Biochemical, spectroscopic, and structural investigations on [FeFe]-hydrogenase maturation and complex metallocluster assembly
Mulder, David Wayne.
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Metals are present in nearly half of all enzymes, often at the active site, where they modulate catalytic function. Some of these metalloenzymes exist with a single bound metal ion while many others contain complex metal clusters. Complex FeS assemblies are associated with the interconversion of the small molecules H 2, CO, CO 2, N 2, and NH 3. One such complex metalloenzyme, [FeFe]-hydrogenase, catalyzes the reversible oxidation of molecular H 2. The active site of [FeFe]-hydrogenases, the Hcluster, exists as a [4Fe-4S]-subcluster bridged by a protein thiolate ligand to a 2Fesubcluster which contains biologically unique CO and CN- ligands and a dithiolate ligand. The H-cluster is synthesized by the activities of the hydrogenase maturation enzymes HydE, HydF, and HydG and until recently little was known concerning the biosynthetic pathway for the H-cluster. The results presented here provide significant insight into the stepwise mechanism of H-cluster biosynthesis. Biochemical and spectroscopic characterization of the structural [FeFe]-hydrogenase enzyme expressed in a genetic background devoid of maturation genes hydE, hydF, and hydG (HydA Delta EFG) indicates by the presence of a [4Fe-4S] cluster required for [FeFe]-hydrogenase activation that the [4Fe-4S]-subcluster and 2Fe-subcluster of the H-cluster are synthesized independently. The determination of the x-ray crystal structure of HydA Delta EFG confirms this by revealing the presence of the [4Fe-4S]-subcluster and an open binding pocket for the 2Fe-subcluster, indicating that H-cluster synthesis is directed in a stepwise manner with synthesis and insertion of the [4Fe-4S]-subcluster occurring first by generalized host cell machinery followed by synthesis and insertion of the 2Fe-subcluster by specialized hyd encoded maturation machinery. The structure also reveals that insertion of the 2Fe-subcluster occurs through a positively charged channel that collapses following incorporation, as a result of conformational changes in two conserved loop regions. By utilizing complementary gene data base searching with these structural studies, new insight is made known into the evolutionarily relationships between [FeFe]-hydrogenases present in microorganisms and the eukaryotic Nar1 family of proteins which function in iron-sulfur cluster biosynthesis. The work presented as a whole, by establishing parallels to complex metal cofactor biosynthesis in nitrogenase, reveals unifying themes in complex metal cluster assembly and fundamental features of metalloenzyme evolution.