A new type 2 copper cysteinate azurin. Involvement of an engineered exposed cysteine in copper binding through internal rearrangement

J Biol Chem. 2002 Nov 15;277(46):44121-30. doi: 10.1074/jbc.M202977200. Epub 2002 Aug 16.

Abstract

The double mutant H117G/N42C azurin exhibits tetragonal type 2 copper site characteristics with Cys(42) as one of the copper ligands as concluded from spectroscopic evidence (UV-visible, EPR, and resonance Raman). Analysis of the kinetics of copper uptake by the apoprotein by means of stopped flow spectroscopy suggests that the solvent-exposed Cys(42) assists in binding the metal ion and carrying it over to the active site where it becomes coordinated by, among others, a second cysteine, Cys(112). A structure is proposed in which the loop from residue 36 to 47 has rearranged to form a tetragonal type 2 copper site with Cys(42) as one of the ligands. The process of copper uptake as observed for the double mutant may be relevant for a better understanding of the way copper chaperones accept and transfer metal ions in the living cell.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Azurin / chemistry*
  • Binding Sites
  • Copper / chemistry*
  • Cysteine / chemistry*
  • Electromagnetic Fields
  • Electron Spin Resonance Spectroscopy
  • Escherichia coli / metabolism
  • Hydrogen-Ion Concentration
  • Kinetics
  • Ligands
  • Models, Molecular
  • Plasmids / metabolism
  • Protein Binding
  • Spectrophotometry
  • Spectrum Analysis, Raman
  • Time Factors
  • Ultraviolet Rays

Substances

  • Ligands
  • Azurin
  • Copper
  • Cysteine