Structural and Biochemical Investigations on Fe-Only Hydrogenases

本课题的主要目的是研究纯铁氢化酶的结构和催化性能。氢化酶催化可逆的氢化氧化，并在生理上发挥作用，要么回收在发酵代谢过程中积累的还原电子载体，要么将氢的氧化与可用于能量生成过程的还原电子载体结合起来。含金属氢化酶根据其金属含量分类，包括既含镍又含铁的氢化酶（含镍氢化酶）和只含铁的氢化酶（含铁氢化酶）。这些酶在许多微生物的代谢中起着至关重要的作用。此外，由于氢正在被探索作为未来的替代燃料来源，人们对这些产氢生物催化剂的细节非常感兴趣。这些研究将主要集中在验证从厌氧土壤微生物巴氏梭菌中分离出的Fe-Only氢化酶（CpI）的详细结构分析所产生的假设。具体目标是：(1)对CpI进行额外的结构和生化研究，探讨关于H簇结构和附属[FeS]簇的作用的一些剩余关键问题；(2)在CpI上进行位点特异性氨基酸取代实验，考察H簇多肽环境在催化和稳定性中的作用；(3)对巴氏杆菌中第二种纯铁氢化酶（CpII）进行结构表征，以确定其一级序列和与其他纯铁氢化酶的结构关系，并探讨H簇和附属FeS簇的环境对纯铁氢化酶中氢氧化和质子还原活性的影响。以上目标将通过结合生化方法、光谱方法和x射线衍射方法的多学科方法来实现，以回答有关纯铁氢化酶的结构和功能的一些关键问题。

The main objective of this projectl is to investigate the structural and catalyticproperties of Fe-Only hydrogenases. Hydrogenases catalyze reversible hydrogenoxidation and function physiologically to either recycle the reduced electron carriers thataccumulate during a fermentative metabolism or couple the oxidation of hydrogen to thegeneration of reduced electron carriers that can be utilized in energy yielding processes.The metal-containing hydrogenases are classified according to their metal content andinclude those that contain both Ni and Fe (NiFe-hydrogenases) and those that contain justFe (Fe-Only hydrogenases). These enzymes are of paramount importance in themetabolism of numerous microorganisms. In addition, since hydrogen is being exploredas a future alternative fuel source, there is considerable interest in the details of thesehydrogen producing biological catalysts. The studies will focus primarily on testing hypotheses thatwere developed as a result of the detailed structural analysis of the Fe-Only hydrogenase(CpI) isolated from the anaerobic soil microorganism Clostridium pasteurianum. The specific objectives are (1) additional structural and biochemical studies on CpI probing a number of remaining key questions with respect to the H Cluster structure and the role of the accessory [FeS] clusters; (2) site-specificamino acid substitution experiments on CpI to examine the role of the polypeptideenvironment of the H Cluster in catalysis and stability; (3) structural characterization ofthe second Fe-Only hydrogenase (CpII) from C. pasteurianum to ascertain its primarysequence and structural relationship to other Fe-Only hydrogenases and to probe thecontributions of the environment of the H Cluster and the accessory FeS clusters indefining hydrogen oxidation versus proton reduction activities in the Fe-Onlyhydrogenases. The above objectives will be pursued by a multidisciplinary approach combiningbiochemical methods, spectroscopic methods and structure determination by X-raydiffraction methods to answer some of the key remaining questions with respect to thestructure and function of Fe-Only hydrogenases.