The unicellular green alga Chlamydomonas reinhardtii is used extensively as a model to study eukaryotic photosynthesis, flagellar functions, and more recently the production of hydrogen as biofuel. Two of these processes, photosynthesis and hydrogen production, are highly dependent on iron-sulfur (Fe-S) enzymes. To understand how Fe-S proteins are assembled in Chlamydomonas, we have analyzed its recently sequenced genome for orthologs of genes involved in Fe-S cluster assembly. We found a total of 32 open reading frames, most single copies, that are thought to constitute a mitochondrial assembly pathway, mitochondrial export machinery, a cytosolic assembly pathway, and components for Fe-S cluster assembly in the chloroplast. The chloroplast proteins are also expected to play a role in the assembly of the H-cluster in ½FeFe-hydrogenases, together with the recently identified HydEF and HydG proteins. Comparison with the higher plant model Arabidopsis indicated a strong degree of conservation of Fe-S cofactor assembly pathways in the green lineage, the pathways being derived from different origins during the evolution of the photosynthetic eukaryote. As a haploid, unicellular organism with available forward and reverse genetic tools, Chlamydomonas provides an excellent model system to study Fe-S cluster assembly and its regulation in photosynthetic eukaryotes.

The genome of the green alga Chlamydomonas reinhardtii encodes two [FeFe]-hydrogenases, HydA1 and HydA2, and the hydrogenase-like protein HYD3. The unique combination of these proteins in one eukaryotic cell allows for direct comparison of their in vivo functions, which have not been established for HydA2 and HYD3. Using an artificial microRNA silencing method developed recently, the expression of HydA1, HydA2 and HYD3 was specifically down-regulated. Silencing of HydA1 resulted in 4-fold lower hydrogenase protein and activity under anaerobic conditions. In contrast, silencing of HydA2 or HYD3 did not affect hydrogen production. Cell lines with strongly (>90%) decreased HYD3 transcript levels grew more slowly than wild-type. The activity of aldehyde oxidase, a cytosolic Fe-S enzyme, was decreased in HYD3-knockdown lines, whereas Fe-S dependent activities in the chloroplast and mitochondria were unaffected. In addition, the HYD3-knockdown lines grew poorly on hypoxanthine, indicating impaired function of xanthine dehydrogenase, another cytosolic Fe-S enzyme. The expression levels of selected genes in response to hypoxia were unaltered upon HYD3 silencing. Together, our results clearly distinguish the cellular roles of HydA1 and HYD3, and indicate that HYD3, like its yeast and human homologues, has an evolutionary conserved role in the biogenesis or maintenance of cytosolic Fe-S proteins.

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