2idt
|
Structure of M98Q mutant of amicyanin, Cu(II)
OverviewOverview
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 (</=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.
About this StructureAbout this Structure
2IDT is a Single protein structure of sequence from Paracoccus denitrificans with CU as ligand. Full crystallographic information is available from OCA.
ReferenceReference
Generation of Novel Copper Sites by Mutation of the Axial Ligand of Amicyanin. Atomic Resolution Structures and Spectroscopic Properties(,)., Carrell CJ, Ma JK, Antholine WE, Hosler JP, Mathews FS, Davidson VL, Biochemistry. 2007 Feb 20;46(7):1900-1912. PMID:17295442
Page seeded by OCA on Wed Nov 21 12:15:58 2007