Sulfide oxidation coupled to ATP synthesis in chicken liver mitochondria.

Chicken liver mitochondria consumed O2 at an accelerated rate when supplied with low concentrations of hydrogen sulfide. Maximum respiration occurred in 10 microM sulfide, and continued more slowly up to concentrations as high as 60 microM. Sulfide oxidation was coupled to adenosine triphosphate (ATP) synthesis, as shown by firefly luciferase luminescence and by measurement of the mitochondrial membrane electrochemical gradient. Synthesis of ATP required low, steady-state concentrations of sulfide (< 5 microM), which were maintained by use of a syringe pump. The ratio of consumed O2 to sulfide changed at low sulfide and O2 concentrations, indicating alternative metabolic reactions and products. In low concentrations of sulfide, presumably most similar to physiological, the O2/sulfide ratio was 0.75. This is the first report of sulfide oxidation linked to ATP synthesis in any organism not specifically adapted to a sulfide-rich environment. We suggest that this may be a widespread mitochondrial trait, and that it is consistent with the hypothesis that mitochondria originated from sulfide-oxidizing symbionts.

Sulfur reduction by human erythrocytes.

Washed human erythrocytes incubated with glucose and S8 and purged with N2 produced H2S at a nearly constant rate of 170 mumol (L cells)-1 min-1, which continued for several hours. In sealed vials up to 25 mM HS- accumulated. Glucose caused the fastest H2S production, although either lactate or glycerol could support slower rates. When glucose was added without S8, anoxic H2S production nonetheless occurred at about 1.5% of the maximum rate, after 24 hr totaling 0.5 mmol H2S (L cells)-1, suggesting the presence of endogenous reducible sulfur. Anaerobic conditions were not required, since oxygenated cells produced H2S from S8 at 80% of the anoxic rate. Using cell lysates, production of H2S occurred after addition of either glutathione, NADH, or NADPH. The observations suggest possible physiological roles for H2S as an electron carrier, and are consistent with an evolutionary relationship between eukaryotic cytoplasm and sulfur-reducing Archaea.

HS-:O2 oxidoreductase activity of Cu,Zn superoxide dismutase.

Cu,Zn-Superoxide dismutase (SOD, EC 1.15.1.1) catalyzes HS- oxidation according to the reaction: HS- + H+ + 02 --> S(0) + H202. At pH 7.8, 20 degrees C, 280 microM 02, and 100 microM HS-, the rate of HS- oxidation is 0.2 mol min-1 (mol SOD)-1. The affinities of the enzyme for HS- and O2 are consistent with previous measurements for these substrates in connection with other enzymic reactions. The rate of HS oxidation by SOD is slower than that provided by other cellular mechanisms, and it is unlikely to be of physiological significance. However, using

Interaction of Cu,Zn superoxide dismutase with hydrogen sulfide.

Addition of HS- enhanced the O(2-)-scavenging activity of bovine erythrocyte Cu,Zn superoxide dismutase (EC 1.15.1.1) by about twofold. The positive effect was measured using a diverse selection of SOD activity assays, and cannot be an artifact restricted to any single technique. Km values for HS- varied in different assay techniques, but we estimate Km approximately 80 microM HS-. In contrast to HS-, other small molecules tested with SOD either had little effect or were inhibitory. Consumption of HS- and O2- occurred in nearly 1:1 mole ratio. The products were H2O2 and sulfane sulfur, such as either elemental sulfur or polysulfide. Binding of HS- to the enzyme was rapid, with k > 10(7) M-1 s-1. The resulting complex exhibited a Cu-to-S charge-transfer absorbance band at 345 nm and an altered Cu(II) EPR spectrum. Taken together, these observations suggest that HS- binds at the catalytic Cu center of SOD and can be a genuine substrate of the enzyme.

Cytoskeleton in the archaebacterium Thermoplasma acidophilum? Viscosity increase in soluble extracts.

Thermoplasma acidophilum has no cell wall, and so its irregular shape implies the presence of a cytoskeleton. When soluble extracts of T. acidophilum were incubated in vitro they increased in viscosity, suggestive of a polymerizable component. Optimal conditions for the viscosity increase coincided with physiological ionic concentrations. Electron micrographs of negatively stained extracts showed a meshlike lattice of elements 10 nm in diameter similar to nuclear lamins. However, immunologically there was no cross-reaction with lamins nor with the other eukaryotic cytoskeletal proteins tested: tubulin, calmodulin, giardin, actin or myosin.

Cytoskeletal origins in sulfur-metabolizing archaebacteria.

Several of the thermophilic acidopholic sulfur-metabolizing archaebacteria lack rigid cell walls. Their irregular shapes were maintained by an internal mechanism, presumably a cytoskeleton. Apparently this is an adaptation for respiration upon elemental sulfur, which requires cell contact since sulfur is insoluble in water. Also, we speculate that there could be additional functions of the cytoskeleton, such as prevention of osmotic cell lysis, thermal stabilization of enzymes, and improvements in metabolic efficiency through specific enzyme positioning. Such a well-developed cytoskeleton, evolving first in thermophilic archaebacteria, could have been a preadaptation for the evolution of eukaryotic cells.

Phenylalanine-to-tyrosine singlet energy transfer in the archaebacterial histone-like protein HTa.

The Archaebacterium Thermoplasma acidophilum has a histone-like protein (HTa) abundantly associated with its deoxyribonucleic acid. Each native tetrameric complex of HTa contains 20 phenylalanine residues, 4 tyrosine residues, and no tryptophan. When the protein was excited by radiation at 252 nm, which is a wavelength absorbed predominantly by phenylalanine, the fluorescent emission was mostly from tyrosine. According to the excitation spectrum for this tyrosine fluorescence, the cause was energy transfer from phenylalanine, which occurred with about 50% efficiency. When the tyrosine residues were removed enzymatically, the excited-state lifetime of the phenylalanine residues nearly doubled. Because of energy transfer, the tyrosine emission had two apparent fluorescence decay lifetimes; one lifetime (3.9 ns) was that of tyrosine while the second (12.1 ns) corresponded to the excited state of phenylalanine.

Crystallization of a Fe,Zn superoxide dismutase from the archaebacterium Thermoplasma acidophilium.

The novel Fe,Zn superoxide dismutase from the archaebacterium Thermoplasma acidophilium has been crystallized in space groups P1, P2(1) and P2(1)2(1)2, with 2,4 and 1/2 of an 84,000 Mr tetramer, respectively, estimated to be in the asymmetric unit of the unit cell. The orthorhombic crystals, which have unit cell dimensions a = 84.2 A, b = 72.7 A, c = 67.8 A, diffract X-rays to at least 2.0 A and are suitable for a determination of the three-dimensional structure of the Fe,Zn superoxide dismutase.

Histone-like protein in the Archaebacterium Sulfolobus acidocaldarius.

The Archaebacterium Thermoplasma acidophilum contains a basic chromosomal protein remarkably similar to the histones of eukaryotes. Therefore, it was of interest to examine a different Archaebacterium for similar proteins. We chose to examine Sulfolobus acidocaldarius because it is thermophilic, like T. acidophilum, but nevertheless the two organisms are not particularly closely related. Two major chromosomal proteins were found in S. acidocaldarius. The smaller of these was soluble in 0.2 M H2SO4 and had a molecular weight of 14500. The larger was acid-insoluble and had a molecular weight of about 36000. Together, the proteins protected about 5% of the DNA against nuclease digestion and stabilized about 50% against thermal denaturation. Overall, the properties of these proteins were intermediate between those of the Escherichia coli protein HU and T. acidophilum protein HTa.

Superoxide dismutase from the Archaebacterium Thermoplasma acidophilum.

Thermoplasma acidophilum is a mycoplasma-like thermophilic organism that has been classified with the archaebacteria. It has a single superoxide dismutase (superoxide : superoxide oxidoreductase, EC 1.15.1.1) which is composed of four identically sized subunits. It has a metal content per molecule of two atoms of iron and probably one of zinc and a molecular weight of 82 000. The amino acid composition is rich in tryptophan and is typical of the manganese or iron superoxide dismutases found in other prokaryotes. However, the enzyme is resistant to denaturation by chloroform plus ethanol, by sodium dodecyl sulfate plus urea or by heat. In these respects it resembles the copper-zinc superoxide dismutase of eukaryotes. It is suggested that the enzyme may belong to a new group of superoxide dismutases.

A histone-like protein (HTa) from Thermoplasma acidophilum. I. Purification and properties.

A histone-like protein (HTa) has been isolated from cell extracts of Thermoplasma acidophilum by column chromatography on DNA-cellulose, hydroxylapatite, and Sephadex G-75, HTa elutes from DNA-cellulose in two fractions, one of which contains an 80-residue form of the protein with an NH2-terminal sequence of Val-Gly. The other fraction apparently contains the 89-residue species, in addition to a 90-residue form of the protein with the NH2-terminal sequence Met-Val. The sequence of 47 residues from the NH2 terminus of the 89-residue protein was established by automated Edman degradation. HTa is characterized by the following properties: 22% of its residues are lysine and arginine; the lysine:arginine ratio is 2.33; the absorption spectrum of the protein is distinctive due to the lack of tryptophan and the presence of 1 tyrosine and 5 phenylalanine residues; and the protein stabilizes DNA against thermal denaturation (Stein, D. B., and Searcy, D. G. (1978) Science 202, 219-221) and condenses DNA into spherical particles. All of these characteristics indicate that HTa resembles eukaryotic histones, but there are distinctive differences.

A histone-like protein (HTa) from Thermoplasma acidophilum. II. Complete amino acid sequence.

The complete amino acid sequence of the DNA-binding histone-like protein (Hta) from Thermoplasma acidophilum has been established by sequence studies directly on the protein and on tryptic, chymotryptic, and thermolysin peptides derived from the protein. The sequence of the 89-residue form of HTa is: H2N-Val-Gly-Ile-Ser-Glu-Leu-Ser-Lys-Glu-Val-Ala-Lys-Lys-Ala-Asn-Thr-Thr-Gln-Lys -Val-Ala-Arg-Thr-Val-Ile-Lys-Ser-Phe-Leu-Asp-Glu-Ile-Val-Ser-Glu-Ala-Asn-Gly-Gl y-Gln-Lys-Ile-Asn-Leu-Ala-Gly-Phe-Gly-Ile-Phe-Glu-Arg-Arg-Thr-Gln-Gly-Pro-Arg-L ys-Ala-Arg-Asn-Pro-Gln-Thr-Lys-Lys-Val-Ile-Glu-Val-Pro-Ser-Lys-Lys-Lys-Phe-Val- Phe-Arg-Ala-Ser-Ser-Lys-Ile-Lys-Tyr-Gln-Gln-COOH The molecular weight calculated from the sequence is 9,934. Another form of HTa probably differs only by the presence of an additional residue (methionine) at the NH2 terminus (the calculated molecular weight of this form is 10,065). HTa resembles eukaryotic histones in several ways, including some sequence homology, HTa also shows sequence homology with the Escherichia coli DNA-binding proteins NS1 (or HU-1) and NS2 (or HU-2).

Nucleoprotein subunit structure in an unusual prokaryotic organism: Thermoplasma acidophilum.

The freeliving thermophilic mycoplasma, Thermoplasma acidophilum, has a small acid-soluble protein tightly bound to its DNA. This protein is similar to eukaryotic histones in both size and amino acid composition. Here we report that the protein condenses DNA into globular particles that are about half the size of eukaryotic nucleosomes. Our conclusions are based primarily upon the following observations: (1) Nuclease digestion produced DNA fragments of 40 base-pairs. (2) The ratio of protein to DNA was such that 4--5 molecules of protein were associated with each 40 base-pair segment of DNA. (3) Protein crosslinking reagents produced tetramers of the histone-like protein. (4) Electron microscopy revealed globular particles 5--6 nm in diameter. (5) Each globular particle reduced the apparent contour length of the DNA by 40 base-pairs. Thus, each nucleoprotein particle is apparently composed of 40 base-pairs of DNA coiled around four molecules of proteins.

Thermoplasma acidophilum histone-like protein. Partial amino acid sequence suggestive of homology to eukaryotic histones.

Thermoplasma acidophilum is a freeliving mycoplasma-like organism that has a small basic protein tightly bound to its DNA. The N-terminal sequence of this protein has been determined. It has a distanct but statistically significant homology to eukaryotic histones H2A, H3, and to Escherichia coli protein HU.

Physiologically important stabilization of DNA by a prokaryotic histone-like protein.

The thermophilic mycoplasma Thermoplasma acidophilum has tightly bound to its DNA a protein that closely resembles the histones of eukaryotes. DNA associated with this protein is more stable than free DNA against thermal denaturation by about 40 degrees C, as shown in both native nucleoprotein and in hybrid nucleoprotein reconstituted in vitro with calf DNA. Since only about 20 percent of the DNA in this organism is associated with the histone-like protein, we suggest that its physiological function is to prevent complete separation of the DNA strands during brief exposures of the organism to denaturing conditions, and thus to facilitate rapid renaturation when normal environmental conditions return.

A restriction enzyme Tha I from the thermophilic mycoplasma Thermoplasma acidophilum.

A type II restriction enzyme (Tha I) has been isolated from the thermophilic mycoplasma Thermoplasma acidophilum. A new method of general application was used to determine the DNA sequence cleaved by the enzyme. Tha I cuts DNA in the centre of the sequence CGCG. Single-stranded DNA is not a substrate. Tha I does not cut T. acidophilum DNA which is presumably modified. This is the first description of a restriction enzyme from a mycoplasma. Because Tha I is easily prepared in large amounts of approximately 10(5) units per gram of cells, it will be a valuable addition to the battery of restriction enzymes used in studies of DNA sequences. It is active at high temperatures and may therefore be useful for special purposes requiring more extreme conditions.

Phylogenetic affinities between eukaryotic cells and a thermophilic mycoplasma.

Thermoplasma acidophilum, a thermophilic mycoplasma, has several unusual features suggesting a possible relationship to eukaryotic cells. One feature is a histone-like protein that is associated with the DNA, condensing it into subunits similar to those in eukaryotic chromatin. A second feature is an association of cytoplasmic proteins that resembles eukaryotic actin and myosin. These two components are widely distributed in different groups of eukaryotic cells, but are typically lacking in prokaryotic cells. Furthermore, T. acidophilum lacks cytochromes and respires by enzymes that apparently are not coupled to oxidative phosphorylation. This primitive type of respiration resembles that of microbodies, another feature which is represented in the cytoplasm of all groups of eukaryotic cells. Furthermore, since T. acidophilum lacks a cell wall and appears to have a primitive correlate of endocytosis, it would appear to be mechanically capable of acquiring a symbiotic mitochondrion. Thus, our observations are consistent with the symbiotic hypothesis for the origin of eukaryotic cells. We suggest that an organism similar to T. acidophilum was the host cell for the original symbiosis, becoming the nucleus and cytoplasm of modern eukaryotic cells.

Thermoplasma acidophilum: intracellular pH and potassium concentration.

Thermoplasma acidophilum is a free-living thermophilic mycoplasma. Although the organism lacks a cell wall, it can grow in medium as dilute as 66 mosM. The intracellular K+ concentration can be as low as 17 mM, but varies according to the osmolality of the culture medium. The internal pH can be measured by taking advantage of the fact that T. acidophilum undergoes lysis when the pH is adjusted to neutrality. Thus, by appropriate analysis of titration curves, it is possible to conclude that the internal pH is near 5.5. This result was confirmed by a second type of experiment in which the internal pH was analyzed by rupturing the cells in a French Pressure Cell.

Age-related increase of cuticle permeability in the nematode Caenorhabditis briggsae.

The external cuticular surface of nematodes, which resembles cellular membranes in certain ways, appears to deteriorate with age. For example, when the permeabilities to radioactive water of young and old nematodes were compared, and the data were corrected for the different surface: volume ratios, the older nematodes were significantly more permeable. In both living and dead nematodes, the same rates of water exchange were observed, indicating that the major route of exchange was probably by passive diffusion through the cuticle rather than by active processes such as swallowing or excreting water.