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

Abstract

The hyperthermophilic archaebacterium Pyrodictium brockii grows optimally at 105°C by a form of metabolism known as hydrogen-sulfur autotrophy, which is characterized by the oxidation of H2 by So to produce ATP and H2S. UV-irradiated membranes were not able to carry out the hydrogen-dependent reduction of sulfur. However, the activity could be restored by the addition of ubiquinone Qlo or ubiquinone Q6 to the UV-damaged membranes. A quinone with thin-layer chromatography migration properties similar to those of Q6 was purified by thin-layer chromatography from membranes of P. brockii, but nuclear magnetic resonance analysis failed to confirm its identity as a ubiquinone. P. brockii quinone was capable of restoring hydrogen-dependent sulfur reduction to UV-irradiated membranes. Hydrogen-reduced-minus-air-oxidized absorption difference spectra on membranes revealed absorption peaks characteristic of c-type cytochromes. A c-type cytochrome with alpha, beta, and gamma peaks at 553, 522, and 421 nm, respectively, was solubilized from membranes with 0.5% Triton X-100. Pyridine ferrohemochrome spectra confirmed its identity as a c-type cytochrome, and heme staining of membranes loaded on sodium dodecyl sulfate gels revealed a single heme-containing component of 13 to 14 kDa. Studies with the ubiquinone analog 2-n-heptyl-4-hydroxyquinoline-N-oxide demonstrated that the P. brockii quinone is located on the substrate side of the electron transport chain with respect to the c-type cytochrome. These first characterizations of the strictly anaerobic, presumably primitive P. brockii electron transport chain suggest that the hydrogenase operates at a relatively high redox potential and that the H2-oxidizing chain more closely resembles those of aerobic eubacterial H2-oxidizing bacteria than those of the H2-metabolizing systems of anaerobes or the hyperthermophile Pyrococcus furiosus.The discovery of bacteria with optimal growth temperatures of 100°C or above has led to a major research effort to understand the biochemical basis for these extreme forms of thermophily. 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).P. brockii was originally isolated from the solfatara fields off the coast of Volcano, Italy, by Stetter and his colleagues (35, 36). P. brockii has an optimal growth temperature of 105°C (36) and an absolute requirement for H2 and C02, and it couples oxidation of H2 to the reduction of sulfur to sulfide (26,35,36). This form of metabolism has been termed hydrogen-sulfur autotrophy (30, 36), a mode of growth that was originally recognized in hyperthermophilic archaebacteria. Even though CO2 is utilized as the primary if not sole source of carbon in this for...

Purification and properties of an extremely thermostable membrane‐bound sulfur‐reducing complex from the hyperthermophilic Pyrodictium abyssi

“…5). When the complex was incubated without (O'Brian and Maier, 1985;Wong and Maier, 1984) and to in-sulfur and under N2 as gas phase, 50% of the H2S-forming activhibit H 2 S formation of the quinone-containing membranes of the ity was lost within 1 h. In the presence of H 2 and absence of S°, aechaeon Pyrodictium brockii (Pihl et al, 1992). N-terminal only about 28% of the activity was lost in the same period of amino acid sequences were obtained from the 82, 65, 45 and time.…”

“…Among chemolithoautotrophic hyPreparation and solubilization of the membrane fraction. perthermophiles, sulfur reduction has been investigated in PyroAll of the following steps were performed at room temperature dictium brockii (Pihl et al, 1992;Pihl and Maier, 1991). A in an anaerobic chamber (Coy Laboratory Products Inc.) under membrane-bound electron-transport chain, consisting of a hy-95% N 2 and 5% H 2 as the gas phase.…”

“…A in an anaerobic chamber (Coy Laboratory Products Inc.) under membrane-bound electron-transport chain, consisting of a hy-95% N 2 and 5% H 2 as the gas phase. Frozen cells (wet mass, drogenase, a cytochrome c and a quinone, which couples H 2 8 g) were suspended in 26 ml 0.1 M 4-(2-hydroxyethyl)-1-piperoxidation to S°reduction has been postulated (Pihl et al, 1992).azinepropane sulfonic acid, pH 8.4 (EPPS), where they lysed So far, the best understood example for membrane-bound sulfur spontaneously. After addition of approximately 80 µg DNAse, reduction is in the mesophilic bacterium Wolinella succinogenes.…”

“…A in an anaerobic chamber (Coy Laboratory Products Inc.) under membrane-bound electron-transport chain, consisting of a hy-95% N 2 and 5% H 2 as the gas phase. Frozen cells (wet mass, drogenase, a cytochrome c and a quinone, which couples H 2 8 g) were suspended in 26 ml 0.1 M 4-(2-hydroxyethyl)-1-piperoxidation to S°reduction has been postulated (Pihl et al, 1992).…”

Effects of elemental sulfur on the metabolism of the deep-sea hyperthermophilic archaeon Thermococcus strain ES-1: characterization of a sulfur-regulated, non-heme iron alcohol dehydrogenase

“…The mechanism of S 0 reduction in one autotrophic hyperthermophile, Pyrodictium brockii, an obligate S 0 -reducing species which grows optimally at 105ЊC, has been investigated. This organism was shown to have a primitive membrane-bound electron transport chain for coupling H 2 oxidation and H 2 S production (52), although the S 0 -reducing entity was not purified.…”