Biosynthesis of the unusual organometallic H-cluster at the active site of the [FeFe]-hydrogenase requires three accessory proteins, two of which are radical AdoMet enzymes (HydE, HydG) and one of which is a GTPase (HydF). We demonstrate here that HydG catalyzes the synthesis of CO using tyrosine as a substrate. CO production was detected by using deoxyhemoglobin as a reporter and monitoring the appearance of the characteristic visible spectroscopic features of carboxyhemoglobin. Assays utilizing (13)C-tyrosine were analyzed by FTIR to confirm the production of HbCO and to demonstrate that the CO product was synthesized from tyrosine. CO ligation is a common feature at the active sites of the [FeFe], [NiFe], and [Fe]-only hydrogenases; however, this is the first report of the enzymatic synthesis of CO in hydrogenase maturation.

What's your poison? Hydrogenases catalyze the reversible formation of dihydrogen from two electrons and two protons. The maturation of the [FeFe]‐hydrogenase active‐site cofactor (H cluster) requires three gene products, HydE, HydF, and HydG. Cyanide has been characterized as one of the products of tyrosine cleavage by the S‐adenosylmethionine‐dependent enzyme HydG, clarifying its role in H‐cluster biosynthesis. DOA=deoxyadenosine.

In anaerobic organisms such as E. coli the tyrosine lyase ThiH is essential for the biosynthesis of the thiazole moiety of the vitamin thiamine. ThiH is a member of the “radical AdoMet” family. The products formed by cleavage of tyrosine in vitro have been identified and suggest a radical‐mediated cleavage resulting in p‐cresol and dehydroglycine which is hydrolyzed to glyoxylate.

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Edited by Barry HalliwellKeywords: Iron-sulfur cluster S-adenosylmethionine Enzyme kinetics Product inhibition a b s t r a c t Members of the radical S-adenosylmethionine (AdoMet) superfamily reductively cleave AdoMet to generate the highly reactive 5 0 -deoxyadenosyl radical (DOA Å ) which initiates biological transformations by abstraction of a hydrogen atom. We demonstrate that three members of the family: biotin synthase (BioB), lipoyl synthase (LipA) and tyrosine lyase (ThiH) are inhibited in vitro by a combination of the products 5 0 -deoxyadenosine (DOA) and methionine. These results suggest the observed inhibition is a common feature of the radical AdoMet proteins that form DOA and methionine as products. Addition of 5 0 -methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) to BioB, LipA or ThiH activity assays removed the product inhibition by catalysing the hydrolysis of DOA and gave an increase in activity.