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

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]_H-subcluster 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.