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The genes that encode the bc-type nitric-oxide reductase from Paracoccus denitrificans have been identified. They are part of a cluster of six genes (norCBQDEF) and arc found near the gene cluster that encodes the cd1-type nitrite reductase, which was identified earlier [de Boer, A. P. N., Reijnders. W. N. M., Kuenen. J. G., Stouthamer, A. H. & van Spanning, R. J. M. (1994) Isolation, sequencing and mutational analysis of a gene cluster involved in nitrite reduction in Paracoccus denitrificans, Antonio Leon wenhoek 66. 111-127]. norC and norB encode the cytochrome-c-containing subunit II and cytochromeb-containing subunit I of nitric-oxide reductase (NO reductase), respectively, norQ encodes a protein with an ATP-binding motif and has high similarity to NirQ from Pseudomonas stutzeri and Pseudomonas aerurginosa and CbbQ from Pseudomonas hydrogenothermophila. norE encodes a protein with five putafive transmembrane α-helices and has similarity to CoxIII, the third subunit of the αα3-type cytochromec oxidases. norF encodes a small protein with two putative transmembrane α-helices. Mutagenesis of norC, norB norQ and nord resulted in cells unable to grow anaerobically. Nitrite reductase and NO reductase (with succinate or ascorbate as substrates) and nitrous oxide reductase (with succinate as sub strate) activities were not detected in these mutant strains. Nitrite extrusion was detected in the medium. indicating that nitrate reductase was active. The norQ and norD mutant strains retained about 16% and 23% of the wild-type level of NorC, respectively. The norE and norF mutant strains had specific growth faits and NorC contents similar to those of the wild-type strain but had reduced NOR and NIR activities. indicating tirol their gene products arc involved in regulation of enzyme activity. Mutant strains containing the norCBQDEF region on the broad-host-range vector pEG400 were able to grow anaerobically, althongh at a lower specific growth rate and with lower NOR activity compared with the wild-type strain. [ABSTRACT FROM AUTHOR]

Investigates molecular cloning of the human glucose-regulated protein ERp57/GRP58, a thiol-dependent reductase. Identification of the secretory form and inducible expression by the oncogenic transformation.

Focuses on prostaglandin-E[sub 2] 9-reductase from corpus luteum of pseudopregnant rabbit, a member of the aldo-keto reductase superfamily featuring 20alpha-hydroxysteroid dehydrogenase activity. Substrate specificity comprising the reduction of adelhydes, ketones and quinones.

Examines the glycine reductase of Clostridium litorale. Cloning and sequencing of a 2.8-kb HindIII fragment from Clostridium litorale; Northern-blot analysis.

Examines the purification and characterization of protein P[sub B] of betaine reductase and its relationship to the corresponding protein glycine reductase and sarcosine reductase from Eubacterium acidaminiphilum. Development of simple complementation assay systems for the substrate-specific proteins P[sub B]; Labile viologen-dependent formate dehydrogenase.

A mechanism for biological nitrogen fixation — Evidence for this proposal — A mechanism for biological nitrogen fixation — Conclusions. [ABSTRACT FROM AUTHOR]

The kinetic aspects of the reduction process in cytochrome c3 from Desulfovibrio gigas have been investigated over a wide range of pH values ranging between pH 5.8 and pH 9.8. The data have been analyzed in the framework of an I2H4 interaction network coupled to a proton-linked equilibrium between two tertiary structures (Cornish­Bowden, A. & Koshland, D. E. Jr (1970) J. Biol. Chem. 245, 6241–6250). The kinetic rate constants for the reduction of the four hemes for the two tertiary conformations have been characterized in the framework of the thermodynamic network obtained from the equilibrium analysis (Coletta, M., Catarino, T., LeGall, J. J. & Xavier, A. V. (1991) Eur. J. Biochem. 202, 1101–1106). The intrinsic reduction rate constants determined by reaction with sodium dithionite for two hemes (namely heme 4 and heme 1) are significantly faster than those for the other two heme residues. In view of the equilibrium redox properties, heme 4 (with the fastest reduction rate) may then work as the kinetic electron-capturing site for the electrons from sodium dithionite. The transfer to hemes 2 and 3 then occurs by virtue of their free-energy levels at equilibrium. At our experimental conditions, there is also transfer of electrons to hemes 2 and 3 from heme 1, which is reduced at a slower rate than heme 4, thus contributing to the biphasic kinetics observed for the overall process. The kinetic parameters obtained are discussed in terms of the mechanism proposed for the coupling between the electron and proton transfer, as induced by the heme/heme cooperativity network. [ABSTRACT FROM AUTHOR]

Putidamonooxin, a conjugated iron-sulfur protein, functions as the oxygen-activating component within the 4-methoxybenzoate monooxygenase from Pseudomonas putida. A [2Fe-2S] cluster and a mononuclear non-heme iron together form the oxygen-activating units within the active sites of putidamonooxin. In the present paper the reduction kinetics of oxidized putidamonooxin were studied in the absence and in the presence of a substrate and/or the mononuclear non-heme iron. The reducing agents used were (a) NADH plus the NADH-putidamonooxin oxidoreductase and (b) artificial reductants, i.e. O2- radicals and sodium dithionite in the presence or in the absence of the NADH-putidamonooxin oxidoreductase. The reduction kinetics of cofactor-iron-depleted putidamonooxin revealed that the addition of a substrate strongly enhanced the rate of reduction of the [2Fe-2S] chromophores by the native reductase but significantly decreased the reduction by artificial reductants. These effects were even more pronounced when the mononuclear non-heme iron was bound to putidamonooxin in addition to the substrate. The presence of the native reductase markedly enhanced the rate of reduction of native putidamonooxin by sodium dithionite, though not to the optimal rate as measured in the presence of both NADH and the NADH- putidamonooxin oxidoreductase. The experimental findings are taken to show that the reduction of oxidized putidamonooxin in the absence and in the presence of its mononuclear non-heme iron, when NADH-putidamonooxin oxidoreductase and artificial reductants are used as electron donors, may occur at two different sites, both of which are influenced by substrate binding. The most powerful reductant of putidamonooxin is the NADH-putidamonooxin oxidoreductase in its partially reduced state (which is characterized by its broad absorption band centered near 600 nm and its typical isotropic electron-paramagnetic-resonance signal with g = 2.0033), rather than in its fully reduced state. [ABSTRACT FROM AUTHOR]

The adenovirus type 2 hexon protein produced in excess during infection was carboxymethylated by reduction and alkylation with iodo[2-14C]acetate in 6 M guanidine hydrochloride. Only one type of polypeptide chain was detected by exclusion chromatography in dissociating buffers. Peptide mapping experiments, sequence analysis of peptides and identification of possible protein terminal residues also suggest that all subunits are similar and probably identical. Structural analysis of [14C]carboxymethylated hexon and of hexon which was labeled in vivo with [35S]cysteine revealed six unique cysteine derivatives in soluble tryptic peptides. A seventh unique cysteine derivative, only partly carboxymethylated, was present in the tryptic core material and was recovered after chymotryptic digestion. Seven unique cysteine derivatives in the hexon subunit, correspond to a minimum molecular weight of about 100 000 for the subunit. This value is consistent with values obtained from exclusion chromatography in guanidine hydrochloride, from mercurial titration of an accessible thiol group in the intact protein and from peptide mapping experiments. Peptides corresponding to more than one tenth of the total number of residues in the protein chain were characterized and, with the inclusion of other peptides studied, about one quarter of all residues are accounted for. The results exclude extensive regions of identity in primary structure within the hexon subunits and the partial resistance towards attack by proteolytic enzymes is structurally explained. [ABSTRACT FROM AUTHOR]

The kinetic aspects of the reduction process in cytochrome c3 from Desulfovibrio gigas have been investigated over a wide range of pH values ranging between pH 5.8 and pH 9.8. The data have been analyzed in the framework of an I2H4 interaction network coupled to a proton-linked equilibrium between two tertiary structures (Cornish­Bowden, A. & Koshland, D. E. Jr (1970) J. Biol. Chem. 245, 6241–6250). The kinetic rate constants for the reduction of the four hemes for the two tertiary conformations have been characterized in the framework of the thermodynamic network obtained from the equilibrium analysis (Coletta, M., Catarino, T., LeGall, J. J. & Xavier, A. V. (1991) Eur. J. Biochem. 202, 1101–1106). The intrinsic reduction rate constants determined by reaction with sodium dithionite for two hemes (namely heme 4 and heme 1) are significantly faster than those for the other two heme residues. In view of the equilibrium redox properties, heme 4 (with the fastest reduction rate) may then work as the kinetic electron-capturing site for the electrons from sodium dithionite. The transfer to hemes 2 and 3 then occurs by virtue of their free-energy levels at equilibrium. At our experimental conditions, there is also transfer of electrons to hemes 2 and 3 from heme 1, which is reduced at a slower rate than heme 4, thus contributing to the biphasic kinetics observed for the overall process. The kinetic parameters obtained are discussed in terms of the mechanism proposed for the coupling between the electron and proton transfer, as induced by the heme/heme cooperativity network. [ABSTRACT FROM AUTHOR]