Purification and characterization of the hydrogen uptake hydrogenase from the hyperthermophilic archaebacterium Pyrodictium brockii

Abstract

Pyrodictium brockii is a hyperthermophilic archaebacterium with an optimal growth temperature of 105cC. P. brockii is also a chemolithotroph, requiring H2 and CO2 for growth. We have purified the hydrogen uptake hydrogenase from membranes of P. brockii by reactive red affinity chromatography and sucrose gradient centrifugation. The molecular mass of the holoenzyme was 118,000 19,000 Da in sucrose gradients. The holoenzyme consisted of two subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The large subunit had a molecular mass of 66,000 Da, and the small subunit had a molecular mass of 45,000 Da. Colorometric analysis of Fe and S content in reactive red-purified hydrogenase revealed 8.7 + 0.6 mol of Fe and 6.2 + 1.2 mol of S per mol of hydrogenase. Growth of cells in 63NiCl2 resulted in label incorporation into reactive red-purified hydrogenase. Growth of cells in 63NiC12 resulted in label incorporation into reactive redpurified hydrogenase. Temperature stability studies indicated that the membrane-bound form of the enzyme was more stable than the solubilized purified form over a period of minutes with respect to temperature. However, the membranes were not able to protect the enzyme from thermal inactivation over a period of hours. The artificial electron acceptor specificity of the pure enzyme was similar to that of the membrane-bound form, but the purified enzyme was able to evolve 12 in the presence of reduced methyl viologen. The Km of membrane-bound hydrogenase for H2 was approximately 19 ,uM with methylene blue as the electron acceptor, whereas the purified enzyme had a higher Km value.

Bioenergetics of sulfur reduction in the hyperthermophilic archaeon Pyrococcus furiosus

“…In contrast, all known hyperthermophiles metabolize SO; in fact, the majority are obligately dependent upon So reduction and use either H2 or organic compounds as electron donors. The best studied is the autotroph Pyrodictium brockii (49), from which several components of its membrane-bound electron transport chain between H2 oxidation and S0 reduction have been characterized (40,41).Only a limited number of hyperthermophiles can grow in the absence of So, and most of the research in this area has been carried out with Pyrococcus furiosus (22). This organism grows optimally at 100'C by a fermentative-type metab-* Corresponding author.…”

“…In contrast, all known hyperthermophiles metabolize SO; in fact, the majority are obligately dependent upon So reduction and use either H2 or organic compounds as electron donors. The best studied is the autotroph Pyrodictium brockii (49), from which several components of its membrane-bound electron transport chain between H2 oxidation and S0 reduction have been characterized (40,41).…”

Hydrogen-oxidizing electron transport components in the hyperthermophilic archaebacterium Pyrodictium brockii

“…The use of Triton X-100 (at 0.5%) was sufficient to solubilize all of the cytochrome c from the membrane (data not shown). It is interesting to note that, in comparison, the solubilization of hydrogenase from P. brockii membranes required 2% Triton X-100 (29). When subjected to pyridine in basic conditions, c-type cytochromes give a characteristic peak at 550 nm in dithionite-reduced-minus-air-oxidized difference spectra (27).…”

“…The characteristics of this chain, involving a NiFeS hydrogenase functioning at a relatively high potential (29), quinone, and a c-type cytochrome, resemble those of aerobic H2-oxidizing bacteria. Although our model may not be the complete electron transport chain, it is the minimum that is consistent with the available data and represents the first description of the electron transport chain of this bacterium.…”

“…To date, most of the work has centered upon trying to elucidate and characterize the various metabolic components that allow bacteria to thrive at high temperatures. The two most intensively studied hyperthermophiles (defined by us [30,31] as organisms that are able to grow at or above 100°C) are the heterotroph Pyrococcus furiosus (2, 5-7, 9-11) and the autotroph Pyrodictium brockii (25,26,(28)(29)(30)(31)(32).…”