Characterization of the membranous denitrification enzymes nitrite reductase (cytochrome cd1) and copper-containing nitrous oxide reductase from Thiobacillus denitrificans

Cytochrome cd ₁-nitrite reductase and nitrous oxide reductase of Thiobacillus denitrificans were purified and characterized by biochemical and immunochemical methods. In contrast to the generally soluble nature of the denitrification enzymes, these two enzymes were isolated from the membrane fraction of T. denitrificans and remained active after solubilization with Triton X-100. The properties of the membrane-derived enzymes were similar to those of their soluble counterparts from the same organism. Nitrous oxide reductase activity was inhibited by acetylene. Nitrite reductase and nitrous oxide reductase cross-reacted with antisera raised against the soluble enzymes from Pseudomonas stutzeri. The nirS, norBC, and nosZ genes encoding the cytochrome cd ₁-nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, respectively, from P. stutzeri hybridized with genomic DNA from T. denitrificans. Cross-reactivity and similar N-terminal amino acid and gene sequences suggest that the primary structures of the Thiobacillus enzymes are homologous to the soluble proteins from P. stutzeri. Received: 18 August 1995 / Accepted: 30 October 1995     

Spectroscopic and functional characterization of Cu-containing nitrite reductase from Hyphomicrobium denitrificans A3151

The Cu-containing nitrite reductase from Hyphomicrobium denitrificans (HydNIR) has been spectroscopically and functionally characterized. The visible absorption spectrum implies that the enzyme has two type 1 Cu ions in one subunit (ca. 50 kDa). The electron paramagnetic resonance (EPR) spectrum of HydNIR is simulated assuming the sum of three distinct S = 1/2 systems: two type 1 Cu signals (axial and rhombic symmetries) and one type 2 Cu signal. The intramolecular electron transfer reaction from the type 1 Cu to the type 2 Cu at pH 6.0 does not occur in the absence of nitrite, but a very slow electron transfer reaction is observed in the presence of nitrite. The apparent first-order rate constants for the intramolecular electron transfer reactions (k(ET(intra))) in the presence of nitrite and also the apparent catalytic rate constants (k(cat)) of HydNIR decrease gradually with increasing pH in the range of pH 4.5-7.5. These pH profiles are substantially similar to each other, suggesting that the intramolecular electron transfer process is linked to the subsequent nitrite reduction process.     

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.     

Isolation of Paracoccus denitrificans cytochrome cd1: comparative kinetics with other nitrite reductases

The dissimilatory nitrite reductase of the cytochrome cd₁ type was purified from Paracoccus denitrificans (ATCC 13543) by a novel procedure that avoided conventional ion-exchange techniques. The characterization of this enzyme was extended to include amino acid composition, extinction coefficients, and kinetic properties not previously reported. Cytochromes cd₁ from Alicaligenes faecalis and Pseudomonas aeruginosa were also isolated and assayed with electron donor proteins. The enzymes from all three sources were shown to obey the same integrated rate law. Cross-reactivities were measured in which a reduced donor protein from one strain was assayed with cytochrome cd₁ from another strain using nitrite as ultimate acceptor. Donors included c-type cytochromes and azurins. In general, the enzymes showed specificity for a donor from the same strain; interspecies cross-reactions were typically slower on the order of 10-fold than corresponding native rates. Notable exceptions were Paracoccus cytochrome cd₁, which alone reacted with eukaryotic horse cytochrome c at appreciable rates, and the Pseudomonas cd₁-Alcaligenesc554 reaction, which was 4-fold faster than the native Alcaligenes cd₁-Alcaligenesc554 reaction. For all three enzymes, competitive kinetics were measured in which the alternative substrates, nitrite and oxygen, competed for enzyme in the same assay. It was found that the competitive kinetics were dominated by nonenzymatic reactions involving an enzyme product, nitric oxide.     

Nitrite reduction by xanthine oxidase family enzymes: a new class of nitrite reductases

Mammalian xanthine oxidase (XO) and Desulfovibrio gigas aldehyde oxidoreductase (AOR) are members of the XO family of mononuclear molybdoenzymes that catalyse the oxidative hydroxylation of a wide range of aldehydes and heterocyclic compounds. Much less known is the XO ability to catalyse the nitrite reduction to nitric oxide radical (NO). To assess the competence of other XO family enzymes to catalyse the nitrite reduction and to shed some light onto the molecular mechanism of this reaction, we characterised the anaerobic XO- and AOR-catalysed nitrite reduction. The identification of NO as the reaction product was done with a NO-selective electrode and by electron paramagnetic resonance (EPR) spectroscopy. The steady-state kinetic characterisation corroborated the XO-catalysed nitrite reduction and demonstrated, for the first time, that the prokaryotic AOR does catalyse the nitrite reduction to NO, in the presence of any electron donor to the enzyme, substrate (aldehyde) or not (dithionite). Nitrite binding and reduction was shown by EPR spectroscopy to occur on a reduced molybdenum centre. A molecular mechanism of AOR- and XO-catalysed nitrite reduction is discussed, in which the higher oxidation states of molybdenum seem to be involved in oxygen-atom insertion, whereas the lower oxidation states would favour oxygen-atom abstraction. Our results define a new catalytic performance for AOR—the nitrite reduction—and propose a new class of molybdenum-containing nitrite reductases.     

Anammox organism KSU-1 expresses a NirK-type copper-containing nitrite reductase instead of a NirS-type with cytochrome cd1

Anaerobic ammonium oxidation (anammox) and denitrification are two distinct microbial reactions relevant to the global nitrogen cycle. The proposed initial step of the anammox reactions, reduction of nitrite to nitric oxide, has been postulated to be identical to that in denitrification catalyzed by the dissimilatory nitrite reductase of the cytochrome cd(1)-type. Here, we characterized the copper-containing nitrite reductase homolog encoded by nirK detected in the genome of an anammox bacterium strain KSU-1. We hypothesize that this NirK-type nitrite reductase, rather than a nitrite reductase of the cytochrome cd(1)-type (NirS), is likely to catalyze nitrite reduction in anammox organism KSU-1.     

Crystal structure of the soluble domain of the major anaerobically induced outer membrane protein (AniA) from pathogenic Neisseria: a new class of copper-containing nitrite reductases

The major anaerobically induced outer membrane protein (AniA) from pathogenic Neisseria gonorrhoeae is essential for cell growth under oxygen limiting conditions in the presence of nitrite and is protective against killing by human sera. A phylogenic analysis indicates that AniA is a member of a new class of copper-containing nitrite reductases. Expression of the soluble domain of AniA yields a protein capable of reducing nitrite with specific activity of 160 units/mg, approximately 50 % of that measured for the nitrite reductase from the strong soil denitrifier Alcaligenes faecalis S-6. The crystal structure of the soluble domain of AniA was solved by molecular replacement and sixfold averaging to a resolution of 2.4 A. The nitrite soaked AniA crystal structure refined to 1.95 A reveals a bidentate mode of substrate binding to the type II copper. Despite low sequence identity (approximately 30 %), the core cupredoxin fold of AniA is similar to that found in copper-containing nitrite reductases from soil bacteria. The main structural differences are localized to two attenuated surface loops that map to deletions in the sequence alignment. In soil nitrite reductases, one of these surface loops is positioned near the type I copper site and contributes residues to the docking surface for proteaceous electron donors. In AniA, the attenuation of this loop results in a restructured hydrophobic binding surface that may be required to interact with a lipid anchored azurin. The second attenuated loop is positioned on the opposite side of AniA and may facilitate a more intimate interaction with the lipid membrane. A unique combination of structural effectors surrounding the type I copper site of sAnia contribute to a unusual visible absorption spectra with components observed previously in either green or blue type I copper sites.     

Preliminary crystallographic studies of two C-terminally truncated copper-containing nitrite reductases from Achromobacter cycloclastes: changed crystallizing behaviors caused by residue deletion

The C-terminal segment of copper-containing nitrite reductase from Achromobacter cycloclastes (AcNiR) has been found essential for maintaining both the quaternary structure and the enzyme activity of AcNiR. C-terminal despentapeptide AcNiR (NiRc-5) and desundecapeptide AcNiR (NiRc-11) are two important truncated mutants whose activities and stability have been affected by residue deletion. In this study, the two mutants were crystallized using the hanging drop vapor diffusion method. Crystals of NiRc-5 obtained at pH 5.0 and 6.2 both belonged to the P2(1)2(1)2(1) space group with unit cell parameters a=99.0 A, b=117.4 A, c=122.8 A (pH 5.0) and a=98.9A, b=117.7A, c=123.0A (pH 6.2). NiRc-11 was crystallized in two crystal forms: the tetragonal form belonged to the space group P4(1) with a=b=96.0A and c=146.6A; the monoclinic form belonged to the space group P2(1) with a=86.0A, b=110.1A, c=122.7A, and beta=101.9 degrees. The crystallizing behaviors of the two mutants differed from that of the native enzyme. Such change in combination with residue deletion is also discussed here.     

Characterization and function of Met150Gln mutant of copper-containing nitrite reductase from Achromobacter cycloclastes IAM1013

The mutant (M150Q-NIR) replacing the Met150 ligand of the type 1 Cu center in Achromobacter cycloclastes nitrite reductase (AcNIR) with Gln has been physicochemically and functionally characterized. The electronic absorption and CD spectra of M150Q-NIR are similar to those of mavicyanin and stellacyanin having the 2His, Cys, and Gln ligands, but the EPR signal has an axial character, although their blue copper proteins show rhombic EPR signals. The mutant has about 80% catalytic activity of AcNIR. Moreover, the midpoint potential (E(1/2)) of M150Q-NIR is +113 mV vs. NHE at pH 7.0, being negatively shifted compared to that of AcNIR (+240 mV). Although the intermolecular electron-transfer process from Achromobacter cycloclastes pseudoazurin (pAz) to M150Q-NIR was not detected, the pAz mutant (M86Q-pAz) replacing the Met86 ligand with Gln transfers one electron to the NIR mutant with an intermolecular electron-transfer rate constant (k(ET)) of 2.3 x 10(5)M(-1)s(-1).     

Conserved active site residues limit inhibition of a copper-containing nitrite reductase by small molecules

The interaction of copper-containing dissimilatory nitrite reductase from Alcaligenes faecalis S-6 ( AfNiR) with each of five small molecules was studied using crystallography and steady-state kinetics. Structural studies revealed that each small molecule interacted with the oxidized catalytic type 2 copper of AfNiR. Three small molecules (formate, acetate and nitrate) mimic the substrate by having at least two oxygen atoms for bidentate coordination to the type 2 copper atom. These three anions bound to the copper ion in the same asymmetric, bidentate manner as nitrite. Consistent with their weak inhibition of the enzyme ( K i >50 mM), the Cu-O distances in these AfNiR-inhibitor complexes were approximately 0.15 A longer than that observed in the AfNiR-nitrite complex. The binding mode of each inhibitor is determined in part by steric interactions with the side chain of active site residue Ile257. Moreover, the side chain of Asp98, a conserved residue that hydrogen bonds to type 2 copper-bound nitrite and nitric oxide, was either disordered or pointed away from the inhibitors. Acetate and formate inhibited AfNiR in a mixed fashion, consistent with the occurrence of second acetate binding site in the AfNiR-acetate complex that occludes access to the type 2 copper. A fourth small molecule, nitrous oxide, bound to the oxidized metal in a side-on fashion reminiscent of nitric oxide to the reduced copper. Nevertheless, nitrous oxide bound at a farther distance from the metal. The fifth small molecule, azide, inhibited the reduction of nitrite by AfNiR most strongly ( K ic = 2.0 +/- 0.1 mM). This ligand bound to the type 2 copper center end-on with a Cu-N c distance of approximately 2 A, and was the only inhibitor to form a hydrogen bond with Asp98. Overall, the data substantiate the roles of Asp98 and Ile257 in discriminating substrate from other small anions.     

Formation of active nitrite reductase (cytochromecd 1) in the strainParacoccus denitrificans HUUG25

Strain HUUG25 ofParacoccus denitrificans has been frequently thought to be devoid of allc-type cytochromes. We show here by means of enzymological and immunological techniques that the mutant synthesizes active nitrite reductase (cytochromecd ₁) upon prolonged exposure to microoxic conditions. The synthesis occurred faster in the presence of exogenous hemin. The time pattern of 5-aminolevulinate synthase activity was also altered by the mutation. These findings suggest a defective regulation of heme supply to the site of nitrite reductase assembly in the periplasm.     

Characterization of the structural gene encoding a copper-containing nitrite reductase and homology of this gene to DNA of other denitrifiers.

A copper-containing nitrite reductase gene (nirU) from Pseudomonas sp. strain G-179 was found in a 1.9-kb EcoRI-BamHI DNA fragment. The coding region contained information for a polypeptide of 379 amino acids. The encoded protein had 78% identity in amino acid sequence to the nitrite reductase purified from Achromobacter cycloclastes. The ligands for type 1 copper- and type 2 copper-binding sites found in A. cycloclastes were also found in Pseudomonas sp. strain G-179, suggesting that these binding sites are conserved. Upstream from the promoter, two putative fnr boxes were found, suggesting that an FNR-like protein may be involved in regulation of the nitrite reductase gene under anaerobic conditions. When the 1.9-kb clone was used to probe Southern blots for similar sequences in DNAs from different denitrifiers, hybridization bands were seen for 15 of 16 denitrifiers known to have nitrite reductase containing copper. Except for Pseudomonas stutzeri JM300, all denitrifiers tested that have nitrite reductases containing heme c,d1 showed no or weak hybridization to this probe. Thus, this structural gene may be useful as a probe to detect denitrifiers with copper-containing nitrite reductases.     

Directing the mode of nitrite binding to a copper-containing nitrite reductase from Alcaligenes faecalis S-6: characterization of an active site isoleucine

Unlike the heme cd(1)-based nitrite reductase enzymes, the molecular mechanism of copper-containing nitrite reductases remains controversial. A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small library of six variants at position 257 in nitrite reductase from Alcaligenes faecalis S-6. The activities of these six variants span nearly two orders of magnitude with one variant, I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8 A) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies identify for the first time that the highly conserved Ile257 in the native enzyme is a key molecular determinant in directing a catalytically competent mode of nitrite binding in the active site. The O-coordinate bidentate binding mode of nitrite observed in native and mutant forms with high activity supports a catalytic model distinct from the heme cd(1) NiRs. (The atomic coordinates for I257V[NO(2)(-)], I257L[NO(2)(-)], I257A[NO(2)(-)], I257T[NO(2)(-)], I257M[NO(2)(-)] and I257G[NO(2)(-)] AfNiR have been deposited in the Protein Data Bank [PDB identification codes are listed in Table 2].)     

Functional analysis of conserved aspartate and histidine residues located around the type 2 copper site of copper-containing nitrite reductase

A heterologous expression system of the blue copper-containing nitrite reductase from Alcaligenes xylosoxidans GIFU1051 (AxgNIR) was constructed, and the purified recombinant enzyme was characterized. All the characteristic spectroscopic properties and enzyme activity of native AxgNIR were retained in the copper-reconstituted recombinant protein expressed in Escherichia coli, indicating the correct coordination of two types of Cu (type 1 and 2) in the recombinant enzyme. Moreover, two conserved noncoordinate residues, Asp98 and His255, located near the type 2 Cu site were replaced to elucidate the catalytic residue(s) of NIR. The Asp98 residue hydrogen-bonded to the water molecule ligating the type 2 Cu was changed to Ala, Asn, or Glu, and the His255 residue hydrogen-bonded to Asp98 through the water molecule was replaced with Ala, Lys, or Arg. The catalytic rate constants of all mutants were decreased to 0.4-2% of those of the recombinant enzyme, and the apparent K(m) values for nitrite were greatly increased in the Asp98 mutants. All the steady-state kinetic data of the mutants clearly demonstrate that both Asp98 and His255 are involved not only in the catalytic reaction but also in the substrate anchoring.     

Periplasmic location of dissimilatory nitrate and nitrite reductases in a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroides forma sp. denitrificans

The localization of dissimilatory nitrate and nitrite reductasesof a denitrifying phototrophic bacterium, Rhodopseudomonas sphaeroidesforma sp. denitrificans, was investigated. Nitrate and nitritereductases were located in the periplasmic space of the bacteriumgrown anaerobically in the presence of nitrate either in lightor in darkness. Chromatophores showed nitrate and nitrite reductaseactivities when dithionite-reduced benzyl viologen was an electrondonor; this suggests that the enzymes were trapped inside thevesicles. ¹Present address: Japanese Red Cross Central Blood Center, Hiroo4-1-31, Shibuyaku, Tokyo 150, Japan. ²Present address: Plant Growth Laboratory, University of California,Davis, California 95616, U.S.A. (Received November 7, 1979; )     

Catalytic roles for two water bridged residues (Asp-98 and His-255) in the active site of copper-containing nitrite reductase

Two active site residues, Asp-98 and His-255, of copper-containing nitrite reductase (NIR) from Alcaligenes faecalis have been mutated to probe the catalytic mechanism. Three mutations at these two sites (D98N, H255D, and H255N) result in large reductions in activity relative to native NIR, suggesting that both residues are involved intimately in the reaction mechanism. Crystal structures of these mutants have been determined using data collected to better than 1. 9-A resolution. In the native structure, His-255 Nepsilon2 forms a hydrogen bond through a bridging water molecule to the side chain of Asp-98, which also forms a hydrogen bond to a water or nitrite oxygen ligated to the active site copper. In the D98N mutant, reorientation of the Asn-98 side chain results in the loss of the hydrogen bond to the copper ligand water, consistent with a negatively charged Asp-98 directing the binding and protonation of nitrite in the native enzyme. An additional solvent molecule is situated between residues 255 and the bridging water in the H255N and H255D mutants and likely inhibits nitrite binding. The interaction of His-255 with the bridging water appears to be necessary for catalysis and may donate a proton to reaction intermediates in addition to Asp-98.