title: Characterization of two type 1 Cu sites of Hyphomicrobium denitrificans nitrite reductase: a new class of copper-containing nitrite reductases.
abstract: We report (1) the amino acid sequence of Hyphomicrobium denitrificans nitrite reductase (HdNIR), containing two type 1 Cu sites and one type 2 Cu site; (2) the expression and preparation of wild-type HdNIR and two mutants replacing the Cys ligand of each type 1 Cu with Ala; and (3) their spectroscopic and functional characterization. The open-reading frame of 50-kDa HdNIR is composed of the 15-kDa N-terminal domain having a type 1 Cu-binding motif like cupredoxins and the 35-kDa C-terminal domain having type 1 Cu-binding and type 2 Cu-binding motifs such as common nitrite reductases (NIRs). Moreover, the amino acid sequences of the N- and C-terminal domains are homologous to those of plastocyanins and NIRs, respectively. The point mutation of the Cys ligand of each type 1 Cu with Ala gives two mutants, C114A and C260A, possessing one type 1 Cu and one type 2 Cu. The spectroscopic data of C114A reveal that the C-terminal NIR-like domain has the green type 1 Cu (type 1 Cu(C)), showing two intense absorption peaks at 455 (epsilon = 2600 M(-1) cm(-1)) and 600 nm (epsilon = 2800 M(-1) cm(-1)) and a rhombic EPR signal like those of the green type 1 Cu of Achromobacter cycloclastes NIR (AcNlR). The spectroscopic data of C260A elucidate that the N-terminal Pc-like domain in HdNIR contains the blue type 1 Cu (type 1 Cu(N)), exhibiting an intense absorption band at 605 nm (epsilon = 2900 M(-1) cm(-1)) and an axial EPR signal like those of the blue type 1 Cu of Alcaligenes xylosoxidans NIR (AxNIR). The sum of the visible absorption or EPR spectra of C114A and C260A is almost equal to the corresponding spectrum of wild-type HdNIR. The spectroscopic characterization of the type 1 Cu indicates that the geometries of the type 1 Cu(N) and Cu(C) sites are slightly distorted tetrahedral (or axially elongated bipyramidal) and flattened tetrahedral, respectively. In the cyclic voltammograms, the midpoint potentials (E(1/2)), probably because of the type 1 Cu ions of C114A and C260A, are observed at +321 and +336 mV versus normal hydrogen electrode (NHE) at pH 7.0, respectively. These values, which are close to each other, are more positive than those ( approximately +0.24-0.28 V at pH 7.0) of the type 1 Cu sites of AcNIR and AxNIR. The electron-accepting capability of C114A from cytochrome c(550) is almost similar to that of wild-type HdNIR, whereas that of C260A is very low. This suggests that the type 1 Cu(C) in the C-terminal domain is essential for the enzyme functions of HdNIR.
authors.affiliation: Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Yamaguchi
authors.forename: Kunishige
authors.initials: K
authors.lastname: Kataoka
authors.forename: Mayuko
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Koushi
authors.initials: K
authors.lastname: Itoh
authors.forename: Atsushi
authors.initials: A
authors.lastname: Fukui
authors.forename: Shinnichiro
authors.initials: S
authors.lastname: Suzuki
mesh_terms: D000595:Amino Acid Sequence; D001400:Azurin / Q000031:analogs & derivatives* / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000145:classification* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D020582:Hyphomicrobium / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009572:Nitrite Reductases / Q000737:chemistry* / Q000145:classification* / Q000235:genetics; D011199:Potentiometry; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
chemical_list: D001426:Bacterial Proteins; C050107:cupredoxin; D001400:Azurin; D003300:Copper; D009572:Nitrite Reductases