Synthesis of the 2Fe subcluster of the [FeFe]-hydrogenase H cluster on the HydF scaffold

Abstract

The organometallic H cluster at the active site of [FeFe]-hydrogenase consists of a 2Fe subcluster coordinated by cyanide, carbon monoxide, and a nonprotein dithiolate bridged to a [4Fe-4S] cluster via a cysteinate ligand. Biosynthesis of this cluster requires three accessory proteins, two of which (HydE and HydG) are radical S-adenosylmethionine enzymes. The third, HydF, is a GTPase. We present here spectroscopic and kinetic studies of HydF that afford fundamental new insights into the mechanism of H-cluster assembly. Electron paramagnetic spectroscopy reveals that HydF binds both [4Fe-4S] and [2Fe-2S] clusters; however, when HydF is expressed in the presence of HydE and HydG (HydF(EG)), only the [4Fe-4S] cluster is observed by EPR. Insight into the fate of the [2Fe-2S] cluster harbored by HydF is provided by FTIR, which shows the presence of carbon monoxide and cyanide ligands in HydF(EG). The thorough kinetic characterization of the GTPase activity of HydF shows that activity can be gated by monovalent cations and further suggests that GTPase activity is associated with synthesis of the 2Fe subcluster precursor on HydF, rather than with transfer of the assembled precursor to hydrogenase. Interestingly, we show that whereas the GTPase activity is independent of the presence of the FeS clusters on HydF, GTP perturbs the EPR spectra of the clusters, suggesting communication between the GTP- and cluster-binding sites. Together, the results indicate that the 2Fe subcluster of the H cluster is synthesized on HydF from a [2Fe-2S] cluster framework in a process requiring HydE, HydG, and GTP.

Fig. 1.

Ribbon representation of Clostridium pasteurianum (CpI) [FeFe] hydrogenase (Protein Data Bank ID code: 3C8Y) with the FeS clusters and H cluster shown as space filling models, and zoom of the H cluster as ball and stick representation. The CpI domains are represented with different colors (C terminus: red, catalytic domain: blue, ferredoxin-like domains: green, purple, and magenta), and the FeS clusters and H cluster are colored to the following scheme: rust (Fe), orange (S), black (C), red (O), blue (N), and magenta (unknown).

Fig. 2.

Low-temperature (12 K) X-band EPR spectroscopic analysis of HydF^ΔEG and HydF^EG. The dashed blue line is photoreduced HydF^ΔEG in the presence of 5.3 mM MgCl₂ (500 μM protein in 500 mM KCl), and the solid blue line is the photoreduced enzyme in the presence of MgCl₂ (5 mM) and GTP (25 mM) (475 μM protein in 500 mM KCl). Reduced HydF^ΔEG spectra comprise a nearly axial signal characteristic of [4Fe-4S]⁺ clusters with g_⊥ = 1.89 and g_∥ = 2.05 overlapping a second signal with g = 2.00 and 1.96 (see text). The dashed black line is photoreduced HydF^EG in the presence of 5.3 mM MgCl₂ (220 μM protein in 440 mM NaCl and 40 mM KCl), and the solid black line is photoreduced HydF^EG in the presence of MgCl₂ (5 mM) and GTP (12.5 mM) (205 μM protein in 400 mM NaCl and 80 mM KCl). Reduced HydF^EG spectra comprise a nearly axial signal characteristic of [4Fe - 4S]⁺ clusters with g_⊥ = 1.89 and g_∥ = 2.05.

Fig. 3.

FTIR spectrum of a HydF^EG sample (36 mg/mL) prepared under anaerobic conditions in a Coy chamber. The major observed bands are indicated. The bands at 1,940 and 1,881 cm^-1 are assigned to the υ(C = O) stretching vibrations of coordinated carbonyl groups whereas the bands at 2,046 and 2,027 cm^-1 are assigned to the υ(C = N) vibrations of bound cyanide (4).

Fig. 4.

H₂ evolution assays to probe the effects of guanosine nucleotides on the activation of HydA^ΔEFG by HydF^EG. All reactions contained 1.1 mM MgCl₂ and 500 mM of an alkali metal salt; the identity of the alkali metal salt and the guanosine nucleotide varied as follows: NaCl and no guanosine nucleotide (solid square); NaCl and 110 μM [β,γ-imido]-GTP (circle); KCl and no guanosine nucleotide (solid triangle and solid diamond); KCl and 110 μM [β,γ-imido]-GTP (down triangle); KCl and 110 μM GTP (left triangle); KCl and 110 μM GDP (solid right triangle); control containing HydF^EG without HydA^ΔEFG (KCl) (solid hexagon).

Fig. 5.

Proposed biosynthetic pathway for the H-cluster of [FeFe] hydrogenases. HydE utilizes an unknown substrate to synthesize the bridging dithiolate ligand on a [2Fe-2S] core on HydF in a step possibly utilizing GTP hydrolysis. This cluster intermediate is further processed by HydG, which utilizes tyrosine to synthesize CO and CN^- ligands, thus completing synthesis of the 2Fe unit of the H-cluster on the HydF scaffold. The activated form of HydF then interacts with HydA^ΔEFG and transfers the 2Fe cluster, thereby affording the holo [FeFe]-hydrogenase.