Comprehensive structural, infrared spectroscopic and kinetic investigations of the roles of the active-site arginine in bidirectional hydrogen activation by the [NiFe]-hydrogenase 'Hyd-2' from Escherichia coli.
Comprehensive structural, infrared spectroscopic and kinetic investigations of the roles of the active-site arginine in bidirectional hydrogen activation by the [NiFe]-hydrogenase 'Hyd-2' from Escherichia coli.
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DOI：
10.1039/d2sc05641k
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发表时间：
2023-08-16
期刊：
影响因子：
8.4
通讯作者：
中科院分区：
文献类型：
--
作者：
[NiFe]-氢化酶的活性位点包含一个严格保守的垂链精氨酸，其胍头基悬浮在Ni和Fe原子的正上方。将这种精氨酸（R479）替换到大肠杆菌的氢化酶-2中，会得到一种酶，这种酶与一端连接在Ni上的非常紧密结合的双原子配体分离，并通过与悬垂赖氨酸的Nζ原子和位于变体活性位点的三个附加水分子之一的氢键稳定。双原子配体在氧化条件下结合，只有在H2和还原的紫甲基长时间还原后才被去除。一旦释放双原子配体，R479K变体催化H2氧化和进化，但与天然酶相比，其速率大大降低。蛋白质膜电化学揭示了关键的动力学特征：最重要的是，H2氧化的活化能非常低，这与H/D同位素效应的增加无关。保留了原有的电催化可逆性。结果表明，观察到赖氨酸变异体的缓慢动力学出现最明显的原因是更有利的低能途径的优势被极其不利的激活熵大量抵消。广泛的努力来建立双原子配体的身份，其紧密结合是一个意想不到的进一步的后果取代垂坠精氨酸，证明是不确定的。将[NiFe]-氢化酶的保守活性位点精氨酸转变为赖氨酸大大降低了催化H2在各个方向上的活化速率，并导致双原子配体的极紧密结合。
The active site of [NiFe]-hydrogenases contains a strictly-conserved pendant arginine, the guanidine head group of which is suspended immediately above the Ni and Fe atoms. Replacement of this arginine (R479) in hydrogenase-2 from E. coli results in an enzyme that is isolated with a very tightly-bound diatomic ligand attached end-on to the Ni and stabilised by hydrogen bonding to the Nζ atom of the pendant lysine and one of the three additional water molecules located in the active site of the variant. The diatomic ligand is bound under oxidising conditions and is removed only after a prolonged period of reduction with H2 and reduced methyl viologen. Once freed of the diatomic ligand, the R479K variant catalyses both H2 oxidation and evolution but with greatly decreased rates compared to the native enzyme. Key kinetic characteristics are revealed by protein film electrochemistry: most importantly, a very low activation energy for H2 oxidation that is not linked to an increased H/D isotope effect. Native electrocatalytic reversibility is retained. The results show that the sluggish kinetics observed for the lysine variant arise most obviously because the advantage of a more favourable low-energy pathway is massively offset by an extremely unfavourable activation entropy. Extensive efforts to establish the identity of the diatomic ligand, the tight binding of which is an unexpected further consequence of replacing the pendant arginine, prove inconclusive. Changing the conserved active-site arginine of [NiFe]-hydrogenases into a lysine greatly lowers the rates of catalytic H2 activation in each direction and results in the extremely tight binding of a diatomic ligand.
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影响因子：
5.5
作者：
Best, Robert B.;Zhu, Xiao;Shim, Jihyun;Lopes, Pedro E. M.;Mittal, Jeetain;Feig, Michael;MacKerell, Alexander D., Jr.
通讯作者：
MacKerell, Alexander D., Jr.
影响因子：
5.2
作者：
Dragelj, Jovan;Karafoulidi-Retsou, Chara;Katz, Sagie;Lenz, Oliver;Zebger, Ingo;Caserta, Giorgio;Sacquin-Mora, Sophie;Mroginski, Maria Andrea
通讯作者：
Mroginski, Maria Andrea
影响因子：
4.3
作者：
Eastman P;Swails J;Chodera JD;McGibbon RT;Zhao Y;Beauchamp KA;Wang LP;Simmonett AC;Harrigan MP;Stern CD;Wiewiora RP;Brooks BR;Pande VS
通讯作者：
Pande VS
影响因子：
3.7
作者：
Szőri-Dorogházi E;Maróti G;Szőri M;Nyilasi A;Rákhely G;Kovács KL
通讯作者：
Kovács KL
影响因子：
8.4
作者：
Ash PA;Kendall-Price SET;Evans RM;Carr SB;Brasnett AR;Morra S;Rowbotham JS;Hidalgo R;Healy AJ;Cinque G;Frogley MD;Armstrong FA;Vincent KA
通讯作者：
Vincent KA