2009 Annual Science Report
Montana State University Reporting | JUL 2008 – AUG 2009
Structure, Function, and Biosynthesis of the Complex Iron-Sulfur Clusters at the Active Sites of Nitrogenases and Hydrogenases
Iron-sulfur clusters are thought to be among the most ancient cofactors in living systems. The iron-sulfur enzyme thrust is focused on examining the structure, mechanism, and biosynthesis of the complex Fe-S enzymes nitrogenase and hydrogenase. Biochemical, biophysical, and structure biology approaches are being employed to provide insights into complex iron-sulfur biosynthesis to establish paradigms for complex iron-sulfur cluster biosynthesis that can be placed in the context of the evolution of iron-sulfur motifs from the abiotic to biotic systems.
During the current funding period we have made significant progress in the area of [FeFe]-hydrogenase H cluster biosynthesis. We have determined that the complex H cluster consisting of a [4Fe-4S] cluster bridged to a unique 2Fe subcluster with unique diatomic (carbon monoxide and cyanide) and dithiolate ligands is synthesized in a stepwise manner. Our results indicate that the [4Fe-4S] cluster moiety is synthesized by generalized host cell machinery in the same manner that [4Fe-4S] clusters involved in various electron transfer reactions in cells are synthesized. The unique 2Fe subcluster synthesis and insertion follows [4Fe-4S] cluster synthesis and insertion and requires specialized biosynthesis machinery unique to [FeFe]-hydrogenases. The 2Fe subcluster synthesis requires two radical SAM enzymes that couple radical chemistry to the production of the non protein ligands and occurs on a scaffold that we have identified as the HydF gene product much in the same manner is the scaffold dependent synthesis of the nitrogenase FeMo-cofactor. We have identified a potential link between this process and the biosynthesis of the evolutionarily unrelated [Fe]-hydrogenases that also possess diatomic non-protein carbon monoxide ligands. Our recent results indicate that a radical SAM enzyme termed HmdB is involved in the mononuclear Hmd hydrogenase active site biosynthesis. The substrates for these radical SAM enzymes or the metabolic sources of the non protein ligands in these systems remain elusive and we are employing a variety of approaches including mass spectrometry to solve this challenging problem. Our most recent results involve the elucidation of the structure of the [FeFe]-hydrogenase expressed in a background devoid of the 2Fe subcluster biosynthetic machinery, that provide significant insights into the mechanism of 2Fe subcluster insertion, the final stage in [FeFe]-hydrogenase maturation and establish fundamental parallels between H-cluster biosynthesis and nitrogenase FeMo-cofactor biosynthesis.
PROJECT INVESTIGATORS:Joan Broderick
Project InvestigatorJohn Peters
PROJECT MEMBERS:Robert Szilagyi
RELATED OBJECTIVES:Objective 3.1
Sources of prebiotic materials and catalysts
Origins and evolution of functional biomolecules
Origins of energy transduction
Effects of environmental changes on microbial ecosystems
Adaptation and evolution of life beyond Earth
Biosignatures to be sought in Solar System materials
Biosignatures to be sought in nearby planetary systems