Purification and characterization of an iron-nickel hydrogenase from Thermococcus celer

Thermococcus celer cells contain a single hydrogenase located in the cytoplasm, which has been purified to apparent homogeneity using three chromatographic steps: Q-Sepharose, DEAE-Fast Flow, and Sephacryl S-200. In vitro assays demonstrated that this enzyme was able to catalyze the oxidation as well as the evolution of H-2. T. celer hydrogenase had an apparent MW of 155,000 +/- 30,000 by gel filtration. When analyzed by SDS polyacrylamide gel electrophoresis a single band of 41,000 +/- 2000 was detected. Hydrogenase activity was also detected in situ in a SDS polyacrylamide gel followed by an activity staining procedure revealing a single band corresponding to a protein of apparent M-r 84,000 +/- 3000. Measurements of iron and acid-labile sulfide in different preparations of T, celer hydrogenase gave values ranging from 24 to 30 g-atoms Fe/mole of protein and 24 to 36 g-atoms of acid-labile sulfide per mole of protein. Nickel is present in 1.9-2.3 atoms per mole of protein. Copper, tungsten, and molybdenum were detected in amounts lower than 0.5 g-atoms per mole of protein. T. celer hydrogenase was inactive at ambient temperature, exhibited a dramatic increase in activity above 70 degreesC, and had an optimal activity above 90 degreesC. This enzyme showed no loss of activity after incubation at 80 degreesC for 28 h, but lost 50% of its initial activity after incubation at 96 degreesC for 20 h. Hydrogenase exhibited a half-life of approximately 25 min in air. However, after treating the air-exposed sample with sodium dithionite, more than 95% of the original activity was recovered. Copper sulfate, magnesium chloride and nitrite were also inactivators of this enzyme.