Isolation, characterization and N-terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii.
Hydrogenase from Chlamydomonas reinhardtii was purified to homogeneity by five column-chromatography steps 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 H2 evolution of 935 mumol.min-1.mg protein-1 at 25 degrees C, using reduced methyl viologen as electron donor. The optimal temperature for hydrogen evolution is 60 degrees C, the optimal pH value is 6.9. The Km value for methyl viologen is 0.83 mM, for ferredoxin, 35 microM. From SDS/PAGE 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 kDa. 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 4 Fe atoms/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.
Ruhr-Universität Bochum, Lehrstuhl für Biochemie der Pflanzen, Germany.
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D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000693:Anaerobiosis; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006864:Hydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007501:Iron / Q000032:analysis; D008969:Molecular Sequence Data; D013696:Temperature
D000596:Amino Acids; D006859:Hydrogen; D007501:Iron; D006864:Hydrogenase