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 H2. 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+/-2,000 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 Mr 84,000+/-3,000. 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 degrees C, and had an optimal activity above 90 degrees C. This enzyme showed no loss of activity after incubation at 80 degrees C for 28 h, but lost 50% of its initial activity after incubation at 96 degrees C 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.
Blamey
J M
JM
Research and Development Department, Recoleta, Santiago, Chile. jblamey@bioscience.cl
Chiong
M
M
Smith
E T
ET
D002455:Cell Division; D019327:Copper Sulfate / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006859:Hydrogen / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D015636:Magnesium Chloride / Q000737:chemistry; D008970:Molecular Weight; D009573:Nitrites / Q000737:chemistry; D021122:Protein Subunits; D013696:Temperature; D019712:Thermococcus / Q000201:enzymology* / Q000254:growth & development
D009573:Nitrites; D021122:Protein Subunits; D015636:Magnesium Chloride; D006859:Hydrogen; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D019327:Copper Sulfate