a possibility that ligand swapping could be responsible for P420 in some cases, we mutated to Gln the 3 His residues (352, 355, and 361) close enough to the proximal side of the heme that could possibly serve as heme ligands. The triple variant forms P420 which indicates that swapping Cys for His is not a requirement for the P450-to-P420 switch.
细胞色素P450*的一个特征是亚铁血红素铁与一氧化碳形成的复合物在450纳米处产生一个强吸收带。P450的这一独特特征归因于与血红素铁配位的近端硫醇盐半胱氨酸配体。各种苛刻处理会使该吸收带移至420纳米，从而产生P420，它通常与酶的无活性形式相关。人们提出了各种解释来阐释从P450到P420的变化，范围包括半胱氨酸血红素配体的质子化、半胱氨酸配体的置换或用组氨酸取代半胱氨酸配体。有两个经过充分研究的细胞色素P450cam的晶体结构，其中P420的比例很高。在一个结构中，P450cam与其氧化还原伴侣恶臭假单胞菌铁氧还蛋白（Pdx）交联，另一个是在没有底物的情况下结晶的P450cam。在这两种结构中，底物口袋的很大一部分是无序的，底物电子密度的质量很差表明存在大量无序。然而，在这两种结构中，半胱氨酸-铁配位或周围结构均未检测到变化。这些结果表明，从P450到P420的转变主要是由于底物结合口袋周围的开放和无序，而不是配体置换或配体交换。由于在某些情况下配体交换仍有可能是P420产生的原因，我们将靠近血红素近端侧、可能充当血红素配体的3个组氨酸残基（352、355和361）突变为谷氨酰胺。三重变体形成P420，这表明用组氨酸替换半胱氨酸不是P450到P420转变的必要条件。
