Carr Stephen B, Evans Rhiannon M, Brooke Emily J, Wehlin Sara A M, Nomerotskaia Elena, Sargent Frank, Armstrong Fraser A, Phillips Simon E V
Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Oxford, Didcot OX11 0FA, U.K.
Department of Chemistry, University of Oxford, Oxford OX1 3QR, U.K.
Biochem Soc Trans. 2016 Jun 15;44(3):863-8. doi: 10.1042/BST20160031.
Hydrogenase-1 (Hyd-1) from Escherichia coli is a membrane-bound enzyme that catalyses the reversible oxidation of molecular H2 The active site contains one Fe and one Ni atom and several conserved amino acids including an arginine (Arg(509)), which interacts with two conserved aspartate residues (Asp(118) and Asp(574)) forming an outer shell canopy over the metals. There is also a highly conserved glutamate (Glu(28)) positioned on the opposite side of the active site to the canopy. The mechanism of hydrogen activation has been dissected by site-directed mutagenesis to identify the catalytic base responsible for splitting molecular hydrogen and possible proton transfer pathways to/from the active site. Previous reported attempts to mutate residues in the canopy were unsuccessful, leading to an assumption of a purely structural role. Recent discoveries, however, suggest a catalytic requirement, for example replacing the arginine with lysine (R509K) leaves the structure virtually unchanged, but catalytic activity falls by more than 100-fold. Variants containing amino acid substitutions at either or both, aspartates retain significant activity. We now propose a new mechanism: heterolytic H2 cleavage is via a mechanism akin to that of a frustrated Lewis pair (FLP), where H2 is polarized by simultaneous binding to the metal(s) (the acid) and a nitrogen from Arg(509) (the base).
来自大肠杆菌的氢化酶-1（Hyd-1）是一种膜结合酶，可催化分子氢的可逆氧化。其活性位点包含一个铁原子和一个镍原子以及几个保守氨基酸，其中包括一个精氨酸（Arg(509)），它与两个保守天冬氨酸残基（Asp(118)和Asp(574)）相互作用，在金属上方形成一个外壳冠层。在活性位点与冠层相对的一侧还有一个高度保守的谷氨酸（Glu(28)）。通过定点诱变剖析了氢活化机制，以确定负责裂解分子氢的催化碱基以及进出活性位点的可能质子转移途径。先前报道的对冠层中残基进行突变的尝试未成功，导致人们认为其仅起结构作用。然而，最近的发现表明它具有催化作用，例如用赖氨酸取代精氨酸（R509K），结构几乎不变，但催化活性下降了100多倍。在一个或两个天冬氨酸处含有氨基酸取代的变体保留了显著活性。我们现在提出一种新机制：异裂氢裂解是通过一种类似于受阻路易斯对（FLP）的机制，其中氢分子通过同时与金属（酸）和来自精氨酸（Arg(509)）的氮（碱）结合而极化。
