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The nitric oxide reductase of Paracoccus denitrificans.

ABSTRACT: 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.

SUBMITTER: Carr GJ

PROVIDER: S-EPMC1131594 | biostudies-other | 1990 Jul

REPOSITORIES: biostudies-other

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Project description:Variant nomenclature: the variants were made in the NorB subunit if not indicated by the superscript c, which are variants in the NorC subunit (e.g. E122A = exchange of Glu-122 in NorB for an Ala, E71cD; exchange of Glu-71 in NorC for an Asp). Bacterial NO reductases (NORs) are integral membrane proteins from the heme-copper oxidase superfamily. Most heme-copper oxidases are proton-pumping enzymes that reduce O2 as the last step in the respiratory chain. With electrons from cytochrome c, NO reductase (cNOR) from Paracoccus (P.) denitrificans reduces NO to N2O via the following reaction: 2NO+2e-+2H+?N2O+H2O. Although this reaction is as exergonic as O2-reduction, cNOR does not contribute to the electrochemical gradient over the membrane. This means that cNOR does not pump protons and that the protons needed for the reaction are taken from the periplasmic side of the membrane (since the electrons are donated from this side). We previously showed that the P. denitrificans cNOR uses a single defined proton pathway with residues Glu-58 and Lys-54 from the NorC subunit at the entrance. Here we further strengthened the evidence in support of this pathway. Our further aim was to define the continuation of the pathway and the immediate proton donor for the active site. To this end, we investigated the region around the calcium-binding site and both propionates of heme b3 by site directed mutagenesis. Changing single amino acids in these areas often had severe effects on cNOR function, with many variants having a perturbed active site, making detailed analysis of proton transfer properties difficult. Our data does however indicate that the calcium ligation sphere and the region around the heme b3 propionates are important for proton transfer and presumably contain the proton donor. The possible evolutionary link between the area for the immediate donor in cNOR and the proton loading site (PLS) for pumped protons in oxygen-reducing heme-copper oxidases is discussed.

Project description:N2O is generated by denitrifying bacteria as a product of NO reduction. In denitrification, N2O is metabolized further by the enzyme N2O reductase (N2OR), a multicopper protein which converts N2O into dinitrogen and water. The structure of N2OR remained unknown until the recent elucidation of the structure of the enzyme isolated from Pseudomonas nautica. In the present paper, we report the crystal structure of a blue form of the enzyme that was purified under aerobic conditions from Paracoccus denitrificans. N2OR is a head-to-tail homodimer stabilized by a multitude of interactions including two calcium sites located at the intermonomeric surface. Each monomer is composed of two domains: a C-terminal cupredoxin domain that carries the dinuclear electron entry site known as Cu(A), and an N-terminal seven-bladed beta-propeller domain which hosts the active-site centre Cu(Z). The electrons are transferred from Cu(A) to Cu(Z) across the subunit interface. Cu(Z) is a tetranuclear copper cluster in which the four copper ions (Cu1 to Cu4) are ligated by seven histidine imidazoles, a hydroxyl or water oxygen and a bridging inorganic sulphide. A bound chloride ion near the Cu(Z) active site shares one of the ligand imidazoles of Cu1. This arrangement probably influences the redox potential of Cu1 so that this copper is stabilized in the cupric state. The treatment of N2OR with H2O2 or cyanide causes the disappearance of the optical band at 640 nm, attributed to the Cu(Z) centre. The crystal structure of the enzyme soaked with H2O2 or cyanide suggests that an average of one copper of the Cu(Z) cluster has been lost. The lowest occupancy is observed for Cu3 and Cu4. A docking experiment suggests that N(2)O binds between Cu1 and Cu4 so that the oxygen of N2O replaces the oxygen ligand of Cu4. Certain ligand imidazoles of Cu1 and Cu2, as well as of Cu4, are located at the dimer interface. Particularly those of Cu2 and Cu4 are parts of a bonding network which couples these coppers to the Cu(A) centre in the neighbouring monomer. This structure may provide an efficient electron transfer path for reduction of the bound N2O.

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The nitric oxide reductase of Paracoccus denitrificans.

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