Compartmentalisation of [FeFe]‐hydrogenase maturation in Chlamydomonas reinhardtii

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.