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Compartmentalisation of [FeFe]‐hydrogenase maturation in Chlamydomonas reinhardtii
Author(s) -
Sawyer Anne,
Bai Yu,
Lu Yinghua,
Hemschemeier Anja,
Happe Thomas
Publication year - 2017
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13535
Subject(s) - chlamydomonas reinhardtii , hydrogenase , chloroplast , gene cluster , mutant , biology , chemistry , biochemistry , microbiology and biotechnology , gene , enzyme
Summary Molecular hydrogen (H 2 ) can be produced in green microalgae by [FeFe]‐hydrogenases as a direct product of photosynthesis. The Chlamydomonas reinhardtii hydrogenase HYDA 1 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 HYDA 1 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 H 2 production. The transformants expressing HYDA 1 from the chloroplast genome displayed levels of H 2 production comparable to the wild type, as did the transformants expressing full‐length HYDA 1 from the nuclear genome. In contrast, cells equipped with cytoplasm‐targeted HYDA 1 produced inactive enzyme, which could only be activated in vitro after reconstitution of the [4Fe4S] cluster. This indicates that the HYDA 1 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 H 2 ‐producing strains, demonstrating that a hydrogenase with a very different structure can fulfil the role of HYDA 1 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 HYDA 1 or CPI expression.