Radical SAM Enzyme HydE Generates Adenosylated Fe(I) Intermediates En Route to the [FeFe]-Hydrogenase Catalytic H-Cluster

Abstract

The H-cluster of [FeFe]-hydrogenase consists of a [4Fe−4S] H -subcluster linked by a cysteinyl bridge to a unique organometallic [2Fe] H -subcluster assigned as the site of interconversion between protons and molecular hydrogen. This [2Fe] Hsubcluster is assembled by a set of Fe−S maturase enzymes HydG, HydE and HydF. Here we show that the HydG product [Fe II (Cys)(CO) 2 (CN)] synthon is the substrate of the radical SAM enzyme HydE, with the generated 5′-deoxyadenosyl radical attacking the cysteine S to form a C5′−S bond concomitant with reduction of the central low-spin Fe(II) to the Fe(I) oxidation state. This leads to the cleavage of the cysteine C3−S bond, producing a mononuclear [Fe I (CO) 2 (CN)S] species that serves as the precursor to the dinuclear Fe(I)Fe(I) center of the [2Fe] H -subcluster. This work unveils the role played by HydE in the enzymatic assembly of the H-cluster and expands the scope of radical SAM enzyme chemistry.

Biosynthesis of the catalytic H-cluster of [FeFe] hydrogenase: the roles of the Fe–S maturase proteins HydE, HydF, and HydG

“…4.2.2 The second rSAM enzyme HydE activates the HydG product synthon on route to the binuclear [2Fe] H subcluster. Recently Tao et al 57 addressed the rst question: what is the mechanism of cysteine cleavage? This work revealed this specic reaction is performed by HydE, the other rSAM enzyme of the HydE/F/G maturase set.…”

“…Tao et al 57 tested a sequential synthesis model, where rst HydG generates its product synthon, which then serves as the substrate for the HydE rSAM enzyme. The rst intermediate characterized by EPR, resulting from the 5 0 dAdoc attack on the cysteine sulfur of the [Fe(II)(CO) 2 CN(cysteine)] provided by the syn-B carrier, forms in about 10 s (Fig.…”

Computational Approaches: An Underutilized Tool in the Quest to Elucidate Radical SAM Dynamics

“…Although the examples presented above highlight the importance of structural changes to effect catalysis, DFT has also been applied to evaluate proposed mechanistic pathways [ 46 , 47 , 48 ], assist in the analysis of spectroscopic data [ 49 ], and compare the thermodynamic reaction profiles of enzymes within the superfamily [ 50 ].…”

S‐Adenosyl‐l‐ethionine is a Catalytically Competent Analog of S‐Adenosyl‐l‐methionine (SAM) in the Radical SAM Enzyme HydG

“…The RS enzyme HydE is proposed to provide the dithiomethylamine (DTMA) ligand that forms a 2Fe H‐cluster precursor on HydF ([2Fe] F ), which is then delivered to HydA to produce the active hydrogenase [25–27] . A recent report provides intriguing evidence that the substrate of HydE is a HydG‐generated cysteine‐coordinated organometallic synthon [28] …”

“…[25][26][27] Ar ecent report provides intriguing evidence that the substrate of HydE is aH ydG-generated cysteine-coordinated organometallic synthon. [28] Reduced HydG with SAM bound to the [4Fe-4S] + cluster furthermore undergoes cryogenic photoinduced electron transfer from the cluster to SAM, resulting in regiospecific cleavage of the SÀCH 3 bond, rather than the SÀC5' bond (as in the case of PFL-AE). [29,30] This generates a CCH 3 radical in the active site,a sc haracterized by electron paramagnetic resonance (EPR) spectroscopy.…”