Departments of Molecular Biology and Biochemistry, Pharmaceutical Sciences, and Chemistry , University of California , Irvine , California 92697-3900 , United States.
Department of Chemistry & Biochemistry , University of California , Santa Cruz , California 95064 , United States.
J Am Chem Soc. 2019 Feb 13;141(6):2678-2683. doi: 10.1021/jacs.8b13079. Epub 2019 Jan 31.
It has become increasingly clear that cytochromes P450 can cycle back and forth between two extreme conformational states termed the closed and open states. In the well-studied cytochrome P450cam, the binding of its redox partner, putidaredoxin (Pdx), shifts P450cam toward the open state. Shifting to the open state is thought to be important in the formation of a proton relay network essential for O-O bond cleavage and formation of the active Fe(IV)═O intermediate. Another important intermediate is the oxy-P450cam complex when bound to Pdx. Trapping this intermediate in crystallo is challenging owing to its instability, but the CN complex is both stable and an excellent mimic of the O complex. Here we present the P450cam-Pdx structure complexed with CN. CN results in large conformational changes including cis/trans isomerization of proline residues. Changes include large rearrangements of active-site residues and the formation of new active-site access channel that we have termed channel 2. The formation of channel 2 has also been observed in our previous molecular dynamics simulations wherein substrate binding to an allosteric site remote from the active site opens up channel 2. This new structure supports an extensive amount of previous work showing that distant regions of the structure are dynamically coupled and underscores the potentially important role that large conformational changes and dynamics play in P450 catalysis.
越来越明显的是，细胞色素 P450 可以在两种极端构象状态之间来回循环，这两种状态分别称为关闭状态和开放状态。在研究充分的细胞色素 P450cam 中，其氧化还原伴侣 putidaredoxin（Pdx）的结合将 P450cam 推向开放状态。向开放状态的转变被认为对于形成质子传递网络至关重要，该网络对于 O-O 键的断裂和活性 Fe(IV)═O 中间物的形成是必需的。另一个重要的中间物是与 Pdx 结合时的氧合-P450cam 复合物。由于其不稳定性，在结晶中捕获这种中间物具有挑战性，但 CN 复合物既稳定又能很好地模拟 O 复合物。在这里，我们展示了与 CN 结合的 P450cam-Pdx 结构复合物。CN 导致构象发生很大变化，包括脯氨酸残基的顺/反异构化。变化包括活性位点残基的大规模重排和新的活性位点进入通道的形成，我们称之为通道 2。在我们之前的分子动力学模拟中也观察到了通道 2的形成，其中底物与远离活性位点的别构位点的结合打开了通道 2。这个新结构支持了大量先前的工作，表明结构的遥远区域是动态耦合的，并强调了大的构象变化和动力学在 P450 催化中的潜在重要作用。
