Sahil Mohammad, Singh Tejender, Ghosh Soumya, Mondal Jagannath
Tata Institute of Fundamental Research, Hyderabad 500046, India.
J Am Chem Soc. 2023 Nov 1;145(43):23488-23502. doi: 10.1021/jacs.3c06144. Epub 2023 Oct 23.
We identified a multisubstrate-bound state, hereby referred as a 3site state, in cytochrome P450cam via integrating molecular dynamics simulation with nuclear magnetic resonance (NMR) pseudocontact shift measurements. The 3site state is a result of simultaneous binding of three camphor molecules in three locations around P450cam: (a) in a well-established "catalytic" site near heme, (b) in a kink-separated "waiting" site along channel-1, and (c) in a previously reported "allosteric" site at E, F, G, and H helical junctions. These three spatially distinct binding modes in the 3site state mutually communicate with each other via homotropic allostery and act cooperatively to render P450cam functional. The 3site state shows a significantly superior fit with NMR pseudo contact shift (PCS) data with a -score of 0.045 than previously known bound states and consists of D251 free of salt-bridges with K178 and R186, rendering the enzyme functionally primed. To date, none of the reported cocomplex of P450cam with its redox partner putidaredoxin (pdx) has been able to match solution NMR data and controversial pdx-induced opening of P450cam's channel-1 remains a matter of recurrent discourse. In this regard, inclusion of pdx to the 3site state is able to perfectly fit the NMR PCS measurement with a -score of 0.08 and disfavors the pdx-induced opening of channel-1, reconciling previously unexplained remarkably fast hydroxylation kinetics with a of 10.2 s. Together, our findings hint that previous experimental observations may have inadvertently captured the 3site state as an in vitro solution state, instead of the catalytic state alone, and provided a distinct departure from the conventional understanding of cytochrome P450.
通过将分子动力学模拟与核磁共振（NMR）伪接触位移测量相结合，我们在细胞色素P450cam中确定了一种多底物结合状态，在此称为三位点状态。三位点状态是三个樟脑分子同时结合在P450cam周围三个位置的结果：（a）在血红素附近一个公认的“催化”位点；（b）在沿通道1的扭结分隔的“等待”位点；（c）在先前报道的E、F、G和H螺旋交界处的“变构”位点。三位点状态下这三种空间上不同的结合模式通过同向变构相互通信，并协同作用使P450cam发挥功能。三位点状态与NMR伪接触位移（PCS）数据的拟合度明显优于先前已知的结合状态，得分0.045，并且由与K178和R186没有盐桥的D251组成，使酶处于功能就绪状态。迄今为止，所报道的P450cam与其氧化还原伙伴恶臭假单胞菌铁氧还蛋白（pdx）的共复合物均无法与溶液NMR数据匹配，并且关于pdx诱导的P450cam通道1开放的争议仍然是一个反复讨论的问题。在这方面，将pdx纳入三位点状态能够以0.08的得分完美拟合NMR PCS测量结果，并且不支持pdx诱导的通道1开放，从而解释了先前无法解释的10.2 s的显著快速羟基化动力学。总之，我们的研究结果表明，先前的实验观察可能无意中捕捉到了作为体外溶液状态的三位点状态，而不仅仅是催化状态，并提供了与细胞色素P450的传统理解明显不同的观点。
