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

Abstract

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.

Draft genome sequence of Thermococcus waiotapuensis WT1 T , a thermophilic sulfur-dependent archaeon from the order Thermococcales

“…The genome is predicted to encode genes for amino acid transferases and glutamate dehydrogenase, which are both necessary for the fermentation of amino acids ( 16 ). Predicted Ni-Fe hydrogenase complexes and sulfhydrogenases are present in the genome, consistent with the general metabolic capability of Thermococcus spp ( 17 – 19 ).…”

Extremophilic Oxidoreductases for the Industry: Five Successful Examples With Promising Projections

“…This type of hydrogenase is only found in archaea and bacteria, where they catalyze either hydrogen oxidation or production. Among them we can find the soluble hydrogenases produced by nitrogenfixing cyanobacteria and bacteria (Bothe et al, 2010), the membrane bound hydrogen-producing enzyme first described in the hyperthermophile Pyrococcus furiosus (Sapra et al, 2003), and the tetrameric hydrogenases found in some hyperthermophiles such as Thermococcus celer (Blamey et al, 2001). Many hydrogenases from this family use NAD(P) or other soluble cofactors such as coenzyme F 420 .…”

Characterization of a Cytosolic NiFe-Hydrogenase from the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

“…In this manuscript, we described the gene analysis, purification, and characterization of a cytosolic NiFe-hydrogenase from T. kodakaraensis KOD1. So far, there have been a number of reports on the purification and characterization of cytosolic hydrogenases from hyperthermophilic archaea belonging to the order Thermococcales (3,5,21,29,47). A comparison of their properties with those of the T. kodakaraensis hydrogenase is shown in Table 1.…”

“…This is particularly the case for hydrogenase, as a soluble, thermostable hydrogenase could potentially provide a stable biocatalyst for hydrogen production (43). Accordingly, cytosolic hydrogenases have been identified and studied from various hyperthermophiles, including the archaea Pyrococcus furiosus (19), Thermococcus litoralis (29), Thermococcus stetteri (47), and Thermococcus celer (3) and the bacterium Thermotoga maritima (40). The archaeal enzymes have all been found to be NiFe-hydrogenases, while the enzyme from T. maritima was an Fe-only hydrogenase.…”