Radical S-Adenosyl-l-methionine Chemistry in the Synthesis of Hydrogenase and Nitrogenase Metal Cofactors

Abstract

Despite the potential deleterious effects of radical reactions and unconstrained metal ions, the exquisitely controlled protein armature of metalloenzymes and the orchestration of their biosynthetic accessory proteins allow for the creation of complex metal centers that accomplish formidable catalysis. N 2 reduction to ammonia occurs at the nitrogenase FeMo cofactor, which can be viewed as a complex bridged metal assembly consisting of a [4Fe-3S] cluster linked to a [Mo-3Fe-3S] cluster by three sulfides and a central carbon (Figs. 1A and 2A) (1, 2). Reversible reduction of protons to H 2 occurs at the [FeFe]-hydrogenase H-cluster, a [4Fe-4S] cubane bridged by a cysteine to a 2Fe subcluster containing CO, CN Ϫ , and dithiomethylamine ligands (Figs. 1B and 2B) (3). The reversible conversion of methylenetetrahydromethanopterin to methenyltetrahydromethanopterin and H 2 is catalyzed at a mononuclear iron active site, the ligands of which include two CO factors and one guanylylpyridinol cofactor (Figs. 1C and 2C) (4).Metal cofactor assembly begins with the synthesis of FeS clusters by either the housekeeping iron-sulfur cluster assembly machinery (5) in the case of [FeFe]-hydrogenase or the Nif (nitrogen fixation)-specific homologs in the case of nitrogenase. The biosynthetic steps necessary for preparation of [FeFe]-hydrogenase and nitrogenase active sites require additional dedicated proteins that include scaffolds for assembly of the nascent cluster, as well as radical S-adenosyl-L-methionine (SAM) 2 enzymes that synthesize unique non-protein ligands. Whereas less is known about the assembly of the iron-guanylylpyridinol (FeGP) cofactor of the [Fe]-hydrogenase, it is clear that radical SAM chemistry is involved (6).It is interesting to note that in all three enzymes discussed here, the metal cofactors are organometallic in nature and exist with only minimal protein coordination (Figs. 1 and 2). The FeMo cofactor of nitrogenase is covalently bound to the protein by only one histidine and one cysteine ligand; the 2Fe subcluster of the H-cluster of [FeFe]-hydrogenase and the FeGP cofactor of [Fe]-hydrogenase have only a single cysteine ligand.Radical SAM chemistry is required for the synthesis of each of these inorganic cofactors, despite the fact that there is no single non-protein ligand shared by all three cofactors. The requirement for radical SAM chemistry to synthesize this diverse array of non-protein ligands reflects the wide variety of chemical transformations that can be initiated by hydrogen atom abstraction (7). Nitrogenase FeMo Cofactor BiosynthesisFound in a variety of bacteria and some methanogenic archaea, nitrogenase plays a critical role in the global nitrogen cycle by reducing N 2 to NH 3 (Fig. 1A). Cleavage of the triple bond in N 2 requires considerable energy, as reflected in the high temperatures, high pressures, and catalysts required to attain practical rates of N 2 reduction industrially (8). In contrast, nitrogenase employs ATP to drive the process at atmospheric pressure and ambient tempe...

Iron Carbide–Sulfide Carbonyl Clusters

“…1 While good progress is being made on hydrogenases, 2 active-site models for many other FeS-based biocatalysts remain elusive, e.g., CO dehydrogenase, radical SAMs, and, of course, the FeMoco cofactor in nitrogenase (Figure 1). 3,4…”

Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation

“… 12 − 14 The classification of HydE and HydG as radical S -adenosylmethionine (SAM) enzyme superfamily members establishes a parallel between H-cluster synthesis and nitrogenase complex metallocofactor biosynthesis, while also further distinguishing it from [NiFe]-hydrogenase active site biosynthesis. 1 , 12 , 15 − 18 Radical SAM enzymes utilize the unique Fe site of a site-differentiated, redox-active [4Fe-4S] cluster to anchor SAM through its carboxylate and amine groups; this interaction leads to inner sphere electron transfer from the [4Fe-4S] cluster into SAM’s sulfonium group, ultimately generating a 5′-deoxyadenosyl radical (5′-dAdo • ) species that performs a spectacular array of substrate-derived H atom abstraction events. 19 …”

Enzymatic Cascade Reactions in Biosynthesis

“…[104] In the conversion of the inactive apo forms of microbial [FeFe]-hydrogenases to the active forms containing both carbon monoxide and cyanide ion as ligands to active-site iron-sulfur clusters,t he common amino acid tyrosine is fragmented to para-cresol and the two desired one-carbon cyanide ion and carbon monoxide molecules (Scheme 40). [105] This is af ascinating radical SAM enzyme cascade.A bout at hird of the more than 100 000 open reading frames predicted to be radical SAM enzymes in protein data bases are also thought to contain vitamin B 12 ,a lso ap otential radical generator as well as am ethyl donor, thus indicating much new radical cascade chemistry to be elucidated. Versus Apparent Friedel-Crafts Enzymatic Bisalkylation…”