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质。氢化酶催化可逆的氢氧化，生理上的功能是回收在发酵代谢过程中积累的还原电子载体，或将氢的氧化与产生可用于能量产生过程的还原电子载体偶联。含金属氢化酶根据其金属含量进行分类，包括既含Ni又含Fe的氢化酶（NiFe-氢化酶）和仅含Fe（仅含Fe氢化酶）的那些。这些酶在许多微生物的代谢中具有极其重要的作用。此外，由于氢正在被探索作为未来的替代燃料来源，有相当大的兴趣在这些制氢生物催化剂的细节。 这些研究将主要集中在测试的假设，开发作为一个结果的详细结构分析的铁只有氢化酶（CpI）分离自厌氧土壤微生物Clostridium pasteurianum。具体目标是：（1）对CpI进行进一步的结构和生物化学研究，探讨一些关于H簇结构和附属[FeS]簇作用的关键问题：（2）对CpI进行位点特异性氨基酸取代实验，以研究H簇的多肽环境在催化和稳定性中的作用;（3）C. pasteurianum的一级序列和结构关系，并探讨H簇和FeS簇的环境组成对Fe-Onlyhydrogenases的氢氧化和质子还原活性的影响。 上述目标将通过多学科方法结合生物化学方法，光谱方法和X射线衍射方法的结构测定来实现，以回答一些关键的剩余问题，关于铁只有氢化酶的结构和功能。

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.