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The soluble, cytoplasmic NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha is a heterotetrameric enzyme (HoxFUYH) and contains two FMN groups. The purified oxidized enzyme is inactive in the H2-NAD+ reaction, but can be activated by catalytic amounts of NADH. It was discovered that one of the FMN groups (FMN-a) is selectively released upon prolonged reduction of the enzyme with NADH. During this process, the enzyme maintained its tetrameric form, with one FMN group (FMN-b) firmly bound, but it lost its physiological activity – the reduction of NAD+ by H2. This activity could be reconstituted by the addition of excess FMN to the reduced enzyme. The rate of reduction of benzyl viologen by H2 was not dependent on the presence of FMN-a. Enzyme devoid of FMN-a could not be activated by NADH. As NADH-dehydrogenase activity was not dependent on the presence of FMN-a, and because FMN-b did not dissociate from the reduced enzyme, we conclude that FMN-b is functional in the NADH-dehydrogenase activity catalyzed by the HoxFU dimer. The possible function of FMN-a as a hydride acceptor in the hydrogenase reaction catalyzed by the HoxHY dimer is discussed. [ABSTRACT FROM AUTHOR]

Human alcohol dehydrogenase 3/glutathione-dependent formaldehyde dehydrogenase was shown to rapidly and irreversibly catalyse the reductive breakdown of S -nitrosoglutathione. The steady-state kinetics of S -nitrosoglutathione reduction was studied for the wild-type and two mutated forms of human alcohol dehydrogenase 3, mutations that have previously been shown to affect the oxidative efficiency for the substrate S -hydroxymethylglutathione. Wild-type enzyme readily reduces S -nitrosoglutathione with a k cat /K m approximately twice the k cat /K m for S -hydroxymethylglutathione oxidation, resulting in the highest catalytic efficiency yet identified for a human alcohol dehydrogenase. In a similar manner as for S -hydroxymethylglutathione oxidation, the catalytic efficiency of S -nitrosoglutathione reduction was significantly decreased by replacement of Arg115 by Ser or Lys, supporting similar substrate binding. NADH was by far a better coenzyme than NADPH, something that previously has been suggested to prevent reductive reactions catalysed by alcohol dehydrogenases through the low cytolsolic NADH/NAD+ ratio. However, the major products of S -nitrosoglutathione reduction were identified by electrospray tandem mass spectrometry as glutathione sulfinamide and oxidized glutathione neither of which, in their purified form, served as substrate or inhibitor for the enzyme. Hence, the reaction products are not substrates for alcohol dehydrogenase 3 and the overall reaction is therefore irreversible. We propose that alcohol dehydrogenase 3 catalysed S -nitrosoglutathione reduction is of physiological relevance in the metabolism of NO in humans. [ABSTRACT FROM AUTHOR]

The enzyme catalysing the reduction of adenosine 5′-phosphosulfate (AdoPS) to sulfite in higher plants, AdoPS reductase, is considered to be the key enzyme of assimilatory sulfate reduction. In order to address its reaction mechanism, the APR2 isoform of this enzyme from Arabidopsis thaliana was overexpressed in Escherichia coli and purified to homogeneity. Incubation of the enzyme with [35S]AdoPS at 4 °C resulted in radioactive labelling of the protein. Analysis of APR2 tryptic peptides revealed 35SO2–3 bound to Cys248, the only Cys conserved between AdoPS and prokaryotic phosphoadenosine 5′-phosphosulfate reductases. Consistent with this result, radioactivity could be released from the protein by incubation with thiols, inorganic sulfide and sulfite. The intermediate remained stable, however, after incubation with sulfate, oxidized glutathione or AdoPS. Because truncated APR2, missing the thioredoxin-like C-terminal part, could be labelled even at 37 °C, and because this intermediate was more stable than the complete protein, we conclude that the thioredoxin-like domain was required to release the bound SO2–3 from the intermediate. Taken together, these results demonstrate for the first time the binding of 35SO2–3 from [35S]AdoPS to AdoPS reductase and its subsequent release, and thus contribute to our understanding of the molecular mechanism of AdoPS reduction in plants. [ABSTRACT FROM AUTHOR]

Investigates the changes in the proton-motive force in Escherichia coli in response to external oxidoreduction potential (ORP). Reduction of proton-motive force under decreasing ORP conditions; Factor affecting the hydrogen-ion concentration decrease; Effect of low ORP values on Escherichia coli.

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]

The oxidation of protein sulphydryls to disulphides and their reduction back to sulphydryls is an early cellular response to oxidative stress. Hydrogen-peroxide-mediated oxidation of class-pi glutathione S-transferase results in the formation of disulphide bonds, which inhibits its catalytic function yet allows it to retain its non-substrate-ligand-binding properties. The overall hydrodynamic volume of the oxidised claa-pi glutathione S-transferase type-1 homodimer (GSTP1-1) is decreased, and its tertiary-structural differences appear to be prominent in domain 1 of oxidized GSTP1-1, in that the exposure of both tryptophan residues is increased, while the electric potential about one of them is altered. Treatment of the oxidized protein with dithiothreitol or glutathione restores its enzymatic capabilities, albeit with lower specific activities for 1-chloro-2, 4-dinitrobenzene and ethacrynic acid. The hydrophobic binding site (H-site) for electrophilic substrates is negatively affected in that the Km and catalytic-efficiency values are diminished significantly with respect to those values obtained for the native protein. The dithiothreitol-treated oxidized GSTP1-1 is able to regain its overall hydrodynamic volume; however, both its secondary-structural and tertiary-structural elements remain modified with respect to the native protein, as do both tryptophanyl environments. Furthermore, the oxidized glutathione S-transferase and dithiothreitol-treated oxidized glutathione S-transferase are less thermostable (tm - 55.5°C and 56.3°C, respectively) than the native enzyme (tm 59°C). These results indicate that the class-pi glutathione S-transferase is unable to regain its native conformation after oxidative inactivation. [ABSTRACT FROM AUTHOR]

Ferredoxin I in spinach chloroplasts fulfils the role of distributing electrons of low redox potential produced by photosystem I to several metabolic routes. NADP+ reduction being the major output. To investigate the role of Glu92, which is conserved in the chloroplast-type ferredoxins, mutations of this residue to either Gin, Ala or Lys were obtained through site-directed mutagenesis. A Glu93 Ala mutant was also designed. The four mutants of ferredoxin I were overproduced in Escherichia coil, purified and characterised. The different migration in nondenaturing gel electrophoresis of wild-type and mutant proteins confirmed that the desired mutation was present in the expressed Proteins. Spectral and physical properties of the mutants were similar to those of wild-type ferredoxin; electron-transfer properties were. however, quite different in the case of the mutants at position 92. Unexpectedly. these mutant ferredoxins were found to be twice as active as the wild-type protein in supporting the NADPH-cytochrome c reductase reaction cutalysed by ferredoxin-NADP+ reductase. However, interactions of the mutant ferredoxins with the isolated thylakoid membranes deprived of endogenous ferredoxin showed that the mutants were less capable of supporting NADP+ photoreduction than the wild-type protein: both V arid the apparent Km for reduced ferredoxin were influenced. On the other hand, the Kd values for the complex between oxidised ferredoxin and the reductase. measured at low ionic strength. were substantially changed only in the case of the Glu → Lys mutation. With this mutant the rate of cross-linking between the two proteins induced by a carbodiimide was also decreased. It was found that the redox potentials of the iron-sulfur cluster of the mutants were more positive by 73-93 mV than that of ferredoxin I [Aliverti. A., Hagen. W. R. & Zanetti, G. (1995) FEBS Lett. 368. 220-2241. Thus, the behavior of the ferredoxin mutants can he rationalised in terms of the effect of the side-chain replacement on the electrochemical properties of the [2Fe-2S] cluster and of an impairment in the interaction with the reductase under physiological conditions. [ABSTRACT FROM AUTHOR]

Anoxic metabolism of many aromatic compounds proceeds via the common intermediate benzoyl-CoA. Benzoyl-CoA is dearomatized by benzoyl-CoA reductase (dearomatizing) in a two-electron reduction step, possibly yielding cychlohex-1,5-diene-1-carboxyl-CoA. This process has to overcome a high activation energy and is considered a biological Birch reduction. The central, aromatic-ring-reducing enzyme was investigated for the first time in the denitrifying bacterium Thauera aromatica strain K172. A spectrophotometric assay was developed which was strictly dependent on MgATP, both with cell extract and with purified enzyme. The oxygen-sensitive new enzyme was purified 35-fold with 20% yield under anaerobic conditions in the presence of 0.25 mM dithionite. It had a native molecular mass of approximately 170 kDa and consisted of four subunits a,b,c,d of 48, 45, 38 and 32 kDa. The oligomer composition of the protein most likely is abcd. The ultraviolet/visible spectrum of the protein as isolated, but without dithionite, was characteristic for an iron-sulfur protein with an absorption maximum at 279 nm and a broad shoulder at 390 nm. The estimated molar absorption coefficient at 390 nm was 35 000 M-1 cm-1. Reduction of the enzyme by dithionite resulted in a decrease of absorbance at 390 nm, and the colour turned from greenish-brown to red-brown. The enzyme contained 10.8±1.5 mol Fe and 10.5±1.5 mol acid-labile sulfur/mol. Besides zinc (0.5 mol/mol protein) no other metals nor selenium could be detected in significant amounts. The enzyme preparation contained a flavin-like compound; the estimated content was 0.3 mol/mol enzyme. The enzyme reaction required MgATP and a strong reductant such as Ti(III). The reaction catalyzed is: benzoyl-CoA + 2 Ti(III) + n ATP → non-aromatic acyl-CoA + 2 Ti(IV) + n ADP + n Pi. The estimated number n of ATP molecules hydrolyzed/ two electrons transferred in benzoyl-CoA reduction is 2–4. In the absence of benzoyl-CoA the enzyme exhibited oxygen-sensitive ATPase activity. The enzyme was specific for Mg2+-ATP, other nucleoside triphosphates being inactive (<1%). Mg2+ could be substituted to some extent by Mn2+, Fe2+ and less efficiently by Co2+. Benzoate was not reduced, whereas sone fluoro, hydroxy, amino and methyl analogues of the activated benzoic acid were reduced, albeit at much lower rate; the products remain to be identified. The specific activity with reduced methyl viologen as the electron donor was 0.55 μmol min-1 mg-1 corresponding to a catalytic number of 1.6 s-1. The apparent Km values under the assay conditions (0.5 mM for both reduced and oxidized methyl viologen) of benzoyl-CoA and ATP were 15 μM and 0.6 mM, respectively. The enzyme was inactivated by ethylene, bipyridyl and, in higher concentrations, by acetylene. Benzoyl-CoA reductase also catalyzed the ATP-dependent two-electro reduction of hydroxylamine (Km 0.15 mM) and azide. Some of the properties lof the enzyme are reminiscent of those of nitrogenase which similarly overcomes the high activation energy for dinitrogen reduction by coupling electron transfer to the hydrolysis of ATP. [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.

The active form of protein R2, the small subunit of ribonucleotide reductase, contains a diferric center and a free radical localized at Tyr122. Hydroxyurea scavenges this radical but leaves the iron center intact. the resulting metR2 protein is inactive. The introduction of a radical into metR2 is dependent on the reduction of the iron center. In Escherichia coli, this is achieved by an enzyme system consisting of a NAD(P)H:flavin oxidoreductase and a poorly defined protein fraction, fraction b. Assuming that the iron center is deeply buried within the protein, electron transfer is suggested to occur over long distances. Site-directed mutagenesis allowed us to identify two invariant residues. Tyr356 at the C-terminal part of the protein and Tyr122 located 0.5 nm away from the closest iron atom, as mediators of this electron transfer. We also found that deazaflavins were excellent catalysts in the photoreduction of the iron center of meR2 and generation of the tyrosyl radical, providing the simplest and most efficient model for the physiological flavin reductase/fraction b activating system. The properties of the model reaction are described. [ABSTRACT FROM AUTHOR]

A preparation of prolamellar bodies from wheat etioplasts was used as a source for NADPH-protochiorophyllide oxidoreductase (pchlide reductase). The enzyme was solubilized with Triton X-100 after reduction of the endogenous photoconvertilale protochlorophyllide a to chlorophyllide a by saturating illumination. Protochlorophylls a and b, protochlorophyllide a and zinc protopheophorbide b were added to the soluble enzyme preparation to determine if they were reduced in the dark or in the light. None of the compounds were reduced (with NADPH) in the dark; however, light-dependent reduction was demonstrated with protochlorophyllide a and zinc protopheophorbide b. The yield was approximately 50% for both substrates. Photoreduction did not occur with the esterified protochlorophylls a and b. Photoreduction of zinc protopheophorbide b, the zinc analogue of protochlorophyllide b, is the first demonstration of the reduction of a chlorophyll-b-related compound by pchlide reductase. [ABSTRACT FROM AUTHOR]

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

Coupled rat liver mitochondria were incubated with [U-14C]hexadecanoate and carnitine which resulted in the formation of acyl-, 2-enoyl- and 3-hydroxyacyl-CoA and carnitine esters. The production of 2-enoyl-CoA and 3-hydroxyacyl-CoA esters was associated with a significant lowering of the NAD+/NADH ratio, in contrast to rat muscle mitochondria [Eaton, S., Bhuiyan, A. K. M. J., Kler, R. S., Turnbull, D. M. & Bartlett, K. (1993) Biochem. J. 289, 161-172], suggesting that control by the respiratory chain is important under normal conditions. When NAD+/NADH ratios were held low by succinate-induced reverse electron flow, 3-enoyl-CoA esters were also detected, probably formed by the action of 3,2-enoyl-CoA isomerase. Measurement of the flux of β-oxidation at different osmolalities showed that flux was strongly dependent on osmolality changes in the physiological range. Measurement of the CoA and carnitine esters resulting from incubators made at different osmolalities showed that there was an increase in the amounts of the saturated acyl-CoA esters with respect to 2-enoyl-CoA and 3-hydroxyacyl-CoA esters, consistent with control by the electron-transfer flavoprotein-ubiquinone segment {Halestrap, A. P. & Dunlop J. L. (1986) Biochem. J. 239, 559-565]. This however could not be the only factor operating as indicated by the continued presence of 2-enoyl-CoA and 3-hydroxyacyl-CoA esters at high osmolalities. [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]

A new method, faster, milder and more efficient than the one previously described [Bryn, K., Hetland, O. & Stormer, F. C. (1971) Eur. J. Biochem. 18, 116-119], for purification of diacetyl(acetoin) reductase from Enterobacter aerogenes is proposed. The experiments carried out with the electrophoretically pure preparations obtained by this procedure show that the enzyme (a) produces t-glycols from the corresponding L-αhydroxycarbonyls by reversible reduction of their oxo groups and also reduces the oxo group of uncharged αdicarbonyls converting them into L-α-hydroxycarbonyls, and (b) is specific for NAD. This is a new enzyme for which we suggest the systematic name of L-glycol:NAD+ oxidoreductase and the recommended name of L-glycol dehydrogenase(NAD). The molecular mass, pI, affinity for substrates and pH profiles of this enzyme are also described. [ABSTRACT FROM AUTHOR]

In order to compare the oxidation and reduction reactions of c-type cytochromes (cytochromes c552 from the green alga Monoraphidium braunii and horse heart cytochrome c) by different flavins (lumiflavins, riboflavin and FMN), laser flash photolysis studies have been carried out using either reduced or oxidized protein in the presence of triplet or semiquinone flavin, respectively. The reaction kinetics clearly demonstrate the cytochrome oxidation is mediated by the flavin triplet state. The rate constants for reduction are 20-100 times smaller than those for oxidation, indicating that the triplet state is a more effective reactant than is the semiquinone. This is attributed to its excited state nature and correspondingly high free energy content. The rate constants for both the reduction and oxidation of cytochrome c552 by riboflavin are significantly smaller than those obtained with lumiflavin, suggesting a steric interference of the ribityl side chain in the flavin &mdah; cytochrome interaction. The comparison between oxidation and reduction indicates that the former process is less affected by steric hindrance than the latter. Both reduction and oxidation of cytochrome c552 by FMN show an ionic strength dependence with the same sign, consistent with a negatively charged reaction site on the cytochrome. The magnitude of the electrostatic effect is slightly smaller for reduction than it is for oxidation. A pattern quite similar to that observed with cytochrome c552 was obtained when parallel experiments were carried out with horse cytochrome c, although differences were observed in the steric and electrostatic properties of the electron transfer site(s) in these two cytochromes. These results suggest that the same or closely adjacent sites on the proteins are involved in the oxidation and reduction reactions. The biochemical implications of this are discussed. [ABSTRACT FROM AUTHOR]

Hydroxypyruvate reductase of a serine-producing methylotroph, Hyphomicrobium methylovorum GM2, was purified to complete homogeneity, crystallized and characterized, the first time for an enzyme from a methylotroph. The enzyme was found to be a dimer composed of identical subunits (38 kDa). the molecular mass of the enzyme being about 70 kDa. The enzyme was stable against heating at 25 C for 10 min at pH values between 5 and 9. Optimal activity was observed at pH 6.8 and around 45 C. The enzyme catalyzed the reduction of hydroxypyruvate with the oxidation of only NADH. Other than hydroxypyruvate. only glyoxylate served as a substrate. The Km values were found to be 0.175 mM for hydroxypyruvate and 10.8 mM for glyoxylate. Taking advantage of the high substrate specificity of this enzyme, a means of enzymatic determination of hydroxypyruvate was established. [ABSTRACT FROM AUTHOR]

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes: it was purified 219 times with a yield of about 11%. It is an Mr-230000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis. [ABSTRACT FROM AUTHOR]

The specificity of laminin chain assembly was investigated using fragments E8 and C8-9, derived from the long arm of the molecule, whose rod-like domain consists of the α-helical regions of the A. B1 and B2 chains. Urea-induced chain separation and unfolding were monitored by transverse urea/polyacrylamide gel electrophoresis (PAGE) and circular dichroism. Separation of the A and disulphide-linked B1-B2 chains occurred at 3.5-4.0 M urea and by 7.0 M urea all residual α-helicity was lost. Removal of urea by dialysis resulted in high recoveries (87-100%) of renatured protein which in its apparent molecular mass, α-helix content, chain composition, degree of association and ultrastructural appearance was indistinguishable from native E8. Reduction or reduction and alkylation of the chains did not lead to a decrease in their ability to reassemble specifically. Reformation of the single interchain disulphide, linking the B1 and B2 chains, clearly demonstrates that these chains are correctly aligned in parallel and in register in E8 renatured from its reduced chains. Renaturation of E8 from its reduced and alkylated chains precludes a role for disulphide formation in determining chain alignment but suggests rather that it is involved in the stabilisation of the correctly assembled molecule. These results, together with recent sequence data, provide evidence for the interaction of the α-helical regions of the A, B1 and B2 chains in the formation of a triple coiled-coil within the long arm of the molecule. The highly specific nature of this interaction suggests that it is the mechanism by which laminin is assembled in vivo. [ABSTRACT FROM AUTHOR]

The 8-hydroxy-5-deazaflavin (coenzyme F420)-reducing and methyl- viologen-reducing hydrogenase of the anaerobic methanogenic archaebacterium Methanosarcina barkeri strain Fusaro has been purified 64-fold to apparent electrophoretic homogeneity. The purified enzyme had a final specific activity of 11.5 mumol coenzyme F420 reduced ∙ min-1 ∙ mg protein-1 and the yield was 4.8% of the initial deazaflavin-reducing activity. The hydrogenase exists in two forms with molecular masses of approximately 845 kDa and 198 kDa. Both forms reduce coenzyme F420 and methyl viologen and are apparently composed of the same three subunits with molecular masses of 48 kDa (α), 33 kDa (β) and 30 kDa (γ). The aerobically purified enzyme was catalytically inactive. Conditions for anaerobic reductive activation in the presence of hydrogen, 2-mercaptoethanol and KCl or methyl viologen were found to yield maximal hydrogenase activity. Determination of the apparent Km of coenzyme F420 and methyl viologen gave values of 25 μM and 3.3 mM, respectively. The respective turnover numbers of the high molecular mass form of the hydrogenase are 353 s-1 and 9226 s-1. [ABSTRACT FROM AUTHOR]

Examines the reduction of lactoperoxidase with sodium dithionite by means of stopped-flow spectrophotometry in an aerobic system. Pseudo-first-order conditions; Influence of ionic strength and lower pH; Existence of a carboxyl group which is essential to the different enzymatic functions of lactoperoxidase.

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]

Studies reduced photosystem I samples, which give the electron paramagnetic resonance signals associated with A, A and B, and A, B and X centers from the Cyanobacterium Chlorogloea fritschii. Use of Mossbauer spectroscopy; Degree of reduction.

Describes the purification of an NAD-linked acetoacetyl-CoA reductase of Zoogloea ramigera to electrophoretic homogeneity. Stereospecificity; Reduction-oxidation reaction; Culture of organism.

The preparation of model compounds 1-(2′,6′-dichlorobenzyl)-3-halogenopyridinium and the study of their properties were achieved. Their chemical reduction to the corresponding 1,4-dihydropyridines is proved by spectroscopic analysis. 3-Iodopyridine—adenine dinucleotide was prepared by enzymic transglycosidation while the 3-chloro, 3-bromo and 3-iodo pyridine—adenine dinucleotides were synthesized from 3-amino-pyridine-adenine dinucleotide. The 3-halogenopyridine-adenine dinucleotides were proved to be active as hydrogen acceptors with alcohol as a substrate. The absorption band at 290 nm of cinnamaldehyde appeared to be a very sensitive tool for studying the enzymic reaction. With the alcohol dehydrogenase from yeast, only slight activity was detected. 3-Halogenopyridine—adenine dinucleotides are competitive inhibitors with respect to nicotinamide—adenine dinucleotide with alcohol dehydrogenase from yeast, lactate dehydrogenase and malate dehydrogenase. The use of 3-iodopyridine—adenine dinucleotide as a heavy-atom derivative for X-ray structure determination is proposed. [ABSTRACT FROM AUTHOR]

(Adenine-14C) or (γ-32P)-labelled 2,2'[1-(9-adenyl)-1'-(tri-, diphosphoryl-oxymethyl)]-dihydroxydiethylether (rroANP) was obtained from AArp by cleavage of the C-2'-C-3' bond by sodium periodate oxidation and subsequent borohydride reduction. Binding of rroANP to rat liver mitochondria revealed carrier-linked (atractyloside-sensitive) and unspecific (atractyloside-insensitive) binding but no transfer across the inner mitochondrial membrane. Kinetic data indicate rroANP as a competitive inhibitor for ANP uptake with Ki = 9.3 × 10-5 M. Experimental rroANP confirmed that an intact adenine base and three anionic charges of the phosphate chain are essential for the recognition between ANP-carrier and nucleotide but insufficient for the induction of a transmembrane ANP exchange. In addition mobilisation of the carrier-nucleotide complex requires an intact ribofuranoside ring system. [ABSTRACT FROM AUTHOR]

1. In the presence of KCN and a saturaring concentration of antimycin the reduction of the b-type cytochromes in submitochondrial particles is biphasic. This phenomenon was explained by suggesting the existence of two kinetic forms of cytochrome b : bA-the active form which was reduced in the rapid phase, and bs - the sluggish form which was reduced in the slow phase. The ratio between these forms and the transformation from one to other was controlled by the red ox state of an unknown component, named "Y", located between cytochromes b and c1. Pre-treatment with ascorbate plus N,N,N¹,N¹-tetramethyl-p-phenylenediamine transforms all the b-type cytochromes to their sluggish form, and the reduction by succinate follows slow monophasic kinetics. The name "dynamic control mechanism" was given to this mechanism [Eisenbach, M. & Gutman, M. (1975) Eur. J. Blochem. 52, 107-116]. 2. Increasing concentrations of antimycin (0-2 nmol/mg) in the presence of KCN increased the fraction of the rapid phase of the reduction but did not affect the calculated absolute rates of the reduction. It is concluded that antimycin delays the reduction of "Y" and thus permits the observation of the biphasic phenomenon, but that it is not essential for the operation of this dynamic control mechanism. 3. Substituting antimycin with 2-heptyl-4-hydroxyquinoline N-oxide (HpHOQnO) as an inhibitor, did not change significantly the pattern of the kinetics; i.e. the reduction was still biphasic. Omission of KCN from the reaction mixture or pre-treatment with ascorbate plus tetramethyiphenylenediamine did not affect the kinetics of the reduction (in contrast to the experiments with antimycin as inhibitor). From these phenomena it is concluded that the inhibition sites of antimycin and HpHOQnO are not identical. Antimycin is located on the substrate side of "Y" and HpHOQnO is located on its oxygen side. This assumption is supported by measuring the effect of an oxidant, added in the presence of antimycin or HpHOQnO, on the rate of the reduction of cytochrome b. After aerobic reduction of submitochondrial particles by succinate in the presence of KCN, either antimycin or HpHOQnO was added, followed by ferricyanide. With antimycin, a rapid reduction was observed after the addition of the oxidant, followed by a slow reaction, In the presence of HpHOQnO, the addition of ferricyanide did not accelerate the reduction. On the contrary it induced a rapid oxidation of cytochrome b followed by a slow monophasic reduction. If the order of the additions was reversed, i.e. first K3Fe(CN)6 and then either antimycin or HpHOQnO, a biphasic reduction was observed in either case. 4. It is suggested, that a redox equilibrium exists between "Y" and cytochrome c1, and that this equilibrium is sensitive to HpHOQnO but not to antimycin. [ABSTRACT FROM AUTHOR]

Homogenates of dark-pretreated leaves yield two particulate fractions in density gradient centrifugation: one contains chlorophyll (chioroplasts) while a second fraction contains ribulose-1 ,5- bisphosphate carboxylase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase and catalase. Addition of a microbody-rich pellet to chloroplasts isolated from dark-pretreated plants largely enhances both oxygen evolution and CO2-fixation into organic compounds. The pathway of CO2 reduction may be part of a membrane system which, under suitable conditions, may separate from the chloroplast as a distinct cytoplasmic entity, having physical properties similar to those of microbodies. [ABSTRACT FROM AUTHOR]

Alkylation at N-1 of the NAD+ adenine ring with 3,4-epoxybutanoic acid, followed by chemical reduction to the alkali-stable NADH form and alkaline Dimroth rearrangement, gave the NADH derivative alkylated at the exocyclic adenine amino group. Enzymic reoxidation of the latter derivative gave nicotinamide-6-(2-hydroxy-3-carboxypropylamino)purine dinucleotide, a functionalized NAD+ analogue carrying an ω-carboxylakyl side-chain at the exocyclic adenine amino group. Carbodiimide coupling of the latter derivative to high-molecular-weight water-soluble (polyethyleneimine, polylysine) and insoluble (aminohexyl-Sepharose)polymers lave the corresponding macromolecularized NAD+ analogues. These derivatives have been shown to be enzymically reducible. The polyethyleneimine and polylysine analogues showed a substantial degree of efficiency relative to free NAD + with rabbit muscle lactate dehydrogenase (60 and 25% respectively) but a lower one with yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase (2-7 %). The polyethyleneimine derivative entrapped in cellulose triacetate fibres together with the lactate dehydrogenase was operationally stable during repetitive use. [ABSTRACT FROM AUTHOR]

Techniques for preparation of enzyme extracts from Chlorella pyrenoidosa chick are described which yield a stable system which converts adenosine 5'-phosphosulfate to sulfide and/or cysteine in the absence of added active thiols. Since these thiols (such as dithiothreitol) have been shown to yield sulfite, among other products, from the adenosine-5'-phosphosulfate sulfotransferase reaction, we have studied the adenosine 5'-phosphusulfate reducing system in the presence and absence of dithiothreitol. In all cases dithiothreitol increases the activity of the reducing system forming cysteine from adenosine 5'-phosphosulfate. Mutant Sat-2, which cannot grow on sulfate, is shown to lack thiosulfonate reductase activity but to retain sulfite reductase activity. Extracts from this mutant will form sulfide and cysteine only in the presence of dithiothreitol, while wild-type extracts will form these products in presence or absence of dithiothreitol, the activity with dithiothreitol being higher. Mutants Sat-1,3-6 which are blocked in the adenosine-5'-phosphosulfate sulfotransferase step are blocked in the formation of sulfide and cysteine from adenosine 5'-phosphosulfate. Binding studies with adenosine 5'-phosphosulfate indicate that the sulfonyl group is transferred to a carrier to form carrier-S-SO-3 and the available evidence suggests that this carrier is a prosthetic group of the thiosulfonate reductase. On the basis of this evidence, two pathways in the cell-free extracts are thought to be operating. One, in the absence of active thiols, is a pathway consisting of bound intermediates in which the sulfonyl group of adenosine 5'-phosphosulfate is transferred via adenosine-5'-phosphosulfate sulfotransferase to carrier on the thiosulfonate reductase where it is reduced to the thiol level by ferredoxin; the thiol is then transferred via O-acetyl-L-serine sulfhydrylase to form cysteine. The other, or free intermediate pathway operates in the presence of dithiothreitol which releases free sulfite from the adenosine-5'-phosphosulfate sulfotransferase reaction. This sulfite is then reduced via sulfite reductase to sulfide which forms cysteine via O-acetyl-L-serine sulfhydrylase. Since mutant Sat-2, which cannot grow on sulfate nor reduce it in vivo, lacks thiosulfonate reductase activity but yields sulfite reductase activity in extracts, we conclude that the bound intermediate pathway is the preferred pathway of sulfate reduction in vivo; the free intermediate pathway is probably operative only when free sulfite is available either from occasional side reactions in vivo or when sulfite is fed to cells from outside. [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]

Crystalline cortisone reductase prepared from Streptomyces hydrogenans has 3α- and 20βhydroxysteroid: NAD oxidoreductase activity. The total rate of reaction for mixtures of I7α,21dihydroxypregn-4-ene-3,11,20-trione with 5α-androstan-3,16-dione, and of 17β-hydroxy-lαmethyl-5α-androstan-3- one with either of these compounds indicated that these substrates do not react independently. 3β-Hydroxy-5α-androstan-16-one inhibited the reduction of both 17α,21dihydroxypregn-4-ene-3,11,20-trione and 5α-androstane-3,16-dione. 20β-Hydroxypregn-4-en-3-one also inhibited the reduction of both these substrates. The findings allow the view that the 3α- and 20β-activities may depend upon a aingle active centre with complex binding characteristics. [ABSTRACT FROM AUTHOR]

The electron paramagnetic resonance spectrum of 17O in O2- generated by reduction of O2 by two related systems has been observed using the rapid freezing method. The systems were: (a) during re-oxidation of xanthine oxidase following reduction with dithionite and (b) during steady-state oxidation of xanthine catalysed by xanthine oxidase. The spectrum was similar to that reported for O2-, adsorbed on MgO. Both the 11-line spectrum kom 17O17O- and the six-line spectrum from 17O16O- were detected. The results provide final confirmation that one-electron reduction of oxygen can occur in biological systems. [ABSTRACT FROM AUTHOR]

Crystalline rabbit muscle lactate dehydrogenase has been shown to catalyze the oxidation of glyoxylate to oxalate and its reduction to glycollate. Both these reactions have been shown to be catalyzed by all the isoenzymes present. Kinetic constants for both these reactions have been calculated. Oxalate inhibition patterns suggest that glyoxylate may be acting in its hydrated form as a substance for oxidation and in its non-hydrated form as a substrate for reduction. [ABSTRACT FROM AUTHOR]

1. The anaerobic reduction of fungal laccase by quinol and ferrocyanide has been studied in a stopped-flow apparatus by measurements of the change in the absorbancy at 610 mμ associated with the reduction of the so-called Type 1 Cu2+. With ferrocyanide the rate of formation of product was also measured. 2. The reoxidation of reduced laccase by molecular oxygen has been studied by the same technique. 3. The kinetics of reduction and reoxidation as well as of product formation has also been investigated in a series of experiments with both the reducing substrate and oxygen present. In addition, the effect of an inhibitor, fluoride, on the kinetics has been studied. 4. The results confirm earlier static experiments showing that laccase contains two electron-accepting sites other than Type 1 and Type 2 Cu2+. Type 1 Cu2+ is reduced rapidly enough to involve it in the catalytic reaction. However, it appears to be rapidly reoxidized only when the other electron-accepting sites are also occupied. A mechanism involving the cooperation of several electron-accepting sites is consistent with the kinetic results. [ABSTRACT FROM AUTHOR]

The reduction of flavins by BH4- is slow, but strongly catalyzed by light. The main product of the reaction is 1 ,5-dihydroflavin, i. e. normal leucoflavin, which is rapidly reoxidized upon admission of air. Under continuous aeration, however, a second reaction, irreversible by oxygen in the dark, becomes dominant yielding a tetrahydroflavin which, upon exhaustive reaction of BH4-, is autoxidized to give a new dihydroflavin of structure I. This compound is stable toward further oxidation in the crystalline state and in solution with organic solvents. Compound I has to be considered as a formal 3,4-dihydroflavin. It is photooxidizable to give starting flavin, reacts very slowly with S2O4- compared to flavoquinone (II), but is easily reduced by EDTA in the presence of light. The compound was also synthesized by an unequivocal route and found identical with the product obtained by BH4--reduction of lumiflavin. The light absorption spectrum of I shows absorption maxima at about 405, 320, 270, and 220 nm and is practically identical with that of the BH4---reduction product of the flavoproteins D- and L-amino acid oxidase. The BH4--reduction products of the amino acid oxidases are catalytically active. From this it follows that position 4 of the flavocoenzyme is not involved in enzymatic dehydrogenation. The first photo-excited triplet state of I has been investigated by optical and electron paramagnetic resonance spectroscopy. The structure of I was elucidated by means of infrared and nuclear magnetic resonance spectroscopy. [ABSTRACT FROM AUTHOR]

17β-Hydroxy-5α-androstan-3-ones and related androstene compounds with structural modifications only in ring A were studied as substrates and inhibitors for the reduction of the 3-oxo group by cortisone reductase. Various structural modifications were consistent with substrate function, and several poor substrates and non-substrates served as inhibitors. It is suggested that the steroids fit loosely into a binding region which is probably hydrophobic around the A-ring. [ABSTRACT FROM AUTHOR]

1. NADH dehydrogenase from Azotobacter chroococcum was solubilised and purified to yield a single band on disc-gel electrophoresis. The molecular weight of the enzyme was approximately 450000 and it contained one subunit of molecular weight approximately 16000. 2. The enzyme transferred electrons from NADH to triphenyl tetrazolium chloride, benzyl viologen, potassium ferricyanide, NAD or horse-heart cytochrome c, and from NADPH to NAD or potassium ferricyanide. The pH optimum for benzyl viologen or triphenyl tetrazolium chloride was between pH 9 and 10, that for potassium ferricyanide or acetyl-pyridine-NAD between pH 6.5 and 8.0. There were two pH optima with cytochrome c: at 8.0 and 9.5. 3. The rate of reduction of tetrazolium salt or benzyl viologen by purified NADH dehydrogenase at pH 7.5 was not proportional to protein concentration; activity at low concentrations was restored by adding either azotoflavin, azotobacter ferredoxin or nitrogenase. 4. NADH-cytochrome-c-reductase activity was more heat-sensitive and more susceptible to inhibition by p-hydroxymercuribenzoate than were NADH-ferricyanide or NADH benzyl viologen reductases. NADH-ferricyanide reductase was inhibited by organic solvents and NADH benzyl viologen or tetrazolium reductase was inhibited by Cu2+ ions. These results are discussed in terms of separate active centres for the reductions. 5. NADH dehydrogenase coupled to nitrogenase to reduce acetylene in the presence of ATP, benzyl viologen and NADH. [ABSTRACT FROM AUTHOR]