Department of Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China.
Phys Chem Chem Phys. 2018 Jul 18;20(28):18938-18948. doi: 10.1039/c8cp00858b.
Previous density functional theory (DFT) studies have shown that the release of the produced carbon dioxide (CO2) from an active-site cluster is a thermodynamically or kinetically difficult step in the enzymatic carbon monoxide (CO) oxidation catalyzed by Mo-Cu carbon monoxide dehydrogenase (Mo-Cu CODH). To better understand the effect of the protein environment on this difficult CO2 release step as well as other reaction steps, we applied hybrid quantum mechanics and molecular mechanics (QM/MM) calculations to the Mo-Cu CODH enzyme. The results show that in the first step, the equatorial Mo[double bond, length as m-dash]O group in the active-site cluster attacks the nearby CO molecule bound to the Cu site. Afterward, a stable thiocarbonate intermediate is formed in which the CO2 molecule is embedded and the copper-S(μ-sulfido) bond is broken. A free CO2 molecule, i.e., the final product, is then released from the active-site cluster, not directly from the thiocarbonate intermediate but via a previously formed intermediate that also contains CO2 but retains the Cu-S(μ-sulfido) bond. In contrast to the previous DFT results, the calculated barrier for this process was low in our QM/MM calculations. An additional QM/MM analysis of the barrier height showed that the effect of the protein environment on this barrier lowering is not very large. We found that the reason for the low barrier obtained by QM/MM is that the barrier for CO2 release is already not high at the DFT level. These results allow us to conclude that the CO oxidation reaction passes through the formation of a thiocarbonate intermediate, and that the subsequent CO2 release is kinetically not difficult. Nevertheless, the protein environment has an important role to play in making the latter process thermodynamically favored. No low-barrier pathway for the product release could be obtained for the reaction of n-butylisocyanide, which is consistent with the experimental fact that n-butylisocyanide inhibits Mo-Cu CODH.
先前的密度泛函理论 (DFT) 研究表明，在钼-铜一氧化碳脱氢酶 (Mo-Cu CODH) 催化的酶促一氧化碳 (CO) 氧化中，从活性位点簇中释放产生的二氧化碳 (CO2) 是热力学或动力学上困难的步骤。为了更好地理解蛋白质环境对这一困难的 CO2 释放步骤以及其他反应步骤的影响，我们将混合量子力学和分子力学 (QM/MM) 计算应用于 Mo-Cu CODH 酶。结果表明，在第一步中，活性位点簇中的赤道 Mo[双键，长度为 m-dash]O 基团攻击附近与 Cu 位结合的 CO 分子。此后，形成稳定的硫代碳酸盐中间体，其中嵌入 CO2 分子，并且铜-S(μ-硫代)键断裂。然后，自由 CO2 分子，即最终产物，从活性位点簇中释放出来，不是直接从硫代碳酸盐中间体释放，而是通过先前形成的中间体释放，该中间体也含有 CO2，但保留 Cu-S(μ-硫代)键。与先前的 DFT 结果相反，我们的 QM/MM 计算得出该过程的计算势垒较低。对势垒高度的额外 QM/MM 分析表明，蛋白质环境对降低此势垒的影响不是很大。我们发现，QM/MM 获得低势垒的原因是在 DFT 水平下 CO2 释放的势垒已经不高。这些结果使我们得出结论，CO 氧化反应经过硫代碳酸盐中间体的形成，随后的 CO2 释放在动力学上并不困难。尽管如此，蛋白质环境在使后者过程在热力学上有利方面起着重要作用。对于 n-丁基异氰化物的反应，无法获得产物释放的低势垒途径，这与 n-丁基异氰化物抑制 Mo-Cu CODH 的实验事实一致。
