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).
Kataoka
Kunishige
K
Department of Chemistry, Faculty of Science, Kanazawa University, Kakuma, Kanazawa 920-1192, Japan.
Yamaguchi
Kazuya
K
Sakai
Shinobu
S
Takagi
Kohichi
K
Suzuki
Shinnichiro
S
D000421:Alcaligenes / Q000201:enzymology*; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D003300:Copper / Q000032:analysis*; D004578:Electron Spin Resonance Spectroscopy; D005973:Glutamine; D007700:Kinetics; D008715:Methionine; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry
D011994:Recombinant Proteins; D005973:Glutamine; D003300:Copper; D008715:Methionine; D009565:Nitrate Reductases; D050901:Nitrate Reductase