Structure and Function of Iron-Hydrogenases in a Hyperthermophilic Bacterium and of an Analogous Protein in Higher Eukaryotes

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Adams This project seeks to expand upon our knowledge of the structure, function and assembly of biological iron-sulfur (FeS) clusters, the most ubiquitous electron carriers in nature. The research concerns the properties and role of hydrogenase, an enzyme that has the unique ability to catalyze H2 oxidation and H2 production. The enzyme will be obtained from the hyperthermophilic bacterium, Thermotoga maritima (Tm), an organism that grows at 90 degrees C and is one of the two most thermophilic bacteria known. Its heterotrimeric (abg) hydrogenase of mass 160 kDa is proposed to contain eleven iron-sulfur clusters. These include the catalytic site, the so-called H cluster, which consists of six iron atoms. The genes encoding the three subunits, which have already been cloned and sequenced, will be expressed in Escherichia coli and the proteins will be characterized individually and in combination, including structural determinations. Two enzymes thought to be involved in the in vivo assembly and activation of hydrogenase, the hydrogenase-activating protein (HAP) and cyanide dihydratase, will be purified and characterized, and their genes will be coexpressed with those of hydrogenase. The recently completed genome of Tm will be used to identify other accessory proteins involved in hydrogenase synthesis, as well as those that participate in the terminal electron transfer pathway of Tm. These various proteins will be identified and their regulation will be studied using proteomics and transcriptional analyses. Finally, eukaryotes including human and Drosophila contain an analog of bacterial Fe-hydrogenase, and this will be purified and characterized using yeast (Saccharomyces cerevisiae) as the model system. The overall objectives of this research are, therefore, to define the pathways of intra- and intermolecular electron transfer in hydrogenase and associated enzymes, respectively, to determine the nature and role of the metal centers involved, and how novel clusters such as that involved in H2 activation are assembled. These goals will be achieved by a combination of techniques including large scale (600 liter) fermentations and large scale anaerobic protein purifications, crystallography, various biochemical, kinetic, potentiometric and molecular biology techniques, together with genomic and proteomic analyses. This project will provide fundamental information on an enzyme which catalyzes the simplest of reactions, the production and consumption of hydrogen gas, reactions of fundamental chemical and biological significance. This will include how the enzyme's novel iron-containing center is able to carry out these reactions, and how the protein is assembled inside the cell. A protein analogous to hydrogenase is found in higher organisms, including humans, but such organisms are not known to metabolize hydrogen gas. This research will lead to an understanding of the nature of this intriguing protein, which may well function in sensing small molecules other than hydrogen, such as carbon monoxide, nitric oxide or cyanide.