Crystal structure of HydF scaffold protein provides insights into [FeFe]-hydrogenase maturation

Abstract

[FeFe]-hydrogenases catalyze the reversible production of H2 in some bacteria and unicellular eukaryotes. These enzymes require ancillary proteins to assemble the unique active site H-cluster, a complex structure composed of a 2Fe center bridged to a [4Fe-4S] cubane. The first crystal structure of a key factor in the maturation process, HydF, has been determined at 3 Å resolution. The protein monomer present in the asymmetric unit of the crystal comprises three domains: a GTP-binding domain, a dimerization domain, and a metal cluster-binding domain, all characterized by similar folding motifs. Two monomers dimerize, giving rise to a stable dimer, held together mainly by the formation of a continuous β-sheet comprising eight β-strands from two monomers. Moreover, in the structure presented, two dimers aggregate to form a supramolecular organization that represents an inactivated form of the HydF maturase. The crystal structure of the latter furnishes several clues about the events necessary for cluster generation/transfer and provides an excellent model to begin elucidating the structure/function of HydF in [FeFe]-hydrogenase maturation.

FIGURE 1.

Topology diagram of the three domains of HydF. Helices and strands are in red and cyan, respectively. The green dashed line in domain I indicates the flexible loop region of amino acids 32–43, close to the GTP-binding site. The green dots in domain III show the positions of Cys-302, Cys-353, and Cys-356, representing the putative anchors of the [2Fe-2S] subcluster.

FIGURE 2.

A, the HydF monomer shown as a cartoon drawing (left panel) and its surface (right panel). The three domains, labeled I, II, and III, are in different shades of green, and the loop connecting domains I and II is in brown. The side chains of the three cysteines involved in subcluster binding are shown in red. (This and the following drawing were done using the program Pymol (34).) B, stereo view of a cartoon representation of the HydF dimer. The two monomers are related by a 2-fold axis, approximately parallel to the plane of the paper in the horizontal direction. Cysteine side chain residues are in red; putative GTP binding regions are shown in dark blue.

FIGURE 3.

A, cartoon representation of the tetramer. One dimer is in green and cyan, and the other is in yellow and orange. B, van der Waals model of the tetramer. Cysteine residues, barely visible, are shown as red spheres, and residues potentially involved in the binding of GTP are shown as blue spheres.

FIGURE 4.

Detailed illustration of the region of the tetramer around putative FeS cluster binding sites. Cartoons of monomers A and D (belonging to two different dimers) are shown in green and cyan, respectively. Side chains of Cys-302, Cys-353, Cys-356, His-304, and His-352 are shown as ball-and-stick representation (red and yellow for monomers A and D, respectively). Cys-302 of monomers A and D form an intermolecular disulfide bridge, whereas Cys-353 and Cys-356 form an intramolecular disulfide bond within respective monomers. Tetramer formation spatially positions the two FeS cluster-binding ligand sets, belonging to different dimers, close to each other. Formation of the intermolecular S-S bond stabilizes the tetramer.