Characterization of hydrogen-uptake activity in the hyperthermophile Pyrodictium brockii.

Abstract

Pyrodictium brockii is a hyperthermophilic archaebacterium with an optimal growth temperature of 1050C. P. brockii is also a chemolithotroph, requiring H2 and CO2 for growth. We have characterized P. brockii hydrogenuptake activity with regard to temperature, ability to couple hydrogen oxidation to artificial electron acceptor reduction, sensitivity to 02, and cellular localization. The hydrogenuptake activity was localized predominantly in a particulate fraction, was reversibly inhibited by 02, and coupled H2 uptake to the reduction of positive potential artificial electron acceptors. Comparisons between these results and those of the well-studied hydrogen-uptake hydrogenase from the mesophile Bradyrhizobium japonicum showed the two enzymes to be similar despite the very different natural environments of the organisms. However, the optimum temperature for activity differed greatly in the two organisms. We have also used immunological and genetic probes specific to the 65-kDa subunit of B. japonicum hydrogenase to assay crude extracts and genomic DNA, respectively, from P. brockii and found the enzymes to be similar in these respects as well. In addition, we report a formulation for artificial seawater capable of sustaining the growth of P. brockii.The discovery of microorganisms capable of growth at extreme thermal conditions has fostered an expanding interest in the effects of temperature on biological systems (1). In addition to several important applications that have been considered for organisms and their associated biomolecules at elevated temperatures (1, 2), a variety of fundamental scientific questions have arisen concerning the biochemical basis for life under extreme conditions. Understanding the intrinsic nature of enzyme thermostability at high temperatures, for example, may be a key to gaining further insight into several issues related to protein structure and function. By comparing enzymes obtained from organisms growing at high temperatures with their mesophilic counterparts, the subtleties of protein thermostability should become clearer.Pyrodictium brockii is a hyperthermophilic (grows above 100'C) sulfur-metabolizing archaebacterium with a reported optimal growth temperature of 105'C (3, 4). To date, this is the highest reported optimal growth temperature for an organism grown in pure culture. P. brockii is a chemolithotroph, fixing CO2 for cell mass and oxidizing H2 for energy generation (5). The necessity of H2 for growth and metabolism suggests that a hydrogen-uptake hydrogenase is present in P. brockii.The work described here involved a comparison between the hydrogen-uptake system of P. brockii with that of the mesophilic bacterium Bradyrhizobium japonicum. Among the issues addressed were optimal temperature for hydrogen uptake, ability to use artificial electron acceptors, sensitivity to oxygen, and cellular localization. Furthermore, the hydrogenases from the two bacteria were compared on a structural (immunological) and genetic (DNA) level. In addition, we describe a formula...

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

“…The P. furiosus hydrogenase seems to represent a new form of hydrogenase, one that is thermodynamically designed to primarily evolve hydrogen under physiological conditions. In contrast to the P. furiosus hydrogenase, the uptake-type hydrogenase from Pyrodictium brockii seems to have much more in common with its eubacterial counterparts (31).…”

“…We have investigated this enzyme in terms of metal content, temperature stability, and kinetics of substrate reaction. We have also investigated the role that membranes play in the thermal stability of the hydrogen uptake hydrogenase, since the hydrogenase is normally associated with membranes (31 The growth medium used was composed of 25 g of NaCl, 3.5 g of MgSO4, 2.75 g of MgCl2, 0.05 g of NaBr, 0.33 g of KCl, and 2 g of yeast extract per liter. Trace elements were added as described previously (31).…”

“…Nearly complete solubilization of hydrogen uptake activity from membranes by 2% Triton X-100 was achieved (Table 1). All purification steps were done aerobically, because previous studies (31) showed that exposure to 02 did not significantly damage the activity in crude extracts.…”

“…Hydrogen uptake activity was determined amperometrically (25,42,43) with the modifications previously described (31). All assays were done at 900C in 50 mM MOPS (pH 7.01-100 mM NaCT-2 mM EDTA.…”

“…An 80% H2-20% CO2 mixture was sparged through the culture flask at a rate of 0.1 culture volume per min. P. brockii starter cultures were grown in bottles as previously described (29,31). Cells used in 63Ni labeling experiments were grown in bottles with artificial seawater (31) to which 50 ,Ci of 63NiC12 (New England Nuclear, Boston, Mass.)…”