title: Electrochemical Investigations of the Mechanism of Assembly of the Active-Site H-Cluster of [FeFe]-Hydrogenases.
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 also 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 34-valence 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 the active H-cluster. The intramolecular steps are relevant to the final stage of biological H-cluster maturation.
authors: Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford , South Parks Road, Oxford OX1 3QR, United Kingdom.
authors: Clare F
authors: CF
authors: Megarity
authors: Julian
authors: J
authors: Esselborn
authors: Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford , South Parks Road, Oxford OX1 3QR, United Kingdom.
authors: Suzannah V
authors: SV
authors: Hexter
authors: Florian
authors: F
authors: Wittkamp
authors: Ulf-Peter
authors: UP
authors: Apfel
authors: Thomas
authors: T
authors: Happe
authors: Inorganic Chemistry Laboratory, Department of Chemistry, University of Oxford , South Parks Road, Oxford OX1 3QR, United Kingdom.
authors: Fraser A
authors: FA
authors: Armstrong
mesh_terms: D020134:Catalytic Domain; D016825:Chlamydomonas reinhardtii / Q000201:enzymology; D055664:Electrochemical Techniques; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D007506:Iron-Sulfur Proteins; C118830:iron hydrogenase; D006864:Hydrogenase