[FeFe]-hydrogenase maturation: insights into the role HydE plays in dithiomethylamine biosynthesis

Abstract

HydE and HydG are radical S-adenosyl-l-methionine enzymes required for the maturation of [FeFe]-hydrogenase (HydA) and produce the nonprotein organic ligands characteristic of its unique catalytic cluster. The catalytic cluster of HydA (the H-cluster) is a typical [4Fe-4S] cubane bridged to a 2Fe-subcluster that contains two carbon monoxides, three cyanides, and a bridging dithiomethylamine as ligands. While recent studies have shed light on the nature of diatomic ligand biosynthesis by HydG, little information exists on the function of HydE. Herein, we present biochemical, spectroscopic, bioinformatic, and molecular modeling data that together map the active site and provide significant insight into the role of HydE in H-cluster biosynthesis. Electron paramagnetic resonance and UV-visible spectroscopic studies demonstrate that reconstituted HydE binds two [4Fe-4S] clusters and copurifies with S-adenosyl-l-methionine. Incorporation of deuterium from D2O into 5'-deoxyadenosine, the cleavage product of S-adenosyl-l-methionine, coupled with molecular docking experiments suggests that the HydE substrate contains a thiol functional group. This information, along with HydE sequence similarity and genome context networks, has allowed us to redefine the presumed mechanism for HydE away from BioB-like sulfur insertion chemistry; these data collectively suggest that the source of the sulfur atoms in the dithiomethylamine bridge of the H-cluster is likely derived from HydE's thiol containing substrate.

Figure 1

[FeFe]-hydrogenase crystal structure with H-cluster. The active site is located in domain IV common to all [FeFe]-hydrogenases. Atoms explicitly drawn have the following color scheme: Iron-brown, Sulfur-yellow, Nitrogen-blue, Oxygen-Red, and Carbon-gray. PDB ID: 3C8Y.

Figure 2

UV-Vis spectra of C. acetobutylicum HydE as-purified (black) and following chemical reconstitution (red). Protein concentrations adjusted to 83.4 µM. As-purified and reconstituted protein contained 3.7 +/− 0.1 and 8.6 +/− 0.1 Fe/protein respectively.

Figure 3

Low temperature EPR spectra of C. acetobutylicum HydE. (A) As-reconstituted enzyme. (B) Dithionite reduced enzyme. (C) Dithionite reduced enzyme plus exogenous SAM. All spectra were collected at 12 K with 275 µM chemically reconstituted HydE containing 7.6 ± 0.1 Fe/protein. In all panels, the experimental data traces appear in black and are overlaid with the composite simulation traces (shown in red, see below). In panel A, the simulated traces for cluster 1 and cluster 2 are depicted in orange and blue, respectively. In panel B, the simulated trace for the N-terminal cluster appears in green, while the simulated trace for the C-terminal cluster is shown in orange; the blue simulated trace represents the SAM coordinated form of the N-terminal cluster. In panel C, the blue trace again represents the SAM coordinated form of the N-terminal cluster, while the orange trace depicts the C-terminal cluster.

Figure 4

(A) Results of analysis for deuterium incorporation into product 5’-deoxyadenosine after assay of HydE with a range of small molecules in D₂O buffer. Mass spectroscopic result for dAdo(H/D) from selected assays are shown on the right, and the ratio of mass peak ratios are shown on left. B. Initial steps in a radical SAM reaction that can lead to D-atom incorporation into dAdo.

Figure 5

Stimulation of 5-deoxyadenosine production by addition of L-Cys, mercaptopyruvate, and coenzyme M (2.5 mM) to a HydE (25 µM) / SAM (500 µM) assay. Assays were quenched after 30 minutes of reaction.

Figure 6

(A) Active site of T. maritima HydE with [4Fe-4S] cluster, SAM, Arg159, Gln107, and L-Cys from in silico modeling. L-cysteine in purple represents the location of the other thiols all in their lowest energy binding conformation. The deoxyadenosyl radical’s unpaired electron that abstracts the H/D-atom from substrates is generated on the 5’ carbon of SAM (green sphere). This carbon is at distances to the sulfur atom of the mercaptans comparable to those of other radical SAM enzymes with crystal structures solved with SAM and substrates.⁽¹³⁾ All distances are in Å. Color Scheme unless otherwise noted: carbon (green), oxygen (red), nitrogen (blue), iron (orange), and sulfur (yellow). PDB ID: 3IIZ. (B) Relative binding energies of molecules from in silico docking experiments.

Figure 7

(A) Cartoon network representing the relationship of HydE family sequences to other well characterized subgroups and families described in the SFLD. The thick lines represent a higher degree of relatedness that thin lines and the unbroken lines along with the same colored boxes represent membership of the same subgroup. The thin, broken lines represent a more distant relationship. No information is inferred by the lengths of the edges. The “Antiviral” node includes Viperin, 3-Me includes NosL/NocL the tryptophan lyases, and SGUF denotes a subgroup of radical SAM enzymes with unknown function. (B) Overlay of PylB (PDB ID: 3T7V) shown in tan and HydE (PDB ID: 3CIW) shown in blue crystal structures.

Figure 8

A bridged genomic context network for HydE enzymes. Circles represent genomic neighbors within 10 genes upstream/downstream of the 563 HydE enzymes as classified by the SFLD. Connections represent BLAST pairwise sequence similarity with E-value more significant than 1e⁻²⁰. The 1,054 proteins are clustered into sets of similar sequences that are visually identifiable. Only clusters that could be assigned with function are shown and accompanied with the relevant titles. Colors represent the phylogenetic origin of the organisms in which enzymes are encoded, as shown in the legend. Clusters are roughly ordered by size and heterogeneous phylogenetic origin (from upper left to lower right). The largest and most phylogenetically diverse clusters are enriched with functions that typify the H cluster biosynthesis pathways, such as HydA, HydF, HydG and TM1266 proteins, strengthening our confidence in the ability of this method to capture biologically relevant signals.