A novel coccoid, anaerobic, Fe2+-oxidizing archaeum was isolated from a shallow submarine hydrothermal system at Vulcano, Italy. In addition to ferrous iron, H2 and sulfide served as electron donors. NO3- was used as electron acceptor. In the presence of H2, also S2O32- could serve as electron acceptor. The isolate was a neutrophilic hyperthermophile that grew between 65 degrees C and 95 degrees C. It represents a novel genus among the Archaeoglobales that we name Ferroglobus. The type species is Ferroglobus placidus (DSM 10642).

Anaerobic respiration with elemental sulfur/polysulfide or organic disulfides is performed by several bacteria and archaea, but has only been investigated in a few organisms in detail. The electron transport chain that catalyzes polysulfide reduction in the Gram‐negative bacterium Wolinella succinogenes consists of a dehydrogenase (formate dehydrogenase or hydrogenase) and polysulfide reductase. The enzymes are integrated in the cytoplasmic membrane with the catalytic subunits exposed to the periplasm. The mechanism of electron transfer from formate dehydrogenase or hydrogenase to polysulfide reductase is discussed. The catalytic subunit of polysulfide reductase belongs to the family of molybdopterin‐dinucleotide‐containing oxidoreductases. From the hyperthermophilic archaeon Pyrodictium abyssi isolate TAG11 an integral membrane complex has been isolated which catalyzes the reduction of sulfur with H2 as electron donor. This enzyme complex, which is composed of a hydrogenase and a sulfur reductase, contains heme groups and several iron‐sulfur clusters, but does not contain molybdenum or tungsten. In methanogenic archaea, the heterodisulfide of coenzyme M and coenzyme B is the terminal electron acceptor of the respiratory chain. In methanogens belonging to the order Methanosarcinales, this respiratory chain is composed of a dehydrogenase, the membrane‐soluble electron carrier methanophenazine, and heterodisulfide reductase. The catalytic subunit of heterodisulfide reductase contains only iron‐sulfur clusters. An iron‐sulfur cluster may directly be involved in the reduction of the disulfide substrate.

The mitochondrial respiratory chain is required for the induction of some yeast hypoxic nuclear genes. Because the respiratory chain produces reactive oxygen species (ROS), which can mediate intracellular signal cascades, we addressed the possibility that ROS are involved in hypoxic gene induction. Recent studies with mammalian cells have produced conflicting results concerning this question. These studies have relied almost exclusively on fluorescent dyes to measure ROS levels. Insofar as ROS are very reactive and inherently unstable, a more reliable method for measuring changes in their intracellular levels is to measure their damage (e.g. the accumulation of 8-hydroxy-2 -deoxyguanosine (8-OH-dG) in DNA, and oxidative protein carbonylation) or to measure the expression of an oxidative stressinduced gene, e.g. SOD1. Here we used these approaches as well as a fluorescent dye, carboxy-H 2 -dichloro-dihydrofluorescein diacetate (carboxy-H 2 -DCFDA), to determine whether ROS levels change in yeast cells exposed to anoxia. These studies reveal that the level of mitochondrial and cytosolic protein carbonylation, the level of 8-OH-dG in mitochondrial and nuclear DNA, and the expression of SOD1 all increase transiently during a shift to anoxia. These studies also reveal that carboxy-H 2 -DCFDA is an unreliable reporter of ROS levels in yeast cells shifted to anoxia. By using two-dimensional electrophoresis and mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), we have found that specific proteins become carbonylated during a shift to anoxia and that some of these proteins are the same proteins that become carbonylated during peroxidative stress. The mitochondrial respiratory chain is responsible for much of this carbonylation. Together, these findings indicate that yeast cells exposed to anoxia experience transient oxidative stress and raise the possibility that this initiates the induction of hypoxic genes.In many organisms, adaptation to changing oxygen concentrations is achieved both by the short term effects of oxygen on energy metabolism and the long term effects of oxygen on the expression of oxygen-responsive genes in the nucleus (1, 2). These oxygen-regulated nuclear genes can be placed into one of the two following groups: aerobic genes, which are transcribed optimally in the presence of air; and hypoxic genes, which are transcribed optimally under anoxic or micro-aerophilic conditions. In some case, proteins have both aerobic and hypoxic isoforms that are interchangeable but functionally different (3, 4). The oxygen-responsive transcription factors that regulate these genes have been identified in a number of different organisms (5-8), but despite a great deal of progress in understanding how these transcription factors function, the nature of the more proximal events involved in oxygen sensing have remained elusive. Two fundamentally different types of model have been proposed. In the first model, oxygen has a direct effect on transcription either by acting on a transcription fac...

Saccharomyces cerevisiae expresses two forms of superoxide dismutase (SOD): MnSOD, encoded by SOD2, which is located within the mitochondrial matrix, and CuZnSOD, encoded by SOD1, which is located in both the cytosol and the mitochondrial intermembrane space. Because two different SOD enzymes are located in the mitochondrion, we examined the relative roles of each in protecting mitochondria against oxidative stress. Using protein carbonylation as a measure of oxidative stress, we have found no correlation between overall levels of respiration and the level of oxidative mitochondrial protein damage in either wild type or sod mutant strains. Moreover, mitochondrial protein carbonylation levels in sod1, sod2, and sod1sod2 mutants are not elevated in cells harvested from mid-logarithmic and early stationary phases, suggesting that neither Mn-SOD nor CuZnSOD is required for protecting the majority of mitochondrial proteins from oxidative damage during these early phases of growth. During late stationary phase, mitochondrial protein carbonylation increases in all strains, particularly in sod1 and sod1sod2 mutants. By using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we have found that specific proteins become carbonylated in sod1 and sod2 mutants. We identified six mitochondrial protein spots representing five unique proteins that become carbonylated in a sod1 mutant and 19 mitochondrial protein spots representing 11 unique proteins that become carbonylated in a sod2 mutant. Although some of the same proteins are carbonylated in both mutants, other proteins are not. These findings indicate that Mn-SOD and CuZnSOD have both unique and overlapping functions in the mitochondrion.

The chemolithoautotrophic archaeon Pyrodictium abyssi isolate TAG 11 gains energy by reducing sulfur with H 2 to H 2 S. From this hyperthermophile, a sulfur-reducing complex catalyzing this reaction was purified 13.5-fold. The native complex exhibited a brownish-yellow colour and showed an apparent molecular mass of 520 kDa. SDS/PAGE revealed the presence of nine different major polypeptides with apparent molecular masses of 82, 72, 65, 50, 47, 42, 40, 30 and 24 kDa. The native complex contained 50Ϫ55 mol acid-labile sulfur, 50Ϫ55 mol iron, 1.6 mol nickel, 1.2 mol copper, 2.8 mol cytochrome b and 0.3 mol cytochrome c (all per mol native complex). The temperature optimum of the H 2 :sulfur oxidoreductase complex was 100°C, which is consistent with the physiological growth optimum of the native organism. The complex is extremely heat stable. During 5 h incubation at 100°C, no decrease in H 2 Sforming activity could be observed.Keywords : oxidoreductase ; membrane bound ; sulfur respiration; hyperthermophilic ; Pyrodictium abyssi.Based on the 16S rRNA sequences, the deepest and shortest al., 1995). Reduction of S°or polysulfides is mediated by a membrane-bound polysulfide reductase, a molybdo-enzyme branches of the universal phylogenetic tree represent hyperthermophiles (Stetter, 1992). Most of them belong to the archaeal consisting of three subunits (Krafft et al., 1992). The mechanism of electron transport from the formate dehydrogenase and hydrodomain (Woese et al., 1990). In the metabolism of many hyperthermophilic Archaea, sulfur plays an important role. The obli-genase to the polysulfide reductase is not clear (Schröder et al., 1988;Krafft et al., 1995). Here, we report the purification and gate heterotrophic strains of Pyrococcus and Thermococcus grow on organic compounds (Fiala and Stetter, 1986; Zillig et characterization of the first extremely thermostable membranebound sulfur-reducing enzyme complex isolated from the al., 1983; Neuner et al., 1990). In the presence of sulfur or polysulfides, H 2S is formed (Neuner et al., 1990; Blumentals et al., chemolithoautotrophic hyperthermophile, Pyrodictium abyssi isolate TAG 11. It is proposed to contain the entire electron-1990; Keller et al., 1995). From Pyrococcus furiosus, a cytosolic sulfide dehydrogenase which uses NADPH to reduce polysul-transport chain required for the reduction of S°with H 2 to H 2 S, including a hydrogenase, a sulfur reductase and electronfides to H 2S (Ma and Adams, 1994), and a cytosolic hydrogenase with sulfur reductase activity are known. transfering components. In this organism, the presence of sulfur increases growth yields (Schicho et al., 1993). However, so far there is no evidence that MATERIALS AND METHODS sulfur reduction in P. furiosus is coupled with ATP synthesis via Growth of organisms. The isolate P. abyssi TAG 11 was sulfur respiration (Schönheit and Schäfer, 1995). Chemolithoaucultivated on elemental sulfur or thiosulfate as described pretotrophic archaea, as Pyrodictium, utilize the redox couple H2/ viously (Stetter...