The Novel Iron-Sulfur Clusters of Hydrogenase

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9405783 Adams This project seeks to expand upon our knowledge of the structure, function and chemical reactivity of biological iron-sulfur (FeS) clusters, the most ubiquitous electron carriers in nature. We are investigating the structure and properties of a novel type of FeS center, termed the H cluster, found at the catalytic site of hydrogenase. In earlier studies, it was shown the novel H cluster in mesophilic hydrogenases is probably comprised of four Fe atoms and has two types of nitrogenous ligands, one of which is displaced when the cluster covalently binds CO, a potent inhibitor. Hydrogenases have now been purified from the "hyperthermophilic" bacterium, Thermotoga maritima (Tm), which grows up to 90 C, and from an archaeon (archaebacterium) termed ES-4 which grows at 110 C, the upper temperature limit for life. The ES-4 enzyme has the lowest iron content of any known hydrogenase. It has an optimum temperature for catalysis above 100 C and is stable at 95 C for at least 24 hours. Extensive biochemical and spectroscopic studies of this hydrogenase are proposed to investigate the nature of its catalytic FeS center. In addition, crystals of ES-4 hydrogenase suitable for structural determination have been obtained that diffract past 2.8 A. The gene for ES-4 hydrogenase will therefore be cloned to provide the amino acid sequence for crystallographic analyses. Attempts will also be made to obtain an heterologous expression system for the gene encoding a hyperthermophilic hydrogenase. Once structural information is available from crystallography, this will enable proposed mechanisms of both "hyperthermostability" and of catalysis by the FeS H cluster to be directly tested by site-directed mutagenesis. An ultimate goal of this research is to construct an H cluster within an exceptionally thermostable 4Fe-ferredoxin that has been purified from Pyrococcus furiosus, an archaeon which grows up to 105 . In the prior funding period, the secondary structure of this ferredoxin wa s determined by 2D-NMR, and its gene was cloned and expressed in Escherichia coli. Modification of its 4Fe-4S cluster through mutagenesis will be explored as a potential model for the H2-activating cluster of the hyperthermophilic hydrogenases. %%% Hydrogen gas (H2) is an important intermediate in a variety of processes in the chemical and energy industry and is considered by some as the fuel of the future. Many microorganisms are also able to either consume or produce H2, and these reactions are catalyzed by an enzyme termed hydrogenase. Hydrogenases have been proposed as the key catalyst in several biologically-based systems for both the production of H2 from renewable resources and for the use of H2 in chemical syntheses. However, the instability of hydrogenases from conventional bacteria severely limit their practical application. In addition, it is not known how these enzymes catalyze H2 activation, as structural information is not available for any hydrogenase. So far it has been established that an unusual type of iron-sulfur-containing center termed the "H" cluster is intimately involved in catalysis, but the structure for the hydrogenase H cluster or its mechanism of action are not known. This proposal focuses on hydrogenases from so-called hyperthermophilic microorganisms, which have the remarkable property of growing near and even above 100 C. We have purified hydrogenase from an hyperthermophile which grows up to 110 C, the current upper temperature limit of life, and shown that the enzyme is stable for 24 hr at 100 C. Moreover, we have obtained crystals of this hydrogenase which are suitable for structural determination. Using biochemical, spectroscopic and crystallographic analyses, our objectives are to obtain a complete description of a hyperthermophilic hydrogenase at the molecular level, including structures for the protein and its H2-activating iron-sulfur cluster, elucidating its catalytic mechanism, and an explanation of its remarkable stability. These goals will be achieved by an intensely collaborative effort involving 600 liter fermentations and large scale anaerobic protein purifications, crystallography, a variety of biochemical, kinetic, potentiometric and molecular biology techniques, and a range of both conventional and developmental spectroscopies. In addition, we will attempt using recombinant DNA techniques to construct a model of the H2 activating H cluster in a small extremely stable protein. The results from the proposed research will extend our knowledge of the structures and roles of biological iron-sulfur clusters, and will provide the first detailed insight into how H2 is activated enzymatically. The availability of hyperthermophilic hydrogenases and stable models of them may also have important practical applications. ***