Structural and functional characterization of the hydrogenase-maturation HydF protein

Abstract

[FeFe] hydrogenase (HydA) catalyzes interconversion between 2H and H at an active site composed of a [4Fe-4S] cluster linked to a 2Fe subcluster that harbors CO, CN and azapropanedithiolate (adt) ligands. HydE, HydG and HydF are the maturases specifically involved in the biosynthesis of the 2Fe subcluster. Using ligands synthesized by HydE and HydG, HydF assembles a di-iron precursor of the 2Fe subcluster and transfers it to HydA for maturation. Here we report the first X-ray structure of HydF with its [4Fe-4S] cluster. The cluster is chelated by three cysteines and an exchangeable glutamate, which allows the binding of synthetic mimics of the 2Fe subcluster. [Fe(adt)(CO)(CN)] is proposed to be the true di-iron precursor because, when bound to HydF, it matures HydA and displays features in Fourier transform infrared (FTIR) spectra that are similar to those of the native HydF active intermediate. A new route toward the generation of artificial hydrogenases, as combinations of HydF and such biomimetic complexes, is proposed on the basis of the observed hydrogenase activity of chemically modified HydF.

Spectroscopic investigations under whole-cell conditions provide new insight into the metal hydride chemistry of [FeFe]-hydrogenase

“…Due to its unique nature, the biosynthesis of the [2Fe] H subsite requires at least three hydrogenase specic maturation enzymes. [16][17][18][19][20] Despite challenges in preparing the enzyme, extensive in vitro work has revealed a number of potential catalytic intermediates (Scheme 1). The oxidized resting state of the enzyme (H ox ) exhibits a mixed-valence [2Fe] H subsite and an oxidized [4Fe-4S] H cluster ([4Fe-4S] H…”

Serine is the molecular source of the NH(CH2)2 bridgehead moiety of the in vitro assembled [FeFe] hydrogenase H-cluster

“…While the [4Fe-4S] H subcluster in the H-cluster can be formed by the housekeeping gene products that are used to assemble such standard Fe-S clusters, the in vivo bioassembly of the unique [2Fe] H subcluster requires three special Fe-S "maturase" proteins: HydE, HydF, and HydG. 7,8 Although the functions of HydE and HydF have not been fully elucidated, [9][10][11][12] recent studies indicate that HydG is a bifunctional 4Fe-4S radical S-adenosyl-L-methionine (SAM) enzyme which lyses tyrosine to generate CO and CN À and forms a [(Cys)Fe(CO) 2 (CN)] organometallic precursor to the H-cluster on a dangler Fe(Cys) site in HydG. [13][14][15][16] More recently, by using a synthetic [(Cys)Fe(CO) 2 (CN)] carrier we have shown that the two sulfur atoms in the adt ligand are derived from the precursor-bound Cys, but that the CH 2 NHCH 2 component is not.…”

The final steps of [FeFe]-hydrogenase maturation

“…HydE, which has been suggested to provide the proton-shuttling azadithiolate (adt) ligand, is the least characterized of the 3 maturases as the chemical nature of its substrate(s) and product are still under debate (26). The homodimeric GTPase, HydF, is in the center of this process, fulfilling the dual role 1) of acting as a scaffold for the [2Fe H ] precursor assembly in which this precursor is docked to the [4Fe-4S] cluster of HydF via one inverted CN − ligand and 2) of being the carrier which inserts the finished precursor into the apo-hydrogenase protein (27,28). For the latter process, a cluster of 7 amino acids was suggested for HydA1 to partake in the electrostatic interaction, which facilitates HydF/apo-hydrogenase complex formation during [2Fe H ] cofactor transfer (29)(30)(31).…”