Purification and some characteristics of nitrous oxide reductase from Paracoccus denitrificans

Nitrous oxide reductase from the denitrifying bacterium Paracoccus denitrificans has been purified very nearly to homogeneity by an anaerobic procedure that results in a product with high specific activity. The enzyme is a dimer of about Mr 144,000 composed of two subunits of apparently equal Mr and contains 4 mol of Cu per mol of subunit. The isoelectric point is 4.3; specific activity at 25 degrees C, pH 7.1, is 122 mumol X min-1 X mg of protein-1; and Km is about 7 microM N2O under the same conditions. The N2O- and O2-oxidized forms of the enzyme had principal absorption bands at 550 and 820 nm; the dithionite-reduced form, at 650 nm. The extinction coefficient at 550 nm for the oxidized enzyme is about 5300 (M subunit)-1 X cm-1. Ferricyanide-oxidized enzyme and enzyme exposed to O2 for a couple of days at 4 degrees C exhibited additional bands at 480, 620, and 780 nm and had very low specific activities. Cu-EPR signals were observed with oxidized and reduced forms of the enzyme with g perpendicular values at 2.042 and 2.055, respectively. The O2-oxidized enzyme had g parallel and A parallel values of about 2.244 and 35 gauss, respectively, based on the observation of four hyperfine lines in the g parallel region. The enzyme may therefore contain at least one Cu atom approximating the "Type 1" class. Spin counts against Cu-EDTA standards suggest that 20-30% of the enzyme-bound Cu is EPR detectable in the O2-oxidized enzyme and 7-15% in the enzyme as prepared and in the reduced enzyme. Much of the Cu thus appears to be EPR silent. Nitrous oxide reductase was observed to undergo turnover-dependent inactivation, and nitrite and fluoride among other anions were found to accelerate this process. In a number of characteristics, the enzyme resembles nitrous oxide reductase recently purified from Pseudomonas perfectomarina and Rhodopseudomonas sphaeroides, particularly the former. Some differences appear related to whether or not purification is carried out entirely under anaerobic conditions.     

The nitric oxide reductase of Paracoccus denitrificans.

The nitric oxide (NO) reductase activity of the cytoplasmic membrane of Paracoccus denitrificans can be solubilized in dodecyl maltoside with good retention of activity. The solubilized enzyme lacks NADH-dependent activity, but can be assayed with isoascorbate plus 2,3,5,6-tetramethylphenylene-1,4-diamine as electron donor and with horse heart cytochrome c as mediator. Reduction of NO was measured with an amperomeric electrode. The solubilized enzyme could be separated from other electron-transport components, including the cytochrome bc1 complex and nitrite reductase, by several steps of chromatography. The purified enzyme had a specific activity of 11 mumols.min-1.mg of protein-1 and the Km(NO) was estimated as less than 10 microM. The enzyme formed N2O from NO with the expected stoichiometry. These observations support the view that NO reductase is a discrete enzyme that participates in the denitrification process. The enzyme contained both b- and c-type haems. The former was associated with a polypeptide of apparent molecular mass 37 kDa and the latter with a polypeptide of 18 kDa. Polypeptides of 29 and 45 kDa were also identified in the purified protein which showed variable behaviour on electrophoresis in polyacrylamide gels.     

Partial purification and characterization of nitrous oxide reductase fromParacoccus denitrificans

We report the first partial purification of nitrous oxide reductase, a unique and labile enzyme of denitrifying bacteria. The procedure, which required anaerobic conditions throughout, resulted in a 60-fold purification relative to crude lysate in the case ofParococcus denitrificans. The molecular weight was estimated by gel exclusion chromatography to be about 85,000. The partially purified enzyme is inactivated rapidly by O₂, dithionite, and mercaptoethanol and is reversibly inhibited by moderate concentrations of common salts. Up to 80% of the original activity can be reconstituted following O₂ inactivation by incubating the enzyme with reduced benzyl viologen for 2 to 3 h. TheV _max pH profile shows a broad maximum at pH 8. The enzyme is irreversibly retained by common anion exchangers in the range pH 7 to 8 but can be eluted in acceptable yield as one of the last components from an imidazole-based anion exchange material by means of a pH gradient. This behavior implies that nitrous oxide reductase is very acidic. Among the several peptides observed by sodium dodecyl sulfate slab electrophoresis, only two, with apparent molecular weights of 58,000 and 25,000, correlated closely with the activity of fractions eluted from the imidazole column. These two peptides together comprised about 30% of the total protein in the fractions with highest specific activity.     

A pathway for protons in nitric oxide reductase from Paracoccus denitrificans

Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N(2)O (2NO+2e(-)+2H(+)-->N(2)O+H(2)O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O(2)-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O(2), we demonstrate that protons are indeed consumed from the 'outside'. First, multiple turnover reduction of O(2) resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O(2) shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O(2) as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O(2) show electron transfer coupled to proton uptake from outside the NOR-liposomes with a tau=15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Adelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.     

Kinetic analysis of substrate inhibition in nitric oxide reductase of Paracoccus denitrificans.

The current kinetic model for the nitric oxide reductase reaction (Girsch, P., and de Vries, S. (1997) Biochim. Biophys. Acta 1318, 202-216) does not involve the concentration of an electron donor. Here we introduce this variable and show, both theoretically and experimentally, its role in determining the extent of substrate inhibition by the excess of nitric oxide. NO is found to inhibit competitively with the electron donor, possibly by binding to the oxidized form of the enzyme. The observed partial character of the inhibition is tentatively explained by a slow reduction of the non-productive NO complex.     

MauE and MauD proteins are essential in methylamine metabolism of Paracoccus denitrificans

Synthesis of enzymes involved in methylamine oxidation via methylamine dehydrogenase (MADH) is encoded by genes present in the mau cluster. Here we describe the sequence of the mauE and mauD genes from Paracoccus denitrificans as well as some properties of mauE and mauD mutants of this organism. The amino acid sequences derived from the mauE and mauD genes showed high similarity with their counterparts in related methylotrophs. Secondary structure analyses of the amino acid sequences predicted that MauE is a membrane protein with five transmembrane-spanning helices and that MauD is a soluble protein with an N-terminal hydrophobic tail. Sequence comparison of MauD proteins from different organisms showed that these proteins have a conserved motif, Cys-Pro-Xaa-Cys, which is similar to a conserved motif found in periplasmic proteins that are involved in the biosynthesis of bacterial periplasmic enzymes containing haem c and/or disulphide bonds. The mauE and mauD mutant strains were unable to grow on methylamine but they grew well on other C₁-compounds. These mutants grown under MADH-inducing conditions contained normal levels of the natural electron acceptor amicyanin, but undetectable levels of the -subunit and low levels of the -subunit of MADH. It is proposed, therefore, that MauE and MauD are specifically involved in the processing, transport, and/or maturation of the -subunit and that the absence of each of these proteins leads to production of a non-functional -subunit which becomes rapidly degraded.     

The identification of a periplasmic nitrate reductase in Paracoccus denitrificans

Abstract A nitrate reductase activity has been identified in periplasmic extracts of Paracoccus denitrificans . The enzyme is relatively insensitive to azide and does not reduce chlorate, features which distinguish it from the well-characterised membrane-associated nitrate reductase. The specific activity of the enzyme was higher in intact cells grown with butyrate rather than succinate as the sole source of carbon.     

Nitric oxide oscillations in Paracoccus denitrificans: the effects of environmental factors and of segregating nitrite reductase and nitric oxide reductase into separate cells

Nitric oxide is a denitrification intermediate which is produced from nitrite and then further converted via nitrous oxide to nitrogen. Here, the effect of low concentrations of the protonophore carbonylcyanide m-chlorophenylhydrazone on the time courses for dissolved gases was examined. While NO was found to oscillate, N(2)O only increased gradually as the reduction of nitrite progressed. The frequency and shape of protonophore-induced NO oscillations were influenced by temperature and the concentration of electron donor N,N,N',N'-tetramethyl-p-phenylene diamine (TMPD) in a manner compatible with the observed differential effects on the two involved enzyme activities. We demonstrated the existence of a pH interval, where [NO] oscillates even without uncoupler addition. Occurrence of nitric oxide oscillations in mixtures of a nitrite reductase mutant with a nitric oxide reductase mutant suggests that they cannot be due to a competition of the enzymes for redox equivalents from one common respiratory chain.     

ESEEM studies of succinate:ubiquinone reductase from Paracoccus denitrificans

Electron spin-echo envelope modulation (ESEEM) spectroscopy has been performed in order to obtain structural information about the environment of the reduced [2Fe-2S] cluster (S-1 center), the oxidized [3Fe-4S] cluster (S-3 center), and the flavin semiquinone radical in purified succinate:ubiquinone reductase from Paracoccus denitrificans. Spectral simulations of the ESEEM data from the reduced [2Fe-2S] yielded nuclear quadrupole interaction parameters that are indicative of peptide nitrogens. We also observed a weak interaction between the oxidized [3Fe-4S] cluster and a peptide 14N. There was no evidence for coordination of any of the Fe atoms to 14N atoms of imidazole rings. The ESEEM data from the flavin semiquinone radical were more complicated. Here, evidence was obtained for interactions between the unpaired electron and only the two nitrogen atoms in the flavin ring.     

Some characteristics of a chromophoric lipid associated with nitric oxide reductase from Paracoccus denitrificans

A lipid with a UV chromophore (lambda max = 274 nm) was purified in small amounts from nitric oxide reductase of Paracoccus denitrificans and determined to have a molecular weight of 686, a most probable formula of C43H78O4N2 and about two phenylhydrazine-reactive carbonyls. On the basis of 1H and 13C-NMR, IR and mass spectrometric studies, the chromophoric lipid was inferred to have something like bilateral symmetry and to contain ketone-like carbonyls, alkene centers and at least three alkyl or olefinic chains. Several likely substructures are discussed and an enaminoketone is considered as a model for the chromophore. Evidence was lacking for phosphate, an aromatic ring, a 1,4-quinone, polyunsaturated fatty acyl groups, isoprenyl/isopranyl chains and simple alkyl acids, esters, amides and ethers. The compound appears to have some novel features. On the basis of reconstitution studies, the chromophoric lipid may be essential for nitric oxide reductase activity.     

The role of ubiquinone in linking nitrate reductase and cytochrome o to the respiratory chain of Paracoccus denitrificans

Three alternatives of the mode of branching in the ubiquinone-cytochrome b region of the anaerobic respiratory chain of Paracoccus denitrificans were experimentally tested. It was found that the view that the constitutive cytochrome b-560 or b-566 serves as an electron donor for the nitrate reductase is incompatible with the proposed scheme of the cyclic electron flow in the bc₁ segment. By means of the extraction procedure, the extent of reduction of ubiquinone was determined in cells utilizing oxygen and nitrate in the presence of antimycin. It was found that the redox response of ubiquinone was consistent with what had been predicted by the pool model of Kröger and Klingenberg, extended for more than one terminal acceptor. Our results are in support of the assumption that in cells of P. denitrificans ubiquinol (QH₂) has a function of an electron donor both for nitrate reductase and cytochrome o.     

Evidence for the role of soluble cytochrome c in the dissimilatory reduction of nitrite and nitrous oxide by cells of Paracoccus denitrificans.

The role of periplasmic cytochrome c in the denitrification pathway has been investigated using a wild-type and/or a cytochrome c deficient strain of Paracoccus denitrificans. The reconstitution experiments with the isolated proteins showed that bacterial cytochrome c-550 restored the electron transport from the cytoplasmic membrane to soluble nitrite reductase (cytochrome cd1). In response to decreased aeration lasting 3 h, the HUUG25 strain synthesized nitrous-oxide reductase significantly starved of electrons from the respiratory chain and only very small amounts of soluble cytochrome c. The membrane-bound part of the respiratory chain catalyzing the reduction of soluble cytochrome c resembled an autologous region in wild-type cells kinetically and by its sensitivity to antimycin. In the periplasmic fraction obtained from anaerobically grown wild-type cells N2O caused the reoxidation of endogenous cytochrome(s) c previously reduced by N,N,N',N' tetramethyl-p-phenylenediamine plus ascorbate. All these results indicate the involvement of soluble cytochrome(s) c as the electron donor(s) for the reduction of NO2- and N2O in the periplasmic space of cells.     

The copper site in nitrous oxide reductase

Summary The properties of the novel copper enzyme nitrous oxide reductase from denitrifyingPseudomonas stutzeri are described. Multifrequency electron paramagnetic resonance spectroscopy is used to characterize the various forms of the enzyme. The features observed at 2.4, 3.4, 4.5, 9.31 and 35 GHz are explained by a mixed-valence s[Cu(1.5)3. Cu(1.5)s]S=12 species with the unpaired electron delocalized between the two Cu nuclei. This site is also present in the catalytically inactive derivative of nitrous oxide reductase which was obtained from a transposon Tn5-induced mutant with defective chromophore biosynthesis. The resemblance of the low-frequency electron paramagnetic resonance spectra to the spectra for the so-called Cu_A of cytochromec oxidase can be taken as a first indication that the Cu_A may have a structural and electronic arrangement similar to the electron-paramagnetic-resonance-detectable copper in nitrous oxide reductase. Results from oxidation/reduction experiments, and from a quantitative determination of sulfhydryl and disulfide residues in the various forms of nitrous oxide reductase, suggest the involvement of the redox-couple cysteine/cystine in the structural organization of the active site of nitrous oxide reductase.     

The respiratory nitrate reductase from Paracoccus denitrificans. Molecular characterisation and kinetic properties

The respiratory nitrate reductase from Paracoccus denitrificans has been purified in the non-ionic detergent Nonidet P-40. The enzyme comprises three polypeptides, alpha, beta and gamma with estimated relative molecular masses of 127 000, 61 000 and 21 000. Duroquinol or reduced-viologen compounds acted as the reducing substrates. The nitrate reductase contained a b-type cytochrome that was reduced by duroquinol and oxidised by nitrate. A preparation of the enzyme that lacked both detectable b-type cytochrome and the gamma subunit was obtained from a trailing peak of nitrate reductase activity collected from a gel filtration column. Absence of the gamma subunit correlated with failure to use duroquinol as reductant; activity with reduced viologens was retained. It is concluded that in the plasma membrane of P. denitrificans the gamma subunit catalyses electron transfer to the alpha and beta subunits of nitrate reductase from ubiquinol which acts as a branch point in the respiratory chain. A new assay was introduced for both nitrate and quinol-nitrate oxidoreductase activity. Diaphorase was used to couple the oxidation of NADH to the production of duroquinol which acted as electron donor to nitrate reductase. Under anaerobic conditions absorbance changes at 340 nm were sensitive to nitrate concentrations in the low micromolar range. This coupled assay was used to determine that the purified enzyme had Km(NO-3) of 13 microM and a Km of 470 microM for ClO-3, an alternative substrate. With viologen substrates Km(NO-3) of 283 microM and Km(ClO-3) of 470 microM were determined; the enzymes possessed a considerably higher Vmax with either NO-3 or ClO-3 than was found when duroquinol was substrate. Azide was a competitive inhibitor of nitrate reduction in either assay system (Ki = 0.55 microM) but 2-n-heptyl-4-hydroxyquinoline N-oxide was effective only with the complete three-subunit enzyme and duroquinol as substrate, consistent with a site of action for this inhibitor on the b-type cytochrome. The low Km for nitrate observed in the duriquinol assay is comparable with the apparent Km(NO-3) recently reported for intact cells of P. denitrificans [Parsonage, D., Greenfield, A. J. & Ferguson, S. J. (1985) Biochim. Biophys. Acta 807, 81-95]. This similarity is discussed in terms of a possible requirement for a nitrate transport system. The nitrate reductase system from P. denitrificans is compared with that from Escherichia coli.     

Characterization of nitrous oxide reductase from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans A3151

A Cu-containing nitrous oxide reductase (HdN2OR) from a methylotrophic denitrifying bacterium, Hyphomicrobium denitrificans A3151, has been aerobically prepared and spectroscopically characterized. Purple and blue forms of HdN2OR have been isolated. Each form is a homodimer comprising monomers with a molecular mass of 65 kDa. The visible absorption spectrum of the purple form (designated as form A) exhibits three absorption bands at 480 nm, 540 nm, and 650 nm, with a shoulder near 780 nm, and that of the blue form (designated as form B) shows only one absorption band at 650 nm. Reversible spectral changes, between those of forms A and B, are observed on treatment of these forms with redox reagents. Forms A and B are oxidized and reduced forms, respectively. The 77-K EPR spectrum of form A indicates a seven-line copper hyperfine structure centered at gparallel (gparallel=2.18, Aparallel=4.5 mT), which is characteristic of a mixed-valence binuclear CuA site (Amv), and that of form B exhibits a broad featureless signal (g=2.06). The various spectral data of HdN2OR suggest that form A contains Amv and a mixed-valence tetranuclear CuZ site (Zmv*), while form B includes reduced CuA (Ared) and Zmv*. The pH profiles of N2OR activity of the two forms are similar to each other, and the specific activity at optimum pH 8.8 was estimated to be 45 +/- 5 and 29 +/- 3 micromol.min(-1).mg(-1) for forms A and B, respectively.     

The role of tryptophan 272 in the Paracoccus denitrificans cytochrome c oxidase

The mechanism of electron coupled proton transfer in cytochrome c oxidase (CcO) is still poorly understood. The P(M)-intermediate of the catalytic cycle is an oxoferryl state whose generation requires one additional electron, which cannot be provided by the two metal centres. The missing electron has been suggested to be donated to this binuclear site by a tyrosine residue. A tyrosine radical species has been detected in the P(M) and F* intermediates (formed by addition of H2O2) of the Paraccocus denitrificans CcO using electron paramagnetic resonance (EPR) spectroscopy. From the study of conserved variants its origin was determined to be Y167 which is surprising as this residue is not part of the active site. Upon inspection of the active site it becomes evident that W272 could be the actual donor of the missing electron, which can then be replenished from Y167 or from the Y280-H276 cross link in the natural cycle. To address the question, whether such a direct electron transfer pathway to the binuclear centre exists two tryptophan 272 variants in subunit I have been generated. These variants are characterised by their turnover rates as well as using EPR and optical spectroscopy. From these experiments it is concluded, that W272 is an important intermediate in the formation of the radical species appearing in P(M) and F* intermediates produced with hydrogen peroxide. The significance of this finding for the catalytic function of the enzyme is discussed.     

The location of dissimilatory nitrite reductase and the control of dissimilatory nitrate reductase by oxygen in Paracoccus denitrificans.

1. A method is described for preparing spheroplasts from Paracoccus denitrificans that are substantially depleted of dissimilatory nitrate reductase (cytochrome cd) activity. Treatment of cells with lysozyme + EDTA together with a mild osmotic shock, followed by centrifugation, yielded a pellet of spheroplasts and a supernatant that contained d-type cytochrome. The spheroplasts were judged to have retained an intact plasma membrane on the basis that less than 1% of the activity of a cytoplasmic marker protein, malate dehydrogenase, was released from the spheroplasts. In addition to a low activity towards added nitrite, the suspension of spheroplasts accumulated the nitrite that was produced by respiratory chain-linked reduction of nitrate. It is concluded that nitrate reduction occurs at the periplasmic side of the plasma membrane irrespective of whether nitrite is generated by nitrate reduction or is added exogenously. 2. Further evidence for the integrity of the spheroplasts was that nitrate reduction was inhibited by O2, and that chlorate was reduced at a markedly lower rate than nitrate. These data are taken as evidence for an intact plasma membrane because it was shown that cells acquire the capability to reduce nitrate under aerobic conditions after addition of low amounts of Triton X-100 which, with the same titre, also overcame the permeability barrier to chlorate reduction by intact cells. The close relationship between the appearance of chlorate reduction and the loss of the inhibitory effect of O2 on nitrate reduction also suggests that the later feature of nitrate respiration is due to a control on the accessibility of nitrate to its reductase rather than on the flow of electrons to nitrate reductase.     

Nitric oxide reductase. Purification from Paracoccus denitrificans with use of a single column and some characteristics.

Nitric oxide reductase was purified from Paracoccus denitrificans very nearly to homogeneity by a simple method that involved the use of octyl glucoside to solubilize the enzyme from membranes and required a single hydroxyapatite column. The enzyme had specific activities of about 10 mumol NO reduced x min-1 x mg-1 at pH 6.5 in an amperometric assay system using phenazine methosulfate/ascorbate as the reducing agent and about 22 mumol NO reduced x min-1 x mg-1 at pH 5.0, which is the optimum pH. These values are based on average rates over kinetically complex progress curves and would be about three times greater if based on maximum rate values. The enzyme appeared to be reversibly inhibited by NOaq and to have a Km too low (probably less than or equal to 1 microM) to measure reliably by the amperometric method. The effective second-order rate constant of the enzyme lay within 1 to 2 orders of magnitude of the diffusion controlled limit. The enzyme was composed of a tight complex of two cytochromes: a cytochrome c (Mr = 17,500) and a cytochrome b (Mr = 38,000). The mole ratios of cytochrome c to cytochrome b and Mr 17,500 peptide to Mr 38,000 peptide were both about 1.7, and the heme content was about 3 mol/73,000 g (38,000 + 2(17,500)). Each subunit therefore contained only one heme group. The Mr 38,000 peptide aggregated when heated in the sample buffer used for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to the ascorbate-based activity, the enzyme showed a little NADH-NO oxidoreductase activity which was not inhibited by antimycin A. The enzyme lost activity with a half-life of about 2 days at 4 degrees C but could be preserved at -20 degrees C and in liquid nitrogen. It seemed not to be inactivated by aerobic solutions. These observations, and the recent ones by Carr and Ferguson (Carr, G.J., and Ferguson, S.J. (1990) Biochem. J. 269, 423-429) with a partially purified preparation of nitric oxide reductase, establish that the enzyme from Pa. denitrificans is a cytochrome bc complex which resembles that from Pseudomonas stutzeri (Heiss, B., Frunzke, K., and Zumft, W.G. (1989) J. Bacteriol. 171, 3288-3297). There would appear to be no functional relationship between nitric oxide reductase and a Mr = 34,000 peptide of Pa. denitrificans membranes reported previously to be present in purified preparations of a nitric oxide reductase (Hoglen, J., and Hollocher, T.C. (1989) J. Biol. Chem. 264, 7556-7563).