A novel type of Fe-hydrogenase in the green alga Scenedesmus obliquus is linked to the photosynthetic electron transport chain*,**

Hydrogen evolution is observed in the green alga Scenedesmus obliquus after a phase of anaerobic adaptation. In this study we report the biochemical and genetical characterization of a new type of Fe-hydrogenase (HydA) in this photosynthetic organism. The monomeric enzyme has a molecular mass of 44.5 kDa. The complete hydA cDNA of 2609 bp comprises an open reading frame encoding a polypeptide of 448 amino acids. The protein contains a short transit peptide that routes the nucleus encoded hydrogenase to the chloroplast. Antibodies raised against the Fe-hydrogenase from Chlamydomonas reinhardtii react with both the isolated and in E. coli overexpressed protein of S. obliquus as shown by western blotting. By analyzing 5 kb of the genomic DNA, the transcription initiation site and 5 introns within hydA were revealed. Northern experiments suggest that hydA transcription is induced during anaerobic incubation. Alignments of S. obliquus HydA with known Fehydrogenases and sequencing of the N-terminus of the purified protein confirm that HydA belongs to the class of Fe-hydrogenases. The C-terminus of the enzyme including the catalytic site (H-cluster) reveals a high degree of identity to Fe-hydrogenases. However, the lack of additional Fe-S clusters in the N-terminal domain indicates a novel pathway of electron transfer. Inhibitor experiments show that the ferredoxin PetF functions as natural electron