Generation of Novel Copper Sites by Mutation of the Axial Ligand of Amicyanin. Atomic Resolution Structures and Spectroscopic Properties^,

Abstract

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.

Spectroscopic Characterization of a Green Copper Site in a Single-Domain Cupredoxin

“…During the last decade, several studies demonstrated the possibility to engineer cupredoxins and to obtain variants with different spectroscopic features [19]. For example, by mutating the methionine ligand, a classic blue copper site can be transformed into a green copper site [20], [21], or into a red one [22]. In other cases, it was even possible to change a blue mononuclear-copper site into a purple binuclear-copper site [23], [24].…”

The axial ligand and extent of protein folding determine whether Zn or Cu binds to amicyanin

“…Metal content of the protein was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) using a Spectro Genesis spectrometer as previously described [3]. Protein samples were incubated with 3 mM EDTA prior to buffer exchange and analysis.…”

“…Native amicyanin [9] and M98Q amicyanin [3] were expressed in E. coli and purified from the periplasmic fraction of the cells as described previously. Two forms of apoamicyanin which are designated ''fully folded'' apoamicyanin and ''partially folded'' apoamicyanin were prepared for metal reconstitution studies.…”

“…This species is known to be completely folded because it was shown by X-ray crystallography that the crystal structure of this apoprotein is essentially identical to that of holoamicyanin with copper absent but the type 1 geometry intact [10]. The second form which was designated partially folded apoamicyanin was prepared as described originally by Diederix et al [6], and was used previously to reconstitute this M98Q amicyanin with copper [3]. Amicyanins were incubated in 10 mM HEPES buffer, pH 8.0, containing 6 M guanidine HCl, 50 mM EDTA and 2 mM dithiothreitol.…”

“…Amicyanin exhibits a very high specificity and affinity for Cu 2+ . We recently characterized an M98Q mutant amicyanin which is isolated with zinc bound rather than copper, but which can be easily reconstituted with copper after denaturation and the removal of zinc [3]. The decreased selectivity for copper over zinc exhibited by M98Q amicyanin provides an opportunity to use this mutant to address a key question in metalloprotein biogenesis.…”