title: Compartmentalisation of [FeFe]-hydrogenase maturation in Chlamydomonas reinhardtii.
abstract: Molecular hydrogen (H2) can be produced in green microalgae by [FeFe]-hydrogenases as a direct product of photosynthesis. The Chlamydomonas reinhardtii hydrogenase HYDA1 contains a catalytic site comprising a classic [4Fe4S] cluster linked to a unique 2Fe sub-cluster. From in vitro studies it appears that the [4Fe4S] cluster is incorporated first by the housekeeping FeS cluster assembly machinery, followed by the 2Fe sub-cluster, whose biosynthesis requires the specific maturases HYDEF and HYDG. To investigate the maturation process in vivo, we expressed HYDA1 from the C. reinhardtii chloroplast and nuclear genomes (with and without a chloroplast transit peptide) in a hydrogenase-deficient mutant strain, and examined the cellular enzymatic hydrogenase activity, as well as in vivo H2production. The transformants expressing HYDA1 from the chloroplast genome displayed levels of H2production comparable to the wild type, as did the transformants expressing full-length HYDA1 from the nuclear genome. In contrast, cells equipped with cytoplasm-targeted HYDA1 produced inactive enzyme, which could only be activated in vitro after reconstitution of the [4Fe4S] cluster. This indicates that the HYDA1 FeS cluster can only be built by the chloroplastic FeS cluster assembly machinery. Further, the expression of a bacterial hydrogenase gene, CPI, from the C. reinhardtii chloroplast genome resulted in H2-producing strains, demonstrating that a hydrogenase with a very different structure can fulfil the role of HYDA1 in vivo and that overexpression of foreign hydrogenases in C. reinhardtii is possible. All chloroplast transformants were stable and no toxic effects were seen from HYDA1 or CPI expression.
authors.affiliation: AG Photobiotechnologie, Lehrstuhl für Biochemie der Pflanzen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum, 44801, Bochum, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Sawyer
authors.affiliation: AG Photobiotechnologie, Lehrstuhl für Biochemie der Pflanzen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum, 44801, Bochum, Germany.|Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Bai
authors.affiliation: Department of Chemical and Biochemical Engineering, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, 361005, China.
authors.forename: Yinghua
authors.initials: Y
authors.lastname: Lu
authors.affiliation: AG Photobiotechnologie, Lehrstuhl für Biochemie der Pflanzen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum, 44801, Bochum, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Hemschemeier
authors.affiliation: AG Photobiotechnologie, Lehrstuhl für Biochemie der Pflanzen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum, 44801, Bochum, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Happe
mesh_terms: D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D002736:Chloroplasts / Q000378:metabolism; D006864:Hydrogenase / Q000235:genetics / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D007506:Iron-Sulfur Proteins; D010940:Plant Proteins; D006864:Hydrogenase
keywords: 
  Chlamydomonas reinhardtii
  ; HYDA1; chloroplast; gene expression; hydrogen; hydrogenase; iron sulphur protein