Experimental approaches to kinetics of gas diffusion in hydrogenase
Experimental approaches to kinetics of gas diffusion in hydrogenase
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DOI：
10.1073/pnas.0803689105
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发表时间：
2008-08-12
影响因子：
11.1
通讯作者：
Leger, Christophe
中科院分区：
文献类型：
--
作者：
Leroux, Fanny;Dementin, Sebastien;Leger, Christophe
氢化酶催化 H-2 到 H+ 的转化，作为许多微生物生物能代谢的一部分，是利用晶体学和分子动力学描述其气体底物隧道的金属酶之一。然而，蛋白质结构和气体扩散动力学之间的相关性尚未探索。在这里，我们介绍两种探测氢化酶内扩散速率的定量方法。一种利用蛋白质膜伏安法来解析竞争性抑制剂CO的结合和释放动力学；另一种是基于对同位素交换测定中产量的解释。我们研究了 NiFe 氢化酶的结构特征突变体，结果表明，两种突变显着缩小了催化中心入口附近的通道，使 CO 和 H-2 向活性位点和从活性位点扩散的速率降低了高达 2 个数量级。这证明了功能通道的存在，该通道与晶体中发现的疏水空腔相匹配。然而，扩散率的变化并不完全与突变引起的阻塞以及从 X 射线结构推断出的阻塞相关。我们的结果证明了测量扩散速率的必要性，并强调了侧链动力学在确定扩散速率中的作用。
Hydrogenases, which catalyze H-2 to H+ conversion as part of the bioenergetic metabolism of many microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described by using crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion kinetics is unexplored. Here, we introduce two quantitative methods for probing the rates of diffusion within hydrogenases. One uses protein film voltammetry to resolve the kinetics of binding and release of the competitive inhibitor CO; the other is based on interpreting the yield in the isotope exchange assay. We study structurally characterized mutants of a NiFe hydrogenase, and we show that two mutations, which significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO and H-2 toward and from the active site by up to 2 orders of magnitude. This proves the existence of a functional channel, which matches the hydrophobic cavity found in the crystal. However, the changes in diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the x-ray structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of side-chain dynamics in determining these.