A Spectroscopically Validated Computational Investigation of Viable Reaction Intermediates in the Catalytic Cycle of the Reductive Dehalogenase PceA
A Spectroscopically Validated Computational Investigation of Viable Reaction Intermediates in the Catalytic Cycle of the Reductive Dehalogenase PceA
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DOI：
10.1021/acs.biochem.1c00271
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发表时间：
2021-06-16
期刊：
影响因子：
2.9
通讯作者：
Brunold, Thomas C.
中科院分区：
文献类型：
--
作者：
Greenhalgh, Elizabeth D.;Kunze, Cindy;Brunold, Thomas C.
产生还原性脱卤酶的生物在称为有机卤化物呼吸的过程中利用卤代芳香族和脂肪族物质作为末端电子受体。这些生物可以将卤代物质的还原与ATP的产生结合起来。四氯乙烯还原脱卤酶（PceA）催化全氯乙烯和三氯乙烯（分别为PCE和TCE）还原脱卤，主要为顺式二氯乙烯（DCE）。PCE到TCE（和随后的DCE）的酶促转化可能通过一种机制进行，其中第一步涉及单电子转移，亲核加成，然后是氯化物消除或质子化，或直接攻击卤素。生产足够量的PceA的困难极大地阻碍了反应机理的直接实验研究。为了克服这些挑战，我们已经产生了计算模型的休息和TCE结合PceA使用量子力学/分子力学（QM/MM）计算和实验数据的基础上验证这些模型。值得注意的是，norpseudo-cob（II）alamin [Co（II）Cbl*]辅因子在底物与酶结合时保持五配位，在下表面上保留松散结合的水。因此，PceA所使用的具有生物学挑战性的Co（II）-> Co（I）Cbl* 还原的机制与腺苷基转移酶所利用的机制根本不同，腺苷基转移酶产生四配位Co（II）Cbl物质以促进接近Co（I）氧化态。然后将相同的QM/MM计算方法应用于PceA催化循环中的可行反应中间体。预测具有最低能量的中间体是从Co（I）Cbl* 到底物的电子转移产生Co（II）Cbl*、氯离子和乙烯基的中间体。
Organisms that produce reductive dehalogenases utilize halogenated aromatic and aliphatic substances as terminal electron acceptors in a process termed organohalide respiration. These organisms can couple the reduction of halogenated substances with the production of ATP. Tetrachloroethylene reductive dehalogenase (PceA) catalyzes the reductive dehalogenation of per- and trichloroethylenes (PCE and TCE, respectively) to primarily cis-dichloroethylene (DCE). The enzymatic conversion of PCE to TCE (and subsequently DCE) could potentially proceed via a mechanism in which the first step involves a single-electron transfer, nucleophilic addition followed by chloride elimination or protonation, or direct attack at the halogen. Difficulties with producing adequate quantities of PceA have greatly hampered direct experimental studies of the reaction mechanism. To overcome these challenges, we have generated computational models of resting and TCE-bound PceA using quantum mechanics/molecular mechanics (QM/MM) calculations and validated these models on the basis of experimental data. Notably, the norpseudo-cob(II)alamin [Co(II)Cbl*] cofactor remains five-coordinate upon binding of the substrate to the enzyme, retaining a loosely bound water on the lower face. Thus, the mechanism for the thermodynamically challenging Co(II) -> Co(I)Cbl* reduction used by PceA differs fundamentally from that utilized by adenosyltransferases, which generate four-coordinate Co(II)Cbl species to facilitate access to the Co(I) oxidation state. The same QM/MM computational methodology was then applied to viable reaction intermediates in the catalytic cycle of PceA. The intermediate predicted to possess the lowest energy is that resulting from electron transfer from Co(I)Cbl* to the substrate to yield Co(II)Cbl*, a chloride ion, and a vinylic radical.