Biochemical, spectroscopic, and structural investigations on [FeFe]-hydrogenase maturation and complex metallocluster assembly

Abstract

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.