Published: January 4, 2011 r2011 American Chemical Society 1282 dx.doi.org/10.1021/ja110103p | J. Am. Chem. Soc. 2011, 133, 1282–1285 COMMUNICATION pubs.acs.org/JACS Formaldehyde;A Rapid and Reversible Inhibitor of Hydrogen Production by [FeFe]-Hydrogenases Annemarie F. Wait, † Caterina Brandmayr, † Sven T. Stripp, ‡ Christine Cavazza, § Juan C. Fontecilla-Camps, § Thomas Happe, ‡ and Fraser A. Armstrong* ,† † Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, U.K. ‡ Lehrstuhl fur Biochemie der Pflanzen, AG Photobiotechnologie, Ruhr-Universit € at, 44780 Bochum, Germany § Laboratoire de Crystallographie et Crystallographie des Prot  eines, Institut de Biologie Structurale, J.P. Ebel, CEA, CNRS, Universit  e Joseph Fourier, 41, rue J. Horowitz, 38027 Grenoble Cedex 1, France b S Supporting Information ABSTRACT: Dihydrogen (H 2 ) production by [FeFe]- hydrogenases is strongly inhibited by formaldehyde (methanal) in a reaction that is rapid, reversible, and specific to this type of hydrogenase. This discovery, using three [FeFe]-hydrogenases that are homologous about the active site but otherwise structurally distinct, was made by protein film electrochemistry, which measures the activity (as electrical current) of enzymes immobilized on an electrode; importantly, the inhibitor can be removed after addition. Formaldehyde causes rapid loss of proton reduction activity which is restored when the solution is exchanged. Inhibition is confirmed by conventional solution assays. The effect depends strongly on the direction of catalysis: inhibition of H 2 oxidation is much weaker than for H 2 production, and formaldehyde also protects against CO and O 2 inactivation. By contrast, inhibition of [NiFe]-hydrogenases is weak. The results strongly suggest that formaldehyde binds at, or close to, the active site of [FeFe]-hydrogenases at a site unique to this class of enzyme;highly conserved lysine and cysteine residues, the bridgehead atom of the dithiolate ligand, or the reduced Fe d that is the focal center of catalysis. H ydrogenases catalyze the very rapid interconversion of protons and dihydrogen (H 2 ) and have potential uses, direct or inspirational, in future energy technologies such as H 2 production. 1 Hydrogenases are unusual enzymes: the deeply buried active sites contain the biologically rare ligands CO and CN - and must (ideally) act selectively on the lightest of chemical species. There are two main classes: [NiFe]-hydrogenases contain Ni and Fe atoms and are predominantly H 2 oxidizers, whereas [FeFe]-hydrogenases, more commonly regarded as better H 2 producers, contain a complex structure known as the “H-cluster” (Figure 1), in which two Fe atoms, bridged by a non-protein dithiolate ligand, are linked to a [4Fe-4S] cluster via a cysteine thiolate. During catalysis, the Fe (Fe d ) that is distal to the [4Fe- 4S] cluster is thought to cycle between Fe(II) and Fe(I) in states known as H ox and H red , respectively. 2 The H-cluster and its immediate environment are highly conserved among [FeFe]- hydrogenases. Much information on hydrogenases stems from their inhibition by small ligands: notably, both CO and O 2 are potent inhibitors through their ability to coordinate to low-spin d-metals by synergic σ-donor and π-acceptor (back-bonding) interactions. 3 In the case of O 2 , the H-cluster is subsequently destroyed. 4,5 We now report that proton reduction by [FeFe]- hydrogenases is rapidly and reversibly inhibited by formaldehyde (H 2 CO, methanal), an agent much more familiar as an electro- phile and comparatively little reported as a ligand. 6 Formaldehyde is a small molecule (molecular mass 30 Da) that is gaseous at room temperature. Formaldehyde dissolves in water (up to a maximum of 37% w/v, known as formalin solu- tion) to form a diol (hydrate, K eqm (hydration) ≈ 2000 at 25 °C 8 ), which tends to form oligomers in solution. Formalin is widely used as a fixative, as it chemically modifies proteins irreversibly, although the first stages of such reactions are reversible, such as the reaction of an aldehyde with a free thiol to form a thioacetal, RS-C(OH)H 2 , or with an amine to form a Schiff-base adduct, >CdN-. Formaldehyde is used to methylate lysine (-NH 2 ) residues by reducing the Schiff base with borohydride. 9 It is difficult to demonstrate the action of formaldehyde as a reversible enzyme inhibitor because the aldehyde must be re- moved in order to check for recovery of activity. Protein film electrochemistry (PFE) solves this problem: the enzyme is immobilized onto a graphite electrode surface as an electroactive film so that the contacting solution can be exchanged, allowing Figure 1. H-cluster of DdHydAB in the reduced H red state, highlighting the bridgehead atom, X (assigned as a N-atom), and conserved nucleophilic residues close to the active site (within 5 Å of component atoms) that are possible targets for formaldehyde. 7 Received: November 10, 2010