RUI: Infrared Spectroscopy as a Probe of Active Site Structure and Molecular Mechanism in the Hydrogenases

本项目将利用红外差示光谱技术研究氢酶的活性中心结构及其氢键结合和活化的分子机制。以前的红外光谱研究表明，[NiFe]氢酶和只含Fe的氢酶在2100-1800 cm-1的光谱区都有很强的红外光谱。负责这些红外波段的基团已被确定为来自与这些酶活性部位的铁离子配位的内在一氧化碳和氰化物分子。本研究的主要目的是：1)利用红外光谱分析普通脱硫弧菌的[NiFe]氢酶的活性中心结构。普通脱硫弧菌的酶被认为在其活性部位有一个NiFe(CO)(CN)(SO)簇，而不是在其他[NiFe]加氢酶中存在的NiFe(CO)(CN)2簇。2)利用红外差示光谱研究了[NiFe]氢酶还原激活过程中的分子变化。3)利用红外光谱特征，结合同位素标记，确定巴氏梭菌只含铁的加氢酶I和II的活性中心结构。4)利用中红外差示光谱技术研究氨基酸在氢活化中的作用。这项研究将涉及本科生攻读化学和化学相关领域的学位。预计这项工作将对理解氢酶将分子氢分解为质子和电子，或反过来从质子和电子产生氢的机制产生重要结果。这些知识对生产氢气的廉价催化剂的设计产生了影响，氢气是一种清洁燃烧的燃料来源。拟议的研究将包括本科生在学年和暑假期间的参与。学生参与这个项目将为本科生提供一个独特的机会来应用他们在课程中学到的技能和知识，同时为现代物理生物化学中一个令人兴奋的项目做出贡献。

In this project infrared difference spectroscopy will be used to study the active site structure and molecular mechanism of hydrogen binding and activation in the hydrogenases. Previous infrared spectroscopic studies have shown that both the [NiFe] hydrogenases and the Fe-only hydrogenases display intense infrared bands in the 2100-1800 cm-1 spectral region. The groups responsible for these infrared bands have been identified as arising from intrinsic carbon monoxide and cyanide molecules coordinated to Fe ions in the active site of these enzymes. The primary objectives of this research are: 1) Utilization of infrared spectroscopy for delineating the active site structure of the [NiFe] hydrogenase from Desulfovibrio vulgaris. The enzyme from Desulfovibrio vulgaris has been proposed to have a NiFe (CO)(CN)(SO) cluster at its active site rather than the NiFe (CO)(CN)2 cluster shown to exist in other [NiFe] Hydrogenases. 2) Utilization of infrared difference spectroscopy to explore the molecular changes accompanying reductive activation in the [NiFe] hydrogenases. 3) Utilization of the infrared spectral signatures, combined with isotopic labeling, for determining the active site structure of Fe-only Hydrogenases I and II from Clostridium pasteurianum. 4) Utilization of mid-infrared difference spectroscopy to examine the role amino acids play in the activation of hydrogen. The research will involve undergraduate students pursuing degrees in chemistry and chemistry-related fields.It is anticipated that this work will have important consequences for understanding the mechanism underlying the hydrogenases' ability to split molecular hydrogen into protons and electrons, or conversely, to produce hydrogen from protons and electrons. Such knowledge has consequences for the design of cheap catalysts for the production of hydrogen, a clean burning fuel source. The proposed research will involve the participation of undergraduate students, both during the academic year and during the summer. Student participation in this project will afford undergraduate students a unique opportunity to apply skills and knowledge they have learned in their coursework, while contributing to an exciting project in modern physical biochemistry.