Amino acid sequence of nitrite reductase: a copper protein from Achromobacter cycloclastes.

The amino acid sequence of the copper-containing nitrite reductase (EC 1.7.99.3) from Achromobacter cycloclastes strain IAM 1013 has been determined by using peptides derived from digestion with Achromobacter protease I (Lys), Staphylococcus aureus V8 protease (Glu), cyanogen bromide, and BNPS-skatole in acetic acid. The subunit contains 340 amino acids. The identity of the first seven amino acids is tentative. The sequence has been instrumental in the X-ray structure determination of this molecule; in conjunction with the X-ray structure, ligands to a type I copper atom and a type II copper atom (one of each per subunit) have been identified. Comparison of the sequence to those of multi-copper oxidases such as ascorbate oxidase, laccase, and ceruloplasmin [Messerschmidt, A., & Huber, R. (1990) Eur. J. Biochem. 187, 341-352] reveals that each of two domains seen in the X-ray structure is similar to the oxidases and also to the small blue copper-containing proteins such as plastocyanin. The combination of sequence and structural similarity to ascorbate oxidase and sequence similarity to ceruloplasmin leads to a plausible model for the domain structure of ceruloplasmin.     

Crystallization of nitrite reductase from Achromobacter cycloclastes

Crystals of a green copper-containing nitrite reductase from Achromobacter cycloclastes, which diffract to high resolution, belong to the cubic space group P213, with a = b = c = 98.4 A. Crystals of a nitrite reductase from Alcaligenes faecalis S-6 have been made, and belong to space group P212121, with a = 77.3 A, b = 94.6 A and c = 141 A. Crystals of the blue copper protein from Ac. cycloclastes have also been obtained: these belong to space group P21212, with cell dimensions a = 34.9 A, b = 91.1 A and c = 36.7 A (1 A = 0.1 nm).     

pH-profile crystal structure studies of C-terminal despentapeptide nitrite reductase from Achromobacter cycloclastes

Crystal structures of C-terminal despentapeptide nitrite reductase (NiRc-5) from Achromobacter cycloclastes were determined from 1.9 to 2.3A at pH 5.0, 5.4, and 6.2. NiRc-5, that has lost about 30% activity, is found to possess quite similar trimeric structures as the native enzyme. Electron density and copper content measurements indicate that the activity loss is not caused by the release of type 2 copper (T2Cu). pH-profile structural comparisons with native enzyme reveal that the T2Cu active center in NiRc-5 is perturbed, accounting for the partial loss of enzyme activity. This perturbation likely results from the less constrained conformations of two catalytic residues, Asp98 and His255. Hydrogen bonding analysis shows that the deletion of five residues causes a loss of more than half the intersubunit hydrogen bonds mediated by C-terminal tail. This study shows that the C-terminal tail plays an important role in controlling the conformations around the T2Cu site at the subunit interface, and helps keep the optimum microenvironment of active center for the full enzyme activity of AcNiR.     

Crystal structure of C-terminal desundecapeptide nitrite reductase from Achromobacter cycloclastes

Monoclinic crystal structure of C-terminal desundecapeptide nitrite reductase (NiRc-11) from Achromobacter cycloclastes was determined at 2.6A. NiRc-11 exists as a loose trimer in the crystal. Deletion of 11 residues eliminates all intersubunit hydrogen bonds mediated by the C-terminal tail. The rigid irregular coil 105-112, which constitutes part of the sidewall of the active site pocket, undergoes conformational changes and becomes highly flexible in NiRc-11. Correspondingly, the linker segments between the two copper sites 95-100 and 135-136 are partly relaxed in conformation, which leads to disrupted active site microenvironments responsible for the activity loss and spectral change of NiRc-11. Comparison with the native structure revealed a bulky residue Met331 fastened by hydrogen bonding, which may play a direct role in keeping the right copper site geometry by protruding its side chain against the irregular coil 105-112. Sequence alignment showed that the bulky residue is conserved at position 331, indicating an equal importance of C-terminal segment in other copper-containing nitrite reductases.     

The subunit structure of nitrite reductase purified from the denitrifier Achromobacter cycloclastes

The copper-containing nitrite reductase of Achromobacter cycloclastes has been considered to be a homotrimer with three identical subunits both in the crystal and in solution. In this study, however, the enzyme was found to be a heterotrimer consisting of two subunits with molecular masses of 37 kDa and 36.2 kDa, and the 37 kDa subunit was 6 amino acid residues longer than the smaller subunit. Signal-peptide cleavage sites in its N-terminal region are discussed.     

Evidence that the type 2 copper centers are the site of nitrite reduction by Achromobacter cycloclastes nitrite reductase.

Methods have been developed for selective depletion and reconstitution of the Type 2 Cu (non-blue) sites in the nitrite reductase from A. cycloclastes, resulting in preparations ranging from 0.5 to 2.6 Type Cu per trimer; the Type 1 Cu content is invariant at 3.0 per trimer. The activity of the enzyme is directly proportional to the Type 2 content as measured by direct metal determination or by analysis of the EPR spectra. These results indicate that an earlier report that the A. cycloclastes enzyme contains only Type 1 Cu sites is incorrect, and that the Type 2 Cu centers constitute the site at which NO2- is reduced. Furthermore, they suggest that other Cu nitrite reductases that are reported to contain only Type 1 Cu sites and exhibit relatively low activity may actually be largely Type 2 Cu-depleted forms of the enzymes.     

Resonance Raman spectra of the copper-sulfur chromophores in Achromobacter cycloclastes nitrite reductase

Resonance Raman spectroscopy at ambient temperature and 77 K has been used to probe the structures of the copper sites in Achromobacter cycloclastes nitrite reductase. This enzyme contains three copper ions per protein molecule and has two principal electronic absorption bands with lambda max values of 458 and 585 nm. Comparisons between the resonance Raman spectra of nitrite reductase and blue copper proteins establish that both the 458 and 585 nm bands are associated with Cu(II)-S(Cys) chromophores. A histidine ligand probably is also present. Different sets of vibrational frequencies are observed with 457.9 nm (ambient) or 476.1 nm (77 K) excitation as compared with 590 nm (ambient) or 593 nm (77 K) excitation. Excitation profiles indicate that the 458 and 585 nm absorption bands are associated with separate [Cu(II)-S(Cys)N(His)] sites or with inequivalent and uncoupled cysteine ligands in the same site. The former possibility is considered to be more likely.     

Achromobacter cycloclastes nitrite reductase. The function of copper, amino acid composition, and ESR spectra.

1. Dialysis against cyanide at pH 7 of Achromobacter cycloclastes nitrite reductase [EC 1.7.99.3] of a dissimilatory type led to the removal of about 50% of the copper from the enzyme molecule, with a concomitant decrease of the enzymatic activities. It was inferred that enzyme-bound copper atoms play an essential role in the catalytic activities of the enzyme. 2. The amino acid composition of the enzyme was determined after acid hydrolysis. 3. ESR spectra of the frozen solution and lyophilized powder of the nitrite reductase predominantly showed the presence of two kinds of copper: Type 1 Cu2+, which had narrow and sharp hyperfine splitting, and Type 2 Cu2+, which had broader hyperfine splitting. The bond between the oxidized enzyme and nitrite seems to be ionic.     

Cytoplasmic expression of the Achromobacter xylosoxidans blue copper nitrite reductase in Escherichia coli and characterisation of the recombinant protein

The gene of the Achromobacter xylosoxidans (DSM 2402) blue copper-containing nitrite reductase was amplified using the polymerase chain reaction. DNA sequence analysis reveals that the amino acid sequence is identical to those of the GIFU1051 and the NCIMB11015 A. xylosoxidans nitrite reductases. The gene encoding the mature coding region for DSM 2402 nitrite reductase was cloned into a pET-vector, overexpressed in the cytoplasm of Escherichia coli BL21(DE3), and the expressed holoprotein was purified to apparent homogeneity by cation-exchange chromatography. The recombinant blue copper-containing nitrite reductase was obtained in high yields of 70mgL(-1) of culture. The specific catalytic activity as well as the electronic absorption and electron paramagnetic resonance spectra agree with corresponding data for the native protein. Mass spectroscopic analysis of the recombinant nitrite reductase gave a molecular weight of 36659.1Da for the apo-protein monomer, in agreement with the expected molecular mass based on the amino acid sequence.     

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).     

Isolation of a high specific activity pink, monomeric nitrous oxide reductase from Achromobacter cycloclastes

Nitrous oxide reductase from Achromobacter cycloclastes has been purified to homogeneity under aerobic conditions via DEAE-cellulose, phenyl-Sepharose, hydroxyapatite, and Sephacryl S-200 chromatography. It consists of a single polypeptide of MW 72 kDa, and contains 3.8 +/- 0.1 copper atoms per molecule. The enzyme is pink as isolated, yet exhibits a specific activity (86 U/mg) that is ca. 40 times greater than that observed for other N2O reductases under similar conditions. Double integration of the anomalous EPR spectrum at 77K showed the presence of 2.0 +/- 0.1 spins per molecule, implying the presence of EPR-silent copper atoms and/or spin-coupled mixed-valent centers.     

Properties and electron transfer specificity of copper proteins from the denitrifier "Achromobacter cycloclastes"

A blue copper protein (Mr 12,000) was purified from cells of "Achromobacter cycloclastes" grown as a denitrifier. When reduced, the blue copper protein transferred electrons to the copper protein nitrite reductase purified from the same cells, whereas a variety of cytochromes from denitrifiers failed to do so. Inclusion of a protease inhibitor, phenylmethylsulfonyl fluoride, in the buffers employed during preparation yielded purified blue copper protein with 18 more amino acid residues and two times more specific enzyme activity than other researchers have found.     

Anaerobic purification, characterization and preliminary mechanistic study of recombinant nitrous oxide reductase from Achromobacter cycloclastes

An overexpression system for nitrous oxide reductase (N(2)OR), an enzyme that catalyzes the conversion of N(2)O to N(2) and H(2)O, has been developed in Achromobacter cycloclastes. Anaerobically purified A. cycloclastes recombinant N(2)OR (AcN(2)OR) has on average 4.5 Cu and 1.2 S per monomer. Upon reduction by methyl viologen, AcN(2)OR displays a high specific activity: 124 U/mg at 25 degrees C. Anaerobically purified AcN(2)OR displays a unique absorption spectrum. UV-visible and EPR spectra, combined with kinetics studies, indicate that the as-purified form of the enzyme is predominately a mixture of the fully-reduced Cu(Z)=[4Cu(I)] state and the Cu(Z)=[3Cu(I).Cu(II)] state, with the latter readily reducible by reduced forms of viologens. CD spectra of the as-purified AcN(2)OR over a range of pH values reveal perturbations of the protein conformation induced by pH variations, although the principal secondary structure elements are largely unaltered. Further, the activity of AcN(2)OR in D(2)O is significantly decreased compared with that in H(2)O, indicative of a significant solvent isotope effect on N(2)O reduction. These data are in good agreement with conclusions reached in recent studies on the effect of pH on catalysis by N(2)OR [K. Fujita, D.M. Dooley, Inorg. Chem. 46 (2007) 613-615].     

Kinetic studies on the nitrite reductase of Wolinella succinogenes.

Calibration relationships were derived for cartilage proteoglycan subunit (PGS) that relate the inverse z-average hydrodynamic radius (Rs) and the weight-average Mr (Mw) to the partition coefficient (Kav.) for PGS when chromatographed on a Sepharose CL-2B column. PGS isolated from chick limb-bud chondrocyte cell cultures was fractionated chromatographically into eight pools, for which Mw and Rs were determined by total-intensity and dynamic light-scattering measurements. These data were found to be related to Kav. through the following empirical equations: log Mw = -(1.65 +/- 0.27)Kav. +(6.58 +/- 0.08); log Rs = -(0.69 +/- 0.04)Kav. +(2.75 +/- 0.01). Application of these relationships to the chromatographic data led to Mw = 1.48 X 10(6) and Rs = 38.7 nm (387 A) for the unfractionated specimens compared with values of Mw = 1.46 X 10(6) and Rs = 38.2 nm (382 A) determined by light-scattering. Our results were found to be consistent with previously proposed phenomenological models for the gel-filtration mechanism. Application of these calibration relationships to Kav. for several unfractionated specimens led to predicted values of Mw and Rs that are accurate to within 10%.     

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.     

Electron paramagnetic resonance studies of heme c and its nitrosyl derivative in Vibrio (Achromobacter) fischeri nitrite reductase

Interactions of Vibrio (formerly Achromobacter) fischeri nitrite reductase were studied by electron paramagnetic resonance spectroscopy. The spectrum of the oxidized enzyme showed a number of features which were attributed to two low-spin ferric hemes. These comprised an unusual derivative peak at g = 3.7 and a spectrum at g = 2.88, 2.26, and 1.51. Neither heme was reactive in the oxidized state with the substrate nitrite and with cyanide and azide. When frozen under turnover conditions (i.e., reduction in the presence of excess nitrite), the enzyme showed the spectrum of a nitrosyl heme derivative. The g = 2.88, 2.26, and 1.51 signals reappeared partially on reoxidation by nitrite, indicating that the nitrosyl species which remained arose from the g = 3.7 heme. The nitrosyl derivative showed a 14N nuclear hyperfine splitting, Az = 1.65 mT. The nitrosyl derivative was produced by treatment of the oxidized nitrite reductase with nitric oxide or hydroxylamine. Exchange of nitric oxide between the nitrosyl derivative and NO gas in solution was observed by using the [15N]nitrosyl compound. A possible reaction cycle for the enzyme is discussed, which involves reduction of the enzyme followed by binding of nitrite to one heme and formation of the nitrosyl intermediate.     

Kinetic and spectroscopic studies of the interaction of copper with dopamine beta-hydroxylase

The question of the stoichiometry of copper bound to dopamine beta-hydroxylase and the number of copper atoms required for maximal activity was addressed in this study. Incubation of tetrameric enzyme from bovine adrenal medulla with 64Cu2+ followed by rapid gel filtration yielded an enzyme containing 8.3-8.9 mol of Cu/mol of tetramer. An identical stoichiometry was obtained by analysis of bound copper by atomic absorption methods. NMR and EPR were used to monitor titrations of the enzyme with Cu2+ and showed that the longitudinal relaxation rate of solvent water protons and the amplitude of the signal at g approximately 2 increased linearly up to a copper to protein ratio of approximately 8. Additional titrations also indicate that an enzyme-Cu2+-tyramine-CN- inhibitory complex was formed when 8 mol of Cu2+ are bound per mol of enzyme. The rate of inactivation of dopamine beta-hydroxylase by the mechanism-based inhibitor 2-Br-3-(p-hydroxyphenyl)-1-propene was measured and used as a method to follow enzymatic catalysis. An increase in rate was observed with increasing Cu2+ up to a protein to Cu2+ ratio of 8 Cu/tetramer. The rate becomes constant after this ratio is achieved. These data indicate that dopamine beta-hydroxylase specifically binds 8 mol of Cu/tetramer and that this stoichiometry is required for maximal activity.