Purification and characterization of hydrogenase from the marine green alga, Chlorococcum littorale

Abstract

Hydrogenase from the marine green alga, Chlorococcum littorale, was purified 1485-fold, resulting in a specific activity for hydrogen evolution of 75.7 μmol/min/mg of protein at 25°C, using reduced methyl viologen as an electron donor. The K m value for methyl viologen was 0.5 mM. The purity of the enzyme was judged by native PAGE. The molecular weight was estimated to be 55 kDa by SDS-PAGE, and 57 kDa by gel filtration. The optimum temperature and pH value for hydrogen evolution were 50°C and 7.5, respectively. The partially purified hydrogenase catalyzed hydrogen evolution from ferredoxin that had been isolated from the same cells, but not from NADH or NADPH. The K m value for ferredoxin was 0.68 μM. The enzyme was extremely oxygen sensitive, losing over 95% of its activity upon exposure to air within minutes, even at 4°C. Two peptide fragments were obtained from the hydrogenase protein digested enzymatically, and their amino acid sequences were determined. No significant homology was found to any other known sequences of hydrogenases.