Pelmenschikov Vladimir, Blomberg Margareta R A, Siegbahn Per E M, Crabtree Robert H
Department of Physics, Stockholm University, Box 6730, S-113 85 Stockholm, Sweden.
J Am Chem Soc. 2002 Apr 17;124(15):4039-49. doi: 10.1021/ja011664r.
The mechanism for methane formation in methyl-coenzyme M reductase (MCR) has been investigated using the B3LYP hybrid density functional method and chemical models consisting of 107 atoms. The experimental X-ray crystal structure of the enzyme in the inactive MCR(ox1)(-)(silent) state was used to set up the initial model structure. The calculations suggest a mechanism not previously proposed, in which the most remarkable feature is the formation of an essentially free methyl radical at the transition state. The reaction cycle suggested starts from a Michaelis complex with CoB and methyl-CoM coenzymes bound and with a squareplanar coordination of the Ni(I) center in the tetrapyrrole F(430) prosthetic group. In the rate-limiting step the methyl radical is released from methyl-CoM, induced by the attack of Ni(I) on the methyl-CoM thioether sulfur. In this step, the metal center is oxidized from Ni(I) to Ni(II). The resulting methyl radical is rapidly quenched by hydrogen-atom transfer from the CoB thiol group, yielding the methane molecule and the CoB radical. The estimated activation energy is around 20 kcal/mol, which includes a significant contribution from entropy due to the formation of the free methyl. The mechanism implies an inversion of configuration at the reactive carbon. The size of the inversion barrier is used to explain the fact that CF(3)-S-CoM is an inactive substrate. Heterodisulfide CoB-S-S-CoM formation is proposed in the final step in which nickel is reduced back to Ni(I). The suggested mechanism agrees well with experimental observations.
已使用B3LYP杂化密度泛函方法和由107个原子组成的化学模型，对甲基辅酶M还原酶（MCR）中甲烷形成的机制进行了研究。处于无活性MCR(ox1)(-)（沉默）状态的酶的实验X射线晶体结构被用于建立初始模型结构。计算结果表明了一种此前未被提出的机制，其中最显著的特征是在过渡态形成了一个基本自由的甲基自由基。所提出的反应循环从一个米氏复合物开始，其中CoB和甲基辅酶M结合，并且四吡咯F(430)辅基中的Ni(I)中心具有平面正方形配位。在限速步骤中，Ni(I)对甲基辅酶M硫醚硫的攻击诱导甲基自由基从甲基辅酶M中释放出来。在这一步中，金属中心从Ni(I)氧化为Ni(II)。产生的甲基自由基通过来自CoB硫醇基团的氢原子转移迅速淬灭，生成甲烷分子和CoB自由基。估计的活化能约为20 kcal/mol，其中包括由于自由甲基形成而产生的显著熵贡献。该机制意味着反应性碳处的构型反转。反转势垒的大小被用于解释CF(3)-S-CoM是一种无活性底物这一事实。在最后一步中提出了异二硫化物CoB-S-S-CoM的形成，其中镍被还原回Ni(I)。所提出的机制与实验观察结果非常吻合。
