Iron-sulfur centers in the Staphylococcus aureus respiratory chain]

Using low temperature EPR spectroscopy, signals of iron-sulfur centers with g-factors of 2.02 and 1.94 were detected in the respiratory chain of St. aureus membranes. According to their relaxation parameters and redox properties, these iron-sulfur centers are similar to iron-sulfur centers S-1 and S-3 corresponding to succinate dehydrogenases of mitochondria and bacterial membranes.     

The effect of gamma-hydroxybutyric acid on the biosynthesis of macromolecules and metabolic paramagnetic centers in hepatocytes in acute radiation injury

It has been shown, that the single injection of gamma-hydroxybutyric acid (GHBA) (100 mg/kg) to mice 30 min before irradiation (6 Gy) prevents whole-body irradiation-induced inhibition of DNA at early post-irradiation period. GHBA stimulates the biosynthesis of macromolecules at 1-2 days after irradiation. GHBA also prevents the increase in the degree of reduction of the mitochondrial and microsomal electron transport chains at early post-irradiation time (up to 2 h.), that takes place only under irradiation. It means, that GHBA inhibits the production of O2 radicals, which induce lipid peroxidation processes at post-irradiation period.     

Effect of an antioxidant complex on the biosynthesis of macromolecules in hepatocytes during acute experimental radiation injury

The radioprotective efficiency of the antioxidant complex of vitamins (AC) has been estimated by incorporation of 3H-thymidine into DNA of irradiated rat hepatocytes after whole-body irradiation. The results obtained indicate that AC is an effective radioprotective drug.     

Iron-sulfur centers in the mitochondrial respiratory chain at different stages of cell culture growth of the hamster Cricetulus griceus

The dramatic diminishing of the concentration of the N-2 iron-sulfur centre of NADH-dehydrogenase of mitochondria during the growth of cell culture of hamster fibroblasts with the subsequent recovery of concentration to the initial level was discovered by means of low-temperature ESR spectroscopy. It was concluded that the results obtained are due mainly to the decrease of the number of respiratory chains, but not to the change of the electron transport chain structure.     

Thermodynamic and EPR characteristics of two ferredoxin-type iron-sulfur centers in the succinate-ubiquinone reductase segment of the respiratory chain.

Two distinct ferredosin-type iron-sulfur centers (designated as Centers S-1 and S-2) are present in the soulble succinate dehydrogenase in approximately equivalent concentrations to that of bound flavin. Both Centers S-1 and S-2 exhibit electron paramagnetic resonance absorbance in the reduced state at the same magnetic field (gz = 2.03, gy = 1.93, and gx = 1.91) with similar line shape. Center S-2 is reducible only chemically with dithionite and remains oxidized under physiological conditions. Thus, its functional role is unknown; however, thermodynamic and EPR characterization of this iron-sulfur center has revealed important molecular events related to this dehydrogenase. The midpoint potentials of Centers S-1 and S-2 determined in the soluble succinate dehydrogenase preparations are -5 +/- 15 mV and -400 +/- 15 mV, respectively, while corresponding midpoint potentials determined in particulate preparations, such as succinate-cytochrome c reductase or succinate-ubiquinone reductase, are 0 +/- 15 mV and -260 +/- 15 mV. Reconstitution of soluble succinate dehydrogenase with the cytochrome b-c1 complex is accompanied by a reversion of the Center S-I midpoint from -400 +/- 15 mV to -250 +/- 15 mV with a concomitant restoration of antimycin A-sensitive succinate-cytochrome c reductase activity. There observations indicate that, during the reconstitution process, Center S-I is restored to its original molecular environment. In the reconstitutively active succinate dehydrogenase, the relaxation time of Center S-2 is much shorter than that of S-1, thus Center S-2 spectra are well discernible only below 20 K (at 1 milliwatt of power), while the resonance absorbance of Center S-1 is detectable at higher temperatures and readily saturates below 15 K. Over a wide temperature range the power saturation of Center S-1 resonance absorbance is relieved by Center S-2 in the paramagnetic state, and the Center S-2 central resonance absorbance is broadened by Center S-1 spins, due to a spin-spin interaction between these centers. These observations indicate an adjacent location of these centers in the enzyme molecule. In reconstitutively inactive enzymes, subtle modification of the enzyme structure appears to shift the temperature dependence of Center S-2 relaxation to the higher temperature. Thus the EPR signals of Center S-2 are also detectable at higher temperature. In this system a splitting of the central peak of the Center S-2 spectrum due to spin-spin interaction was observed at extremely low temperatures, while this was not observed in reconstitutively active enzymes or in paritculate preparations. This spin-spin interaction phenomena of inactive enzymes disappeared upon chemical reactivation with concomitant appearance of the reconstitutive activity. These observations provide a close correlation between the molecular integrity of the enzyme and its physiological function.     

Iron-sulfur centers and activities of the photosynthetic electron transport chain in iron-deficient cultures of the blue-green alga aphanocapsa

Cultures of the blue-green alga, Aphanocapsa, were grown under iron-limiting conditions and changes in concentration of redox components of the photosynthetic electron transport chain, particularly iron-sulfur centers, were monitored by spectroscopic methods. A moderate iron depletion (1/10 of the normal concentration) had little effect on photosynthetic electron transport reactions and growth. Nevertheless, the amount of membrane-bound non-heme iron decreased sharply, and ferredoxin was nearly totally replaced by a flavin-containing protein, flavodoxin. Severe iron-deficiency (1/100 of the normal concentration) was accompanied by growth inhibition and decreased rates of photosynthetic electron flow. The Photosystem I reaction center was most affected by iron depletion as evidenced by a decrease in the amounts of iron-sulfur centers A, B, and X. However, formation of other redox proteins, even those that do not contain iron, was also inhibited by severe iron deficiency.     

Deoxyribonucleotide biosynthesis in yeast (Saccharomyces cerevisiae). A ribonucleotide reductase system of sufficient activity for DNA synthesis

Ribonucleotide reductase, the central enzyme of DNA precursor biosynthesis, has been isolated and characterized from baker's yeast. The enzyme activity, measured in extracts from three different, exponentially growing yeast strains, is high enough to meet the substrate requirement of DNA replication, in contrast to very low activities found in most other organisms. In thymidylate-permeable yeast cells ribonucleotide reductase activity is stimulated under both starvation and excess of intracellular dTMP. On the other hand growth of yeast in presence of 20 mM hydroxyurea did not increase enzyme activity. Yeast ribonucleotide reductase is composed of two non-identical subunits, inactive separately, of which one binds to immobilized dATP. The relative molecular mass of the holoenzyme is about 250 000. The enzyme reduces all four natural ribonucleoside diphosphates with comparable efficacy. GDP reduction requires dTTP as effector, ADP reduction is stimulated by dGTP, whereas pyrimidine nucleotide reduction is stimulated by any deoxyribonucleotide and ATP. Enzyme activity is independent of exogenous metal ions and is insensitive towards chelating agents. Hydroxyurea inactivates yeast ribonucleotide reductase in a slow reaction; half-inhibition (I50) is reached only at 2-6 mM hydroxyurea concentration. Up to 50% reactivation occurs spontaneously after removal of the inhibitor. In accord with previous attempts by others, extensive purification of the yeast enzyme has failed owing to its extreme instability in solution; the half-life of about 11 h could not be influenced by any protective measure. Taken together, yeast ribonucleotide reductase combines features known from Escherichia coli and mammalian enzymes with differing, individual properties.     

Characterization of an NADH-linked cupric reductase activity from the Escherichia coli respiratory chain.

Previous results from our laboratory have shown that NADH-supported electron flow through the Escherichia coli respiratory chain promotes the reduction of cupric ions to Cu(I), which mediates damage of the respiratory system by hydroperoxides. The aim of this work was to characterize the NADH-linked cupric reductase activity from the E. coli respiratory chain. We have used E. coli strains that either overexpress or are deficient in the NADH dehydrogenase-2 (NDH-2) to demonstrate that this membrane-bound protein catalyzes the electron transfer from NADH to Cu(II), but not to Fe(III). We also show that purified NDH-2 exhibits NADH-supported Cu(II) reductase activity in the presence of either FAD or quinone, but is unable to reduce Fe(III). The K(m) values for free Cu(II) were 32 +/- 5 pM in the presence of saturating duroquinone and 22 +/- 2 pM in the presence of saturating FAD. The K(m) values for NADH were 6.9 +/- 1.5 microM and 6.1 +/- 0.7 microM in the presence of duroquinone and FAD, respectively. The quinone-dependent Cu(II) reduction occurred through both O(*-)(2)-mediated and O(*-)(2)-independent pathways, as evidenced by the partial inhibitory effect (30-50%) of superoxide dismutase, by the reaction stoichiometry, and by the enzyme turnover numbers for NADH and Cu(II). The cupric reductase activity of NDH-2 was dependent on thiol groups which were accessible to p-chloromercuribenzoate at low, but not at high, ionic strength of the medium, a fact apparently connected to a conformational change of the protein. To our knowledge, this is the first protein with cupric reductase activity to be isolated and characterized in its biochemical properties.     

Manganese-dependent ribonucleotide reductase ofPropionibacterium freudenreichii subsp.shermanii: Partial purification, characterization, and role in DNA biosynthesis

LikeLactobacillus leichmanii, Rhizobium meliloti, andEuglena gracilis, P. freudenreichii implicates cobalamin in DNA anabolism via adenosylcobalamin-dependent ribonucleotide reductase. However, in the absence of corrinoids,P. freudenreichii is able to synthesize DNA with the involvement of an alternative ribonucleotide reductase, which is independent of adenosylcobalamin. This enzyme is localized in both the cytoplasm (80% of activity) and the cytoplasmic membrane (20% of activity), being loosely bound to the latter. Experiments with partially purified ribonucleotide reductase isolated from extracts of corrinoid-deficient cells showed that manganese specifically stimulates this enzyme and that it is composed of two protein components, a feature that is typical of all metal-containing reductases activated by molecular oxygen. Low concentrations of manganese ions enhanced DNA synthesis in corrinoid-deficient manganese-limited cells. This effect was prevented by the addition of 80 mM hydroxyurea, a specific inhibitor of metal-containing aerobic ribonucleotide reductases. It was concluded that, in adenosylcobalamin-deficientP. freudenreichii cells, DNA synthesis is provided with deoxyribosyl precursors through the functioning of manganese-dependent aerobic ribonucleotide reductase composed of two subunits.     

An osmotic-remedial, temperature-sensitive mutation in the allosteric activity site of ribonucleotide reductase in Neurospora crassa

An osmotic-remedial, temperature-sensitive conditional mutant (un-24) was generated by Repeat Induced Point mutation (RIP) from a cross between a wild-type N. crassa strain and a strain carrying a approximately 250-kb duplication of the left arm of linkage group II (LGII). The mutation was mapped to the duplicated segment, within 2.6 map units of the heterokaryon incompatibility locus het-6. DNA transformation identified a 3.75-kb fragment that complemented the temperature-sensitive phenotype. A large ORF within this fragment was found to have a high degree of sequence identity to the large subunit of ribonucleotide reductase (RNR) from diverse organisms. Conserved amino acids at the active site and the allosteric activity sites are also evident. An unusual feature of the Neurospora sequence is a large insertion near the C-terminus relative to otherwise homologous sequences from other organisms. Three transition mutations, indicative of RIP, were identified in the N-terminal region of the temperature-sensitive mutant allele. One of these mutations results in a non-conservative amino acid substitution within the four-helix bundle that is important in the allosteric control of ribonucleotide reductase activity. This substitution appears to disrupt proper folding of the allosteric activity site during synthesis of the protein.     

Deoxyribonucleotide biosynthesis in yeast: assay and properties of ribonucleotide reductase in permeabilized Saccharomyces cerevisiae cells

Yeast cells permeabilized by freeze-thaw cycles in a sorbitol-containing medium provide an experimentally favorable system for the study of ribonucleotide reduction in a small number of cells or in mutant strains. Ribonucleotide reductase activities determined in such cells are about twice those found in cell extracts but properties of the enzyme, except pH optimum, are closely comparable in both assay procedures. In contrast with other organisms, the activities measured in permeabilized cells from both diploid or haploid strains exceed the demand for deoxyribonucleotide formation during replication of the yeast genome. The method has been applied to yeast cultures growing in the presence of the ribonucleotide reductase inhibitor hydroxyurea and a twofold increase of enzyme activity has been established in such cells. On the other hand, analysis of a series of hus mutants, selected for hydroxyurea sensitivity in the laboratory of Singer and Johnston did not reveal obvious alterations of the enzyme vs the parental strains, suggesting that the hus phenotype may be due to lesions other than in ribonucleotide reductase.     

Enzymes of DNA biosynthesis in developing sea urchins. Changes in ribonucleotide reductase, thymidine, and thymidylate kinase activities.

The pattern of ribonucleotide reductase, thymidine kinase, and thymidylate kinase activities during development of Paracentrotus lividus eggs and the effect of actinomycin on these enzymatic activities have been studied. Ribonucleotide reductase activity is detectable, though at a low level, in the unfertilized egg; the activity increases sharply soon after fertilization and reaches a peak at the morula stage. Thereafter it decreases and remains at a lower level than that of the unfertilized egg. Actinomycin, at a concentration sufficient to inhibit messenger RNA (mRNA) synthesis does not affect the level of enzymatic activity, indicating that preexisting maternal mRNA is used for the synthesis of this enzyme. Thymidine kinase is present at a low level in the egg; it increases sharply after the hatching blastula until the pluteus stage. Actinomycin does not affect the enzyme activity from fertilization until blastula but prevents the increase in enzyme activity that is observed between blastula and pluteus. Thymidylate kinase activity shows an increase after fertilization, followed by fluctuations throughout development with a considerable decrease at the blastula stage and at the end of gastrulation. Actinomycin has no effect on the activity of thymidylate kinase regardless of when the drug is added to the embryo suspension. Possible regulatory mechanisms of DNA synthesis in sea urchin embryos are discussed: The presence in the unfertilized egg of the most important enzymes controlling the cellular flow of DNA precursors and the availability of dTTP suggest that the block in DNA synthesis observed in the unfertilized egg is due to some particular mechanism that is switched on at fertilization.