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Electrochemical Investigations of the Mechanism of Assembly of the Active-site H-Cluster of [FeFe]-hydrogenases Clare F. Megarity, Julian Esselborn, Suzannah V. Hexter, Florian Wittkamp, Ulf-Peter Apfel, Thomas Happe, and Fraser A. Armstrong J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b09366 • Publication Date (Web): 24 Oct 2016 Downloaded from http://pubs.acs.org on October 24, 2016
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Journal of the American Chemical Society
Electrochemical Investigations of the Mechanism of Assembly of the Active-site H-Cluster of [FeFe]hydrogenases Clare F. Megarity,† Julian Esselborn,§ Suzannah V. Hexter,† Florian Wittkamp,|| Ulf-Peter Apfel,|| Thomas Happe§ and Fraser A. Armstrong† †
Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, United Kingdom §Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie and || Lehrstuhl für Anorganische Chemie I, Ruhr-Universität Bochum, 44801 Bochum, Germany ABSTRACT: Protein film electrochemistry (PFE) has been used to study the assembly of the complex 6Fe active site of [FeFe]hydrogenases (known as the H-cluster) from its precursors − the [4Fe-4S] domain that is already coordinated within the host and the 2Fe domain that is presented as a synthetic water-soluble complex stabilized by an additional CO. Not only does PFE allow control of redox states via the electrode potential, but the immobilized state of the enzyme facilitates control of extremely low concentrations of the 2Fe complex. Results for two enzymes, CrHydA1 from Chlamydomonas reinhardtii and CpI from Clostridium pasteurianum, are very similar, despite large differences in size and structure. Assembly begins with very tight binding of the 34valence electron 2Fe complex to the apo-[4Fe-4S] enzyme, well before the rate-determining step. The precursor is trapped under highly reducing conditions (< -0.5 V vs SHE) that prevent fusion of the [4Fe-4S] and 2Fe domains (via cysteine-S), since the immediate product would be too electron-rich. Relaxing this condition allows conversion to active H-cluster. The intramolecular steps are relevant to the final stage of biological H-cluster maturation.
from its simpler components through the action of two other maturation proteins HydE and HydG.2,3 Two [FeFe]hydrogenases have been under particular scrutiny regarding Hcluster assembly, namely CrHydA1 from the green algae Chlamydomonas reinhardtii and CpI from the fermentative anaerobe Clostridium pasteurianum. Whereas CrHydA1 contains only the H-cluster, CpI is a more complex enzyme that also contains a relay system of four additional FeS clusters to mediate intramolecular electron transfer to and from the Hcluster. Crystallographic comparisons between CpI and apo[4Fe-4S] CpI show that insertion of the 2Fe subcomplex does not greatly change the overall protein structure: in contrast, the only structure so far obtained for CrHydA1 is of the apo-[4Fe4S] form which has an open channel leading to the [4Fe-4S] cluster.4,5 Hence it is possible, but uncertain at this stage, that both apo-[4Fe-4S] enzymes exist as open and closed states in equilibrium. It was recently discovered6 that a synthetic CO-adduct of the [2Fe] subcluster [Fe2[µ-(SCH2)2NH](CN)2(CO)4]2− (abbreviated[2Fe]MIM, II) originally synthesized by Rauchfuss and coworkers7 reacts directly with apo-[4Fe-4S] enzymes to yield active hydrogenases,8 providing a new investigative route for H-cluster assembly. It is now recognized that [FeFe]-hydrogenases have exceptional electrocatalytic activity when attached to a suitable electrode.9-15 Protein film electrochemistry (PFE)
INTRODUCTION The active site of [FeFe]-hydrogenases, known as the ‘Hcluster’ (I, constructed from pdb 2C8Y) consists of two components – an unusual [2Fe] complex, containing diatomic ligands CO, CN− and a bridging azadithiolate, which is the site of formation or oxidation of H2, and a [4Fe-4S] cluster, coordinated by four cysteine thiolates, which can store one electron during catalysis.1 The two subcomplexes are connected by one of the cysteine thiolates (µ-SCys) which also provides the only covalent link between the [2Fe] complex and the protein: the broken line shows the division between the two components.
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During biosynthesis the 2Fe complex is transferred to its awaiting recipient, the [4Fe-4S] ‘apo-enzyme’, from a scaffold protein, HydF, which houses the 2Fe complex as assembled
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is a suite of techniques that allows detailed investigations of redox enzymes under conditions of controlled electrode potential, and the minuscule sample requirement (
