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Electron-paramagnetic-resonance studies of the mechanism of leaf nitrite reductase. Signals from the iron-sulphur centre and haem under turnover conditions.

ABSTRACT: Low-temperature e.p.r. spectra are presented of nitrite reductase purified from leaves of vegetable marrow (Cucurbita pepo). The oxidized enzyme showed a spectrum at g=6.86, 4.98 and 1.95 corresponding to high-spin Fe(3+) in sirohaem, which disappeared slowly on treatment with nitrite. The midpoint potential of the sirohaem was estimated to be -120mV. On reduction with Na(2)S(2)O(4) or Na(2)S(2)O(4)+Methyl Viologen a spectrum at g=2.038, 1.944 and 1.922 was observed, due to a reduced iron-sulphur centre. The midpoint potential of this centre was very low, about -570mV at pH8.1, decreasing with increasing pH. On addition of cyanide, which binds to haem, and Na(2)S(2)O(4), the iron-sulphur centre became further reduced. We think that this is due to an increased midpoint potential of the iron-sulphur centre. Other ligands to haem, such as CO and the reaction product NH(3), had similar but less pronounced effects, and also changed the lineshape of the iron-sulphur signal. Samples were prepared of the enzyme frozen during the reaction with nitrite, Methyl Viologen and Na(2)S(2)O(4) in various proportions. Signals were interpreted as due to the reduced iron-sulphur centre (with slightly different g values), a haem-NO complex and reduced Methyl Viologen. In the presence of an excess of nitrite, the haem-NO spectrum was more intense, whereas in the presence of an excess of Na(2)S(2)O(4) it was weaker, and disappeared at the end of the reaction. A reaction sequence is proposed for the enzyme, in which the haem-NO complex is an intermediate, followed by other e.p.r.-silent states, leading to the production of NH(4) (+).

SUBMITTER: Cammack R

PROVIDER: S-EPMC1183994 | biostudies-other | 1978 Jun

REPOSITORIES: biostudies-other

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The sulphur oxygenase reductase from Acidianus ambivalens is a multimeric protein containing a low-potential mononuclear non-haem iron centre.

Project description:The SOR (sulphur oxygenase reductase) is the initial enzyme in the sulphur-oxidation pathway of Acidianus ambivalens. Expression of the sor gene in Escherichia coli resulted in active, soluble SOR and in inclusion bodies from which active SOR could be refolded as long as ferric ions were present in the refolding solution. Wild-type, recombinant and refolded SOR possessed indistinguishable properties. Conformational stability studies showed that the apparent unfolding free energy in water is approx. 5 kcal x mol(-1) (1 kcal=4.184 kJ), at pH 7. The analysis of the quaternary structures showed a ball-shaped assembly with a central hollow core probably consisting of 24 subunits in a 432 symmetry. The subunits form homodimers as the building blocks of the holoenzyme. Iron was found in the wild-type enzyme at a stoichiometry of one iron atom/subunit. EPR spectroscopy of the colourless SOR resulted in a single isotropic signal at g=4.3, characteristic of high-spin ferric iron. The signal disappeared upon reduction with dithionite or incubation with sulphur at elevated temperature. Thus both EPR and chemical analysis indicate the presence of a mononuclear iron centre, which has a reduction potential of -268 mV at pH 6.5. Protein database inspection identified four SOR protein homologues, but no other significant similarities. The spectroscopic data and the sequence comparison led to the proposal that the Acidianus ambivalens SOR typifies a new type of non-haem iron enzyme containing a mononuclear iron centre co-ordinated by carboxylate and/or histidine ligands.

| S-EPMC1133771 | biostudies-literature

Kinetics of leaf nitrite reductase with Methyl Viologen and ferredoxin under controlled redox conditions.

Project description:Some factors that influence the activity of nitrite reductase (EC 1.7.7.1) were investigated, the enzyme from Curcurbita pepo (vegetable marrow) being used. The activity with ferredoxin or Methyl Viologen as electron donor was inhibited by certain salts, including NaCl. The steady-state kinetic parameters measured in a commonly used open-tube (aerobic) system were compared with a closed-cell (anaerobic) system in which the redox potential, and thus the concentrations of oxidized and reduced donor, could be controlled. This showed that in the open-tube system the apparent Km values determined were overestimated (by a factor of 10 for reduced Methyl Viologen), owing to incomplete mediator reduction and competitive inhibition by the oxidized form of the mediator.

| S-EPMC1158468 | biostudies-other

Active Intermediates in Copper Nitrite Reductase Reactions Probed by a Cryotrapping-Electron Paramagnetic Resonance Approach.

Project description:Redox active metalloenzymes catalyse a range of biochemical processes essential for life. However, due to their complex reaction mechanisms, and often, their poor optical signals, detailed mechanistic understandings of them are limited. Here, we develop a cryoreduction approach coupled to electron paramagnetic resonance measurements to study electron transfer between the copper centers in the copper nitrite reductase (CuNiR) family of enzymes. Unlike alternative methods used to study electron transfer reactions, the cryoreduction approach presented here allows observation of the redox state of both metal centers, a direct read-out of electron transfer, determines the presence of the substrate/product in the active site and shows the importance of protein motion in inter-copper electron transfer catalyzed by CuNiRs. Cryoreduction-EPR is broadly applicable for the study of electron transfer in other redox enzymes and paves the way to explore transient states in multiple redox-center containing proteins (homo and hetero metal ions).

| S-EPMC7497095 | biostudies-literature

Impact of residues remote from the catalytic centre on enzyme catalysis of copper nitrite reductase.

Project description:Enzyme mechanisms are often probed by structure-informed point mutations and measurement of their effects on enzymatic properties to test mechanistic hypotheses. In many cases, the challenge is to report on complex, often inter-linked elements of catalysis. Evidence for long-range effects on enzyme mechanism resulting from mutations remains sparse, limiting the design/redesign of synthetic catalysts in a predictable way. Here we show that improving the accessibility of the active site pocket of copper nitrite reductase by mutation of a surface-exposed phenylalanine residue (Phe306), located 12 Å away from the catalytic site type-2 Cu (T2Cu), profoundly affects intra-molecular electron transfer, substrate-binding and catalytic activity. Structures and kinetic studies provide an explanation for the lower affinity for the substrate and the alteration of the rate-limiting step in the reaction. Our results demonstrate that distant residues remote from the active site can have marked effects on enzyme catalysis, by driving mechanistic change through relatively minor structural perturbations.

| S-EPMC4104443 | biostudies-literature

Identification of a tyrosine switch in copper-haem nitrite reductases.

Project description:There are few cases where tyrosine has been shown to be involved in catalysis or the control of catalysis despite its ability to carry out chemistry at much higher potentials (1 V versus NHE). Here, it is shown that a tyrosine that blocks the hydrophobic substrate-entry channel in copper-haem nitrite reductases can be activated like a switch by the treatment of crystals of Ralstonia pickettii nitrite reductase (RpNiR) with nitric oxide (NO) (-0.8 ± 0.2 V). Treatment with NO results in an opening of the channel originating from the rotation of Tyr323 away from AspCAT97. Remarkably, the structure of a catalytic copper-deficient enzyme also shows Tyr323 in the closed position despite the absence of type 2 copper (T2Cu), clearly demonstrating that the status of Tyr323 is not controlled by T2Cu or its redox chemistry. It is also shown that the activation by NO is not through binding to haem. It is proposed that activation of the Tyr323 switch is controlled by NO through proton abstraction from tyrosine and the formation of HNO. The insight gained here for the use of tyrosine as a switch in catalysis has wider implications for catalysis in biology.

| S-EPMC6038957 | biostudies-literature

The effect of haem ligands on the redox states of the hexa-haem nitrite reductase from Wolinella succinogenes.

Project description:The nitrite reductase of Wolinella succinogenes containing six covalently bound haem groups has one haem group that will not reduce fully in the presence of excess Na2S2O4. The effect of the extrinsic ligands CO and cyanide on the redox state of this haem was studied by e.p.r. and magnetic c.d. spectroscopy. It was found that both ligands increased the extent of reduction of this haem group, and that in the case of CO binding the level of reduction was correlated with the extent of CO saturation of the enzyme. Stopped-flow studies of the effect of cyanide binding on the rate of dithionite reduction showed that the rate of reduction of the ligand-binding site was increased in the presence of cyanide. This suggests that reduction of the haem groups at the active site is thermodynamically unfavourable in the absence of an extrinsic ligand. The role of the 'non-reducing' haem group and the effect of ligands on this centre and on the rate of reduction are discussed in relation to the reduction of nitrite by this enzyme.

| S-EPMC1149541 | biostudies-other

Electron paramagnetic resonance studies on Pseudomonas nitrosyl nitrite reductase. Evidence for multiple species in the electron paramagnetic resonance spectra of nitrosyl haemoproteins.

Project description:The e.p.r. spectra of reduced 14NO- and 15NO-bound Pseudomonas nitrite reductase have been investigated at pH 5.8 and 8.0 in four buffer systems. At pH 8.0, absorption spectra indicated that only the haem d1 was NO-bound, but, although quantification of the e.p.r. signals in all cases accounted for NO bound the the haem d1 in both subunits of the enzyme, the precise form of the signals varied with buffer and temperature. A rhombic species, with gx = 2.07, gz = 2.01 and gy = 1.96, represented in the low-temperature spectra seen in all the buffers was converted at high temperatures (approx. 200K) into a form showing a reduced anisotropy. Hyperfine splitting on the gz component of this rhombic signal indicated a nitrogen atom trans to NO and it is proposed that histidine provides the endogenous axial ligand for haem d1. At pH 5.8, absorption spectra indicated NO binding to both haems c and d1 and e.p.r. quantifications accounted for NO-bound haems c and d1 in both enzyme subunits. The e.p.r. spectra at pH 5.8 were generally similar to those at pH 8.0 with respect to g-values and hyperfine coupling constants, but were broader with less well defined hyperfine splittings. As at pH 8, rhombic signals present in spectra at low temperatures were converted to less anisotropic forms at high temperatures. The results are discussed in relation to work on model nitrosyl-protohaem complexes [Yoshimura, Ozaki, Shintani & Watanabe (1979) Arch. Biochem, Biophys. 193, 301-313]. No. e.p.r. signal was observed from oxidized NO-bound Pseudomonas nitrite reductase at pH 6.0, over the temperature range 6-100K.

| S-EPMC1161999 | biostudies-other

Studies by electron-paramagnetic-resonance spectroscopy of the molybdenum centre of spinach (Spinacia oleracea) nitrate reductase.

Project description:The molybdenum centre of spinach (Spinacia oleracea) nitrate reductase has been investigated by e.p.r. spectroscopy of molybdenum(V) in reduced forms of the enzyme. The resting enzyme gives no signals attributable to Mo(V). However, on reduction with NADH, Mo(V) signals appeared at relatively short reaction times but decreased again on prolonged exposure to excess of the substrate as the enzyme was further reduced. On brief treatment of such samples with nitrate, Mo(V) signals reappeared but disappeared again on longer exposure to excess nitrate as the enzyme became fully reoxidized. Detailed investigation of the signals carried out in both 1H2O and 2H2O revealed the presence of two signal-giving species, referred to as 'signal A' and 'signal B', analogous to corresponding signals from nitrate reductase from Escherichia coli and from liver sulphite oxidase. Signal A has gav. 1.9767 and shows coupling to a single proton, exchangeable with the solvent, with A(1H)av. 1.3mT, whereas signal B shows no more than weak coupling to protons. Investigation of interconversion between the two species indicated that decreasing the pH from 8.0 to 6.7 had little effect, but that signal A was favoured by the presence of Cl-. This suggests, by analogy with recent work on sulphite oxidase by Bray, Gutteridge, Lamy & Wilkinson [Biochem. J. (1983) 211, 227-236] that Cl- is a ligand of molybdenum in the species giving signal A.

| S-EPMC1152100 | biostudies-other

Boletus edulis Nitrite Reductase Reduces Nitrite Content of Pickles and Mitigates Intoxication in Nitrite-intoxicated Mice.

Project description:Pickles are popular in China and exhibits health-promoting effects. However, nitrite produced during fermentation adversely affects health due to formation of methemoglobin and conversion to carcinogenic nitrosamine. Fruiting bodies of the mushroom Boletus edulis were capable of inhibiting nitrite production during pickle fermentation. A 90-kDa nitrite reductase (NiR), demonstrating peptide sequence homology to fungal nitrite reductase, was isolated from B. edulis fruiting bodies. The optimum temperature and pH of the enzyme was 45?°C and 6.8, respectively. B. edulis NiR was capable of prolonging the lifespan of nitrite-intoxicated mice, indicating that it had the action of an antidote. The enzyme could also eliminate nitrite from blood after intragastric administration of sodium nitrite, and after packaging into capsule, this nitrite-eliminating activity could persist for at least 120?minutes thus avoiding immediate gastric degradation. B. edulis NiR represents the first nitrite reductase purified from mushrooms and may facilitate subsequent applications.

| S-EPMC4597360 | biostudies-literature

Transcriptome of Nitrosomonas europaea with a disrupted nitrite reductase (nirK) gene

Project description:Global gene expression was compared between Nitrosomonas europaea wild-type and a nitrite reductase-deficient mutant using a genomic microarray. Keywords: wild-type to mutant global transcriptome comparison

2006-03-23 | GSE4517 | GEO

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