title: A Redox Active [2Fe-2S] Cluster on the Hydrogenase Maturase HydF.
abstract: [FeFe]-hydrogenases are nature's most prolific hydrogen catalysts, excelling at facilely interconverting H2 and protons. The catalytic core common to all [FeFe]-hydrogenases is a complex metallocofactor, referred to as the H-cluster, which is composed of a standard [4Fe-4S] cluster linked through a bridging thiolate to a 2Fe subcluster harboring dithiomethylamine, carbon monoxide, and cyanide ligands. This 2Fe subcluster is synthesized and inserted into [FeFe]-hydrogenase by three maturase enzymes denoted HydE, HydF, and HydG. HydE and HydG are radical S-adenosylmethionine enzymes and synthesize the nonprotein ligands of the H-cluster. HydF is a GTPase that functions as a scaffold or carrier for 2Fe subcluster production. Herein, we utilize UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopic studies to establish the existence of redox active [4Fe-4S] and [2Fe-2S] clusters bound to HydF. We have used spectroelectrochemical titrations to assign iron-sulfur cluster midpoint potentials, have shown that HydF purifies with a reduced [2Fe-2S] cluster in the absence of exogenous reducing agents, and have tracked iron-sulfur cluster spectroscopic changes with quaternary structural perturbations. Our results provide an important foundation for understanding the maturation process by defining the iron-sulfur cluster content of HydF prior to its interaction with HydE and HydG. We speculate that the [2Fe-2S] cluster of HydF either acts as a placeholder for HydG-derived Fe(CO)2CN species or serves as a scaffold for 2Fe subcluster assembly.
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Shepard
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States.
authors.forename: Amanda S
authors.initials: AS
authors.lastname: Byer
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States.
authors.forename: Jeremiah N
authors.initials: JN
authors.lastname: Betz
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States.
authors.forename: John W
authors.initials: JW
authors.lastname: Peters
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University , Bozeman, Montana 59717, United States.
authors.forename: Joan B
authors.initials: JB
authors.lastname: Broderick
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002942:Circular Dichroism; D003013:Clostridium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D006859:Hydrogen / Q000737:chemistry* / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism; D013455:Sulfur / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D013455:Sulfur; D012436:S-Adenosylmethionine; D006859:Hydrogen; D007501:Iron; C118830:iron hydrogenase; D006864:Hydrogenase