Elucidating the Molecular Mechanism of a Ni Hydrogenase: An Infrared Spectroscopic Approach

阐明镍氢化酶的分子机制：红外光谱方法

• 批准号: 9509064
• 负责人: Kimberly Bagley
• 金额: $ 1.8万
• 依托单位: SUNY State University of New York Central Administration
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-06-15 至 1997-11-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9509064&HistoricalAwards=false
• 关键词: Elucidating; Molecular; Mechanism; Ni; Hydrogenase

9509064 Bagley This research details the utilization of static and time resolved Fourier transform infrared spectroscopy for delineating the molecular mechanism of hydrogen binding and activation in Ni hydrogenases. The research objectives are: (1) Determination of whether the anomalous infrared bands detected between 2100 and 1900 cm-1 are general features of Ni hydrogenases. To this end a series of infrared studies will be carried out on a well-characterized hydrogenase from Desulfovibrio gigas. (2) Identification of the structure responsible for these unusual infrared signatures using infrared spectroscopy coupled with chemical modification of the enzyme and the study of isotopically labeled enzyme. (3) Utilization of the three infrared signatures for further study of the various redox states of the enzyme. This approach provides a unique opportunity; Ni hydrogenases display at least two EPR-silent species which are distinguishable by their infrared signatures in the 2100 and 1900 cm-1 spectral region. (4) Utilization of mid- infrared spectroscopy to examine the role individual amino acids play in the activation of hydrogen. (5) Examination of the oxidation state of the Ni center in various redox states of the enzymes. This approach used the infrared detectable carbon monoxide stretching frequency, arising from Ni bound carbon monoxide, as a probe of the oxidation state of the Ni. %%% Ni Hydrogenases are enzymes that catalyze the simplest reaction in nature and the simplest conversion of electrical energy into chemical energy of universal utility: 2e- + 2H+ = H2. This research involves a spectroscopic study of the anomalous infrared absorption bands that are characteristic of these enzymes. Infrared spectroscopic studies will be made on the well-characterized Ni hydrogenases from Desulfovibrio gigas. It is a anticipated that this work will have important consequences for understanding the mechanism underlying Ni hydrogenase's ability to split molecular hydrogen into protons and electrons. The work also involves the development of methodologies for handling infrared samples under strictly anaerobic conditions, and should prove of general utility in infrared studies of other oxygen sensitive metallo-proteins. ***

9509064 Bagley这项研究详细介绍了静态和时间解决的傅立叶变换红外光谱的利用，用于描述Ni氢化酶中氢结合和激活的分子机制。 研究目标是：（1）确定在2100至1900 cm-1之间检测到的异常红外带是NI氢化酶的一般特征。 为此，将对来自Desulfovibrio Gigas的良好表征氢化酶进行一系列红外研究。 （2）使用红外光谱与化学修饰酶和同位素标记的酶的研究结合的红外光谱鉴定负责这些不寻常的红外特征的结构。 （3）利用三个红外特征，以进一步研究酶的各种氧化还原状态。 这种方法提供了独特的机会； NI氢化酶至少显示了两个EPR-Si-Si-Siment物种，它们在2100和1900 cm-1光谱区域中可通过其红外签名而区分。 （4）利用中红外光谱检查单个氨基酸在氢气激活中所起的作用。 （5）检查酶各种氧化还原状态中NI中心的氧化态。 该方法使用由Ni结合的一氧化碳引起的红外可检测到的一氧化碳拉伸频率作为Ni的氧化态的探针。 %% ni氢化酶是酶，可以催化自然界中最简单的反应，并将电能最简单转化为通用实用程序的化学能：2e- + 2h + = h2。 这项研究涉及对这些酶特征的异常红外吸收带的光谱研究。 红外光谱研究将对来自Desulfovibrio Gigas的良好表征的Ni氢化酶进行。 预计这项工作将对理解Ni氢化酶将分子氢分为质子和电子的能力的机制产​​生重要的后果。 这项工作还涉及在严格的厌氧条件下处理红外样品的方法，并应证明在其他氧气敏感金属蛋白的红外研究中具有一般效用。 ***