Evaluation of biosynthetic pathways for the unique dithiolate ligand of the FeFe hydrogenase H-cluster

FeFe hydrogenases [1] catalyze the reversible oxidation of dihydrogen at a remarkable rate [2, 3]. Their active site, termed the H-cluster, comprises a classic [4Fe–4S] cubane that is covalently linked via a bridging cysteine residue to a biologically unprecedented organometallic binuclear 2Fesubcluster. The two clusters are electronically [4] and magnetically [5] inseparable. The low-spin and lowvalence iron centers of the 2Fe-subcluster are stabilized by diatomic carbonyl (CO) and cyanide (CN) strong-field ligands and they are bridged by a dithiolate group (SCH2XCH2S) , whose central atom (bridgehead) has not yet been unequivocally determined. On the basis of X-ray crystallography, Fourier transform IR spectroscopy, N hyperfine sublevel correlation spectroscopy, and theoretical calculations, the dithiolate ligand has been proposed to be 1,3-propanedithiolate (PDT; X is CH2) [6], dithiomethylamine (DTMA; X is NH) [7, 8], or dithiomethyl ether (DTME; X is O) [9]. This unprecedented coordination environment of the 2Fe-subcluster has stimulated an intensive biochemical research to identify the source and the exact biosynthetic pathway leading to the formation of these biologically unique ligands. However, the proposed hypotheses regarding the biosynthesis of the dithiolate bridging ligand are based only on chemical intuition [10, 11]. Therefore, in this communication we report a density-functional-theorybased, comparative evaluation of the energetic and mechanistic feasibility of various pathways. For the sake of completeness, all three possible candidates of dithiolates (X is CH2, NH, or O) were examined. Staying within the boundaries of known biochemical processes, we have found that a pathway that includes known S-adenosylmethionine (SAM)-catalyzed reactions is both energetically and mechanistically more favorable. Although we describe the pathway for the formation of PDT, it can be readily adopted for the biosynthesis of the DTMA and DTME analogues, provided that the proper precursor molecule is available. The enzymes HydE, HydF, and HydG have been identified as necessary and likely sufficient for the biosynthesis of the 2Fe-subcluster of the H-cluster. HydF shows GTPase activity and serves as the scaffold protein where the 2Fesubcluster is assembled [12, 13]. HydE and HydG belong to the radical-SAM superfamily of enzymes [14]. These enzymes generate the highly reactive 5-adenosyl radical (50-dA!) that can drive a variety of biochemical processes, including hydrogen atom abstraction, carbon–carbon bond cleavage, and sulfur insertion [15]. A biosynthetic hypothesis suggested that glycine could serve as the source of the diatomic ligands and it was supported by a computational investigation that is conceptually similar to the one presented here [10]. This hypothesis was recently experimentally verified, as the presence of CO and CN was detected when HydG was treated with tyrosine, through a mechanism that likely involves the formation of a glycyl radical [16, 17]. Therefore, HydE is most likely responsible for the biosynthesis of the dithiolate bridge on a [2Fe–2S] cluster located in the scaffold protein HydF. This [2Fe–2S] cluster is coordinated by three highly conserved cysteine residues, whereas the fourth ligand is readily exchangeable [13]. Moreover, the radical-SAM enzymes biotin synthase Electronic supplementary material The online version of this article (doi:10.1007/s00775-010-0698-y) contains supplementary material, which is available to authorized users.