[FeFe]‐Hydrogenase Cyanide Ligands Derived From S‐Adenosylmethionine‐Dependent Cleavage of Tyrosine

Abstract

Hydrogenases catalyze the reversible reduction of protons to yield molecular hydrogen (H 2 ) and occur in three evolutionarily unrelated forms termed the [Fe]-, [FeFe]-, and [NiFe]hydrogenases. [1,2] [FeFe]-hydrogenases all contain a complex active-site cofactor termed the H cluster (1, Scheme 1) that consists of a regular [4Fe4S] cluster bridged by a shared cysteine thiolate sulfur atom to a 2Fe subcluster with biologically unique carbon monoxide, cyanide, and dithiolate ligands. [3,4] The H cluster is biosynthesized in a stepwise process in which generalized host cell machinery [5] is directed towards the synthesis of a [4Fe4S] subcluster with subsequent synthesis and insertion of the 2Fe subcluster by specialized hyd encoded proteins, HydE, HydF, and HydG. [1,6] HydE and HydG are radical S-adenosylmethionine (AdoMet) enzymes thought to be responsible for the synthesis and proper incorporation of the nonprotein ligands. [1,[7][8][9] HydF has been proposed to function as a scaffold for assembly of the H cluster 2Fe subcluster and also to mediate its subsequent insertion into HydA. [6,10] HydG has recently been shown to catalyze radical-mediated tyrosine cleavage and generate p-cresol, [11] which is a known fermentation product of several anaerobes. [12,13] The HydG-catalyzed reaction is proposed to be similar to that catalyzed by the thiamine biosynthetic enzyme ThiH and to yield dehydroglycine as an intermediate. [14][15][16] It was hypothesized by Pilet et al. [11] that two molecules of dehydroglycine condense at an FeS cluster on HydG and result in generation of the dithiolate ligand. Herein, we demonstrate that cyanide is a product of HydGcatalyzed tyrosine cleavage, a result which clarifies the role of HydG and indicates that tyrosine is the source of the cyanide ligands in the H cluster.To investigate the reaction products formed by HydG, enzyme activity assays containing chemically reconstituted HydG (on average 5.1 AE 0.5 Fe per HydG), tyrosine, AdoMet, and sodium dithionite were prepared. At selected time points, assays were stopped by acidification, and the precipitated protein was removed by centrifugation. HPLC-based analysis methods were then used to measure the concentration of reaction products in the supernatant, thus confirming the turnover of AdoMet to yield deoxyadenosine (DOA), whilst tyrosine was cleaved to yield p-cresol. [11] Cyanide was detected and quantified after derivatization by a modification of the method of Tracqui et al. [17] in which the cyanide anion reacts with naphthalene-2,3-dicarbaldehyde (NDA) 5 and a primary amine (either taurine (6) or N 1 ,N 1 -dimethylethane-1,2-diamine (7)), thus generating the fluorescent 1-cyanobenz[f]isoindole (CBI) derivatives 8 and 9 (Figure 1 A). After derivatization, HPLC analysis showed a fluorescent peak which coeluted with a standard of the CBI derivative (Figure 1 B). The components required for cyanide-forming activity were tyrosine, AdoMet, a reducing agent, and HydG. Omission of any of these components resulted in loss of activity, consi...

Hydrogenases and the Global H2 Cycle

“…The intrinsic cyanide ligands of [NiFe]-hydrogenase starts from carbamoyl phosphate [15] whereas that of [FeFe]-hydrogenases starts from tyrosine. [16] Also the intrinsic CO ligands in the three types of hydrogenases are of different origin, although at present only the origin of the CO in [FeFe]-hydrogenases from tyrosine is really known. [17,18] (3) Evolution: A current hypothesis is that pre-biotic chemistry started out with H 2 and CO 2 , with iron and nickel catalysts and iron-sulfur clusters as electron carriers.…”

Elements of Metabolic Evolution

“…The only vestiges of the short but glorious past of HCN are seen in the cyano ligands of [FeNi]-hydrogenase and [FeFe]-hydrogenase and these owe their existence not to environmental HCN, but to chemical synthesis. [69,70] CO is the oxygen analogue of HCN, with which it is chemically related by NH 3 -H 2 O exchange via HCONH 2 . However, its pattern of reactivity is distinctly different.…”

Unraveling the Biosynthesis of Nature’s Fastest Hydrogenase

“…By using a cell-free approach, Roach and co-workers demonstrated that HydG converts tyrosine into the cyanide needed for the biosynthesis of the H cluster. [4] In a further illustration of the special capabilities of radical SAM enzymes, tyrosine was degraded into p-cresol and dehydroglycine, which released CN À into the medium, where it was detected by conversion into a fluorescent derivative. Dehydroglycine is also involved in the formation of thiamin in some bacteria, where it is also generated by radical SAM enzymes (Scheme 3).…”

“…This situation is changing rapidly now with the elucidation of the pathway to the cyanide ligands. [3,4] Cyanide is widely considered by inorganic chemists as the ultimate "strong ligand". It is featured in the first synthetic coordination compound, Prussian Blue.…”