Altarsha Muhannad, Benighaus Tobias, Kumar Devesh, Thiel Walter
Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470 Mülheim an der Ruhr, Germany.
J Am Chem Soc. 2009 Apr 8;131(13):4755-63. doi: 10.1021/ja808744k.
Proton transfer reactions play a vital role in the catalytic cycle of cytochrome P450cam and are responsible for the formation of the iron-oxo species called Compound I (Cpd I) that is supposed to be the active oxidant. Depending on the course of the proton transfer, protonation of the last observable intermediate (ferric hydroperoxo complex, Cpd 0) can lead to either the formation of Cpd I (coupling reaction) or the ferric resting state (uncoupling reaction). The ratio of these two processes is drastically affected by mutation of the Thr252 residue. In this work, we study the effect of Thr252X (X = serine, valine, alanine, glycine) mutations on the formation of Cpd I by means of hybrid quantum mechanical/molecular mechanical (QM/MM) calculations and classical simulations. In the wild-type enzyme, the coupling reaction is favored since its rate-limiting barrier is 13 kcal/mol lower than that for uncoupling. This difference is reduced to 7 kcal/mol in the serine mutant. In the case of valine, alanine, and glycine mutants, an additional water molecule enters the active site and lowers the activation energy of the uncoupling reaction significantly. With the additional water molecule, coupling and uncoupling have similar barriers in the valine mutant, and the uncoupling reaction becomes favored in the alanine and glycine mutants. These findings agree very well with experimental results and thus confirm the assumption that uncontrolled proton delivery by solvent water networks is responsible for the uncoupling reaction. The present study provides a detailed mechanistic understanding of the role of the Thr252 residue.
质子转移反应在细胞色素P450cam的催化循环中起着至关重要的作用，并且负责形成被称为化合物I（Cpd I）的铁氧物种，该物种被认为是活性氧化剂。根据质子转移的过程，最后一个可观测中间体（铁氢过氧络合物，Cpd 0）的质子化可以导致Cpd I的形成（偶联反应）或铁的静止状态（解偶联反应）。这两个过程的比例受到Thr252残基突变的显著影响。在这项工作中，我们通过混合量子力学/分子力学（QM/MM）计算和经典模拟研究了Thr252X（X = 丝氨酸、缬氨酸、丙氨酸、甘氨酸）突变对Cpd I形成的影响。在野生型酶中，偶联反应占优势，因为其限速势垒比解偶联反应的低13 kcal/mol。在丝氨酸突变体中，这种差异减小到7 kcal/mol。在缬氨酸、丙氨酸和甘氨酸突变体的情况下，一个额外的水分子进入活性位点并显著降低了解偶联反应的活化能。有了这个额外的水分子，缬氨酸突变体中偶联和解偶联具有相似的势垒，而在丙氨酸和甘氨酸突变体中解偶联反应变得占优势。这些发现与实验结果非常吻合，从而证实了溶剂水网络不受控制的质子传递导致解偶联反应的假设。本研究提供了对Thr252残基作用的详细机理理解。
