Mechanistic Exploitation of a Self-Repairing, Blocked Proton Transfer Pathway in an O2-Tolerant [NiFe]-Hydrogenase
Mechanistic Exploitation of a Self-Repairing, Blocked Proton Transfer Pathway in an O2-Tolerant [NiFe]-Hydrogenase
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DOI：
10.1021/jacs.8b04798
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发表时间：
2018-08-15
影响因子：
15
通讯作者：
Armstrong, Fraser A.
中科院分区：
文献类型：
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作者：
Evans, Rhiannon M.;Ash, Philip A.;Armstrong, Fraser A.
[NiFe]-氢化酶的催化远程质子转移一直与位于活性位点4埃内的高度保守的谷氨酸(E)有关。从大肠杆菌中提取的耐o -2 [NiFe]-氢化酶-1取代谷氨酰胺(Q)产生一种具有独特性质的变体（E28Q），该变体已通过蛋白质膜电化学、蛋白质膜红外电化学和x射线晶体学进行了研究。在ph7和中等电位下，E28Q显示出大约1%的天然酶活性，足够高，可以在稳态条件下进行详细的红外测量。原子水平的晶体结构揭示了氢氧化物离子在酰胺侧链上的部分位移，氢氧化物离子的占有随着pH值或氧化条件的增加而增加，支持了位于附近的不寻常的近端[4Fe-3S]簇的超氧化状态的形成。在这些特殊条件下，由H-2氧化产生的至少一个H+离子的基本退出途径（假定在E28Q变体中被阻断）被部分修复。在中性pH下的稳态H-2氧化过程中（即，当H+通过Q28出口的屏障几乎完全关闭时），催化循环以还原态“Ni-a-R”和“Ni-a-C”为主，即使在高氧化条件下也是如此。因此，E28不参与H-2的初始活化/去质子化，而是促进H+在催化循环后期的退出，以再生初始氧化活性态，假设为Ni-a-SI。因此，由于Ni-a-SI （Ni-B的前体）不能积累，E28Q在高电位H-2存在下不能产生氧化的无活性静息态“Ni-B”。这些结果对于理解[NiFe]-氢化酶的催化机制以及氢化酶和其他酶的远程质子偶联电子转移控制具有重要意义。
Catalytic long-range proton transfer in [NiFe]-hydrogenases has long been associated with a highly conserved glutamate (E) situated within 4 angstrom of the active site. Substituting for glutamine (Q) in the O-2-tolerant [NiFe]-hydrogenase-1 from Escherichia coli produces a variant (E28Q) with unique properties that have been investigated using protein film electrochemistry, protein film infrared electrochemistry, and X-ray crystallography. At pH 7 and moderate potential, E28Q displays approximately 1% of the activity of the native enzyme, high enough to allow detailed infrared measurements under steady-state conditions. Atomic level crystal structures reveal partial displacement of the amide side chain by a hydroxide ion, the occupancy of which increases with pH or under oxidizing conditions supporting formation of the superoxidized state of the unusual proximal [4Fe-3S] cluster located nearby. Under these special conditions, the essential exit pathway for at least one of the H+ ions produced by H-2 oxidation, and assumed to be blocked in the E28Q variant, is partially repaired. During steady-state H-2 oxidation at neutral pH (i.e., when the barrier to H+ exit via Q28 is almost totally closed), the catalytic cycle is dominated by the reduced states "Ni-a-R" and "Ni-a-C", even under highly oxidizing conditions. Hence, E28 is not involved in the initial activation/deprotonation of H-2, but facilitates H+ exit later in the catalytic cycle to regenerate the initial oxidized active state, assumed to be Ni-a-SI. Accordingly, the oxidized inactive resting state, "Ni-B", is not produced by E28Q in the presence of H-2 at high potential because Ni-a-SI (the precursor for Ni-B) cannot accumulate. The results have important implications for understanding the catalytic mechanism of [NiFe]-hydrogenases and the control of long-range proton-coupled electron transfer in hydrogenases and other enzymes.