Structure-based engineering of Alcaligenes xylosoxidans copper-containing nitrite reductase enhances intermolecular electron transfer reaction with pseudoazurin

J Biol Chem. 2004 Dec 17;279(51):53374-8. doi: 10.1074/jbc.M410198200. Epub 2004 Oct 7.

Abstract

The intermolecular electron transfer from Achromobacter cycloclastes pseudoazurin (AcPAZ) to wild-type and mutant Alcaligenes xylosoxidans nitrite reductases (AxNIRs) was investigated using steady-state kinetics and electrochemical methods. The affinity and the electron transfer reaction constant (k(ET)) are considerably lower between AcPAZ and AxNIR (K(m) = 1.34 mM and k(ET) = 0.87 x 10(5) M(-1) s(-1)) than between AcPAZ and its cognate nitrite reductase (AcNIR) (K(m) = 20 microM and k(ET) = 7.3 x 10(5) M(-1) s(-1)). A negatively charged hydrophobic patch, comprising seven acidic residues around the type 1 copper site in AcNIR, is the site of protein-protein interaction with a positively charged hydrophobic patch on AcPAZ. In AxNIR, four of the negatively charged residues (Glu-112, Glu-133, Glu-195, and Asp-199) are conserved at the corresponding positions of AcNIR, whereas the other three residues are not acidic amino acids but neutral amino acids (Ala-83, Ala-191, and Gly-198). Seven mutant AxNIRs with additional negatively charged residues surrounding the hydrophobic patch of AxNIR (A83D, A191E, G198E, A83D/A191E, A93D/G198E, A191E/G198E, and A83D/A191E/G198E) were prepared to enhance the specificity of the electron transport reaction between AcPAZ and AxNIR. The k(ET) values of these mutants become progressively larger as the number of mutated residues increases. The K(m) and k(ET) values of A83D/A191E/G198E (K(m) = 88 microM and k(ET) = 4.1 x 10(5) M(-1) s(-1)) are 15-fold smaller and 4.7-fold larger than those of wild-type AxNIR, respectively. These results suggest that the introduction of negatively charged residues into the docking surface of AxNIR facilitates both the formation of electron transport complex and the electron transfer reaction.

MeSH terms

  • Achromobacter denitrificans / metabolism*
  • Amino Acids / chemistry
  • Azurin / analogs & derivatives*
  • Azurin / chemistry*
  • Binding Sites
  • Copper / chemistry
  • Electrochemistry
  • Electron Transport
  • Electrons
  • Electrophysiology
  • Kinetics
  • Models, Chemical
  • Models, Molecular
  • Mutagenesis
  • Mutation
  • Nitrite Reductases / chemistry*
  • Nitrite Reductases / genetics
  • Plasmids / metabolism
  • Protein Conformation
  • Protein Engineering / methods*
  • Protein Structure, Tertiary
  • Spectrophotometry

Substances

  • Amino Acids
  • pseudoazurin
  • Azurin
  • Copper
  • Nitrite Reductases
  • nitrite reductase, copper-containing