Generation of novel copper sites by mutation of the axial ligand of amicyanin. Atomic resolution structures and spectroscopic properties.
Publication Type:Journal Article
Source:Biochemistry, Volume 46, Issue 7, p.1900-12 (2007)
Keywords:Bacterial Proteins, Binding Sites, Copper, Crystallography, X-Ray, Electron Spin Resonance Spectroscopy, Ligands, Metalloproteins, Models, Molecular, Oxidation-Reduction, Paracoccus denitrificans, Protein Conformation, Solutions, Spectrophotometry, Temperature
<p>Amicyanin from Paracoccus denitrificans is a type 1 copper protein with three strong equatorial copper ligands provided by nitrogens of His53 and His95 and the sulfur of Cys92, with an additional weak axial ligand provided by the sulfur of Met98. Met98 was replaced with either Gln or Ala. As isolated, the M98A and M98Q mutant proteins contain zinc in the active site. The zinc is then removed and replaced with copper so that the copper-containing proteins may be studied. Each of the mutant amicyanins exhibits a marked decrease in thermal stability relative to that of native amicyanin, consistent with the weaker affinity for copper. Crystal structures were obtained for the oxidized and reduced forms of M98A and M98Q amicyanins at atomic resolution (<or=1.0 A). The crystal structure of oxidized M98A amicyanin exhibits a type 1 ligation geometry but with the axial ligand provided by a water, which fills the void left by the mutation of Met to Ala. The protein undergoes a reversible switch in ligation geometry when going from the aqueous to the frozen state. The visible absorption spectrum in solution is characteristic of type 1 copper, consistent with the crystal structure. On freezing, the blue color is lost, and EPR spectroscopy reveals that the copper is primarily type 2. The crystal structure of reduced M98A amicyanin exhibits an unprecedented ligation geometry in which the His95-Cu coordination is broken, and copper is left with only two ligands from His53 and Cys92 in an almost linear coordination. The replacement of Met98 with Gln yielded a type 1 copper site with increased rhombicity evident from its EPR and visible absorption spectra, and an increase in distance from Cu to the trigonal equatorial plane seen in the crystal structure. Gln98 coordinates more strongly with copper than Met, and the oxidized and reduced forms each exhibit two alternate conformers. EPR and metal analysis of oxidized M98Q amicyanin indicate that a small population of the protein contains weakly bound type 2 copper, which may be removed by washing with EDTA. These results demonstrate that the identity as well as position and rigidity of the axial ligand of the type 1 copper site has a profound influence in the uptake specificity of metal ions, protein stability, and determination of the active site geometry and its spectroscopic properties.</p>