Gilead Sciences & IOCB Research Center, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Praha 6, Czech Republic.
Faraday Discuss. 2011;148:41-53; discussion 97-108. doi: 10.1039/c004476h.
The key step in the reaction mechanism of multicopper oxidases (MCOs)--the cleavage of the O-O bond in O2--has been investigated using combined quantum mechanical and molecular mechanical (QM/MM) methods. This process represents a reaction pathway from the peroxy intermediate after it accepts one electron from the nearby type-1 Cu site to the experimentally-observed native intermediate, which is the only fully oxidised catalytically relevant state in MCOs. Scans of the QM(DFT)/MM potential energy surface have allowed us to obtain estimates of the activation energies. Furthermore, vacuum calculations on a smaller model of the active site have allowed us to estimate the entropy contributions to the barrier height and to obtain further insight into the reaction by comparing the small cluster model with the QM/MM model, which includes the entire protein. Owing to the complicated electronic structure of these low-spin exchange coupled systems, multireference quantum chemical calculations at the complete-active space second-order perturbation theory (CASPT2) were used in an attempt to benchmark the barrier heights obtained at the DFT(B3LYP) level. Our best estimate of the activation barrier is deltaG = 60-65 kJ mol(-1), in good agreement with the experimental barrier of approximately 55 kJ mol(-1), which can be inferred from the experimental rate constant of k > 350 s(-1). It has also been shown that the reaction involves protonation of the O2 moiety before bond cleavage. The proton likely comes from a nearby carboxylate residue which was recently suggested by the experiments.
多铜氧化酶（MCOs）反应机制中的关键步骤——O2 中 O-O 键的断裂——已经使用量子力学和分子力学（QM/MM）相结合的方法进行了研究。该过程代表了过氧基中间体接受来自附近的 1 型 Cu 位的一个电子后到实验观察到的天然中间体的反应途径，这是 MCOs 中唯一完全氧化的催化相关状态。QM(DFT)/MM 势能面的扫描使我们能够估计活化能。此外，对活性部位较小模型的真空计算允许我们估计熵对势垒高度的贡献，并通过将小团簇模型与包括整个蛋白质的 QM/MM 模型进行比较，进一步深入了解反应。由于这些低自旋交换耦合体系的复杂电子结构，在完全活性空间二阶微扰理论（CASPT2）下进行了多参考量子化学计算，试图对在 DFT(B3LYP)水平获得的势垒高度进行基准测试。我们对活化势垒的最佳估计为 deltaG = 60-65 kJ mol(-1)，与实验得到的约 55 kJ mol(-1)的势垒非常吻合，这可以从实验得到的大于 350 s(-1)的速率常数推断出来。还表明反应涉及 O2 部分在键断裂前的质子化。质子可能来自附近的羧酸盐残基，这是最近的实验所提出的。
