Mechanism of Pyrroloquinoline Quinone-Dependent Hydride Transfer Chemistry from Spectroscopic and High-Resolution X-ray Structural Studies of the Methanol Dehydrogenase from Methylococcus capsulatus (Bath)
Mechanism of Pyrroloquinoline Quinone-Dependent Hydride Transfer Chemistry from Spectroscopic and High-Resolution X-ray Structural Studies of the Methanol Dehydrogenase from Methylococcus capsulatus (Bath)
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DOI：
10.1021/jacs.0c11414
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发表时间：
2021-02-25
影响因子：
15
通讯作者：
Chen, Chun-Jung
中科院分区：
文献类型：
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作者：
Chan, Sunney I.;Chuankhayan, Phimonphan;Chen, Chun-Jung
甲醇脱氢酶（MDH）的活性部位在相邻的半胱氨酸残基之间含有一个罕见的二硫键。作为邻位二硫化物，结构高度紧张，这表明它可能与吡咯喹啉醌（PQQ）辅基和Ca 2+离子在甲醇（CH 3OH）氧化过程中的催化周转。我们从荚膜甲基球菌（Bath）中纯化MDH，其中二硫键断裂成两个巯基。对这种形式的MDH的光谱和高分辨率X射线晶体学研究表明，二硫键具有氧化还原活性。我们观察到一个内部的氧化还原过程中的全息MDH，产生二硫化物自由基阴离子伴随着同伴PQQ自由基，证明了在408 nm处的光吸收和磁偶极耦合的双自由基的EPR光谱。这些观察结果证实了电子密度之间的变化的两个半胱氨酸硫的二硫键，以及之间的绑定的Ca 2+离子和O 5-C5键的PQQ在高分辨率的X射线结构。在这些发现的基础上，我们提出了一种在醇氧化中从CH 3OH进行氢化物转移期间控制两个电子重新分配的机制，而不形成还原的PQQ乙二醇，这是一种双自由基机制，允许可能回收氢化物以转移到外部。在PQQ辅因子再生中的外部NAD（+）氧化剂用于多次催化周转。为了支持这一机制，在CH 3OH和NAD（+）存在下，MDH周转期间观察到稳态水平的二硫自由基阴离子。
The active site of methanol dehydrogenase (MDH) contains a rare disulfide bridge between adjacent cysteine residues. As a vicinal disulfide, the structure is highly strained, suggesting it might work together with the pyrroloquinoline quinone (PQQ) prosthetic group and the Ca2+ ion in the catalytic turnover during methanol (CH3OH) oxidation. We purify MDH from Methylococcus capsulatus (Bath) with the disulfide bridge broken into two thiols. Spectroscopic and high-resolution X-ray crystallographic studies of this form of MDH indicate that the disulfide bridge is redox active. We observe an internal redox process within the holo-MDH that produces a disulfide radical anion concomitant with a companion PQQ radical, as evidenced by an optical absorption at 408 nm and a magnetically dipolar-coupled biradical in the EPR spectrum. These observations are corroborated by electron-density changes between the two cysteine sulfurs of the disulfide bridge as well as between the bound Ca2+ ion and the O5-C5 bond of the PQQ in the high-resolution X-ray structure. On the basis of these findings, we propose a mechanism for the controlled redistribution of the two electrons during hydride transfer from the CH3OH in the alcohol oxidation without formation of the reduced PQQ ethenediol, a biradical mechanism that allows for possible recovery of the hydride for transfer to an external NAD(+) oxidant in the regeneration of the PQQ cofactor for multiple catalytic turnovers. In support of this mechanism, a steady-state level of the disulfide radical anion is observed during turnover of the MDH in the presence of CH3OH and NAD(+).