Isolation, characterization and N‐terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii

Abstract

Hydrogenase from Chlamydomonas reinhardtii was purified to homogeneity by five columnchromatography deps under strict anaerobic conditions. The cells were disrupted by mild treatment with detergent. The enzyme was purified 6100-fold, resulting in a specific activity for H, evolution of 935 pmol . min-' . mg protein-' at 25"C, using reduced methyl viologen as electron donor. The optimal temperature for hydrogen evolution is 60°C, the optimal pH value is 6.9. The K, value for methyl viologen is 0.83 mM, for ferredoxin, 35 pM. From SDSFAGE gels, the protein was judged to be pure. On non-denaturing gels, run under nitrogen, a single band was detected after activity staining. This band corresponded to the single band observed on denaturing SDS gels, which had an apparent molecular mass of 48 m a . If the band was cut out of the native gel and incubated with reduced methyl viologen, hydrogen evolution could be measured. The purified enzyme contains 4Fe atorns/mol. The amino acid composition and the N-terminal amino acid sequence (24 residues) of the protein were determined. No significant amino acid sequence homologies could be found to any sequences from prokaryotic hydrogenases.Many prokaryotes and several eukaryotes have in common an enzyme complex catalyzing the reversible reaction 2 H + + 2 e = H 2 .

Genomic and metagenomic surveys of hydrogenase distribution indicate H2 is a widely utilised energy source for microbial growth and survival

“…Clostridium pasteurianum (Peters et al, 1998) Desulfovibrio desulfuricans (Nicolet et al, 1999) Chlamydomonas reinhardtii (Happe and Naber, 1993) 1FEH 1HFE 3LX4 (Figure 2). Genome architecture is also able to discriminate the sometimes poorly bootstrapped lineages of the group 4 enzymes (Supplementary Figure S4) and therefore is a valuable hydrogenase classification tool.…”

Discovery of Two Novel Radical S-Adenosylmethionine Proteins Required for the Assembly of an Active [Fe] Hydrogenase

“…Hydrogenase activity in C. reinhardtii is induced by anaerobiosis (4,5,20,30,31). This is achieved either in the dark by using an inert gas (or exogenous reductant) to purge O 2 from sealed cultures or in the light by depriving sealed cultures of sulfur, which results in attenuated rates of photosynthetic O 2 evolution (4, 5, 32).…”

“…As expected, the partially purified WT sample (see "Experimental Procedures") shows only a single anaerobically induced band with an electrophoretic mobility of ϳ47-48 kDa due to co-migration of the two hydrogenases. Although full-length HydA1 and HydA2 hydrogenase enzymes from C. reinhardtii have predicted masses of 53.1 and 53.7 kDa, respectively, HydA1 undergoes N-terminal proteolytic processing of a chloroplast transit peptide sequence, resulting in a mature 47.5-kDa protein localized in the chloroplast (31). The HydA2 protein is predicted to undergo similar processing, resulting in an estimated 47.3-kDa mature protein (20).…”

A Novel Type of Iron Hydrogenase in the Green AlgaScenedesmus obliquus Is Linked to the Photosynthetic Electron Transport Chain

“…From the unicellular green alga Chlamydomonas reinhardtii a monomeric iron hydrogenase with high specific activity has been isolated (30,31). In contrast, a nickel-iron hydrogenase was described for another well examined green alga, Scenedesmus obliquus (32,33).…”