Hydrogen bonding networks tune proton-coupled redox steps during the enzymatic six-electron conversion of nitrite to ammonia.
Hydrogen bonding networks tune proton-coupled redox steps during the enzymatic six-electron conversion of nitrite to ammonia.
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DOI：
10.1021/bi500854p
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发表时间：
2014-09-09
期刊：
影响因子：
2.9
通讯作者：
Elliott, Sean J.
中科院分区：
文献类型：
--
作者：
Judd, Evan T.;Stein, Natalia;Pacheco, A. Andrew;Elliott, Sean J.
多电子多质子反应在生物系统和化学反应中起着重要的作用，涉及能量存储和操纵。自然界在实现这种复杂化学过程中采用的关键策略是使用质子偶联氧化还原步骤。细胞色素c亚硝酸盐还原酶（ccNiR）催化亚硝酸盐还原为氨的六电子七质子还原。虽然基于结合计算和晶体学的研究提出了ccNiR的催化机制，但很少有研究直接解决预测沿着亚硝酸盐还原途径发生的质子耦合事件的性质。在这里，我们使用蛋白质膜伏安法直接询问在ccNiR还原亚硝酸盐过程中发生的质子耦合步骤。我们发现，通过吸附到石墨电极的ccNiR将亚硝酸盐转化为氨由两个不同的相定义;一个是质子耦合的，另一个不是。关键活性位点残基（H257、R103和Y206）的突变调节这些阶段，并特异性改变检测到的质子依赖性步骤的性质，但不会抑制ccNiR将亚硝酸盐完全还原为氨的能力。我们的结论是，活性位点的残基检查负责调整的质子化步骤，发生在催化过程中，可能通过一个广泛的氢键网络，但不一定需要的反应进行。这些结果提供了重要的洞察酶如何可以具体调整质子和电子转移步骤，以实现高营业额在生理pH值范围内。
Multielectron multiproton reactions play an important role in both biological systems and chemical reactions involved in energy storage and manipulation. A key strategy employed by nature in achieving such complex chemistry is the use of proton-coupled redox steps. Cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron seven-proton reduction of nitrite to ammonia. While a catalytic mechanism for ccNiR has been proposed on the basis of studies combining computation and crystallography, there have been few studies directly addressing the nature of the proton-coupled events that are predicted to occur along the nitrite reduction pathway. Here we use protein film voltammetry to directly interrogate the proton-coupled steps that occur during nitrite reduction by ccNiR. We find that conversion of nitrite to ammonia by ccNiR adsorbed to graphite electrodes is defined by two distinct phases; one is proton-coupled, and the other is not. Mutation of key active site residues (H257, R103, and Y206) modulates these phases and specifically alters the properties of the detected proton-dependent step but does not inhibit the ability of ccNiR to conduct the full reduction of nitrite to ammonia. We conclude that the active site residues examined are responsible for tuning the protonation steps that occur during catalysis, likely through an extensive hydrogen bonding network, but are not necessarily required for the reaction to proceed. These results provide important insight into how enzymes can specifically tune proton- and electron transfer steps to achieve high turnover numbers in a physiological pH range.
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影响因子：
15
作者：
Gwyer, JD;Richardson, DJ;Butt, JN
通讯作者：
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影响因子：
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发表时间：
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影响因子：
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影响因子：
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作者：
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通讯作者：
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