pH dependent copper binding properties of a CuA azurin variant with both bridging cysteines replaced with serines

Abstract: A double mutant of CuA azurin was prepared in which both bridging cysteine thiolate ligands of the binuclear CuA center were replaced by serine. The copper binding properties of this protein were investigated, and shown to be pH dependent. At lower pH (5.2±0.1), the protein binds one copper per protein molecule as demonstrated by electrospray ionization mass spectrometry. Copper titrations resulted in electronic absorptions at 730nm (peak) and ca. 330nm (shoulder) in the UV–Vis spectrum. EPR data show a four line pattern with hyperfine A ∥ =150G and g ∥ and g ⊥ values 2.32 and 2.03, characteristic of a type II (T2) copper. Superhyperfines to two nitrogen atoms were also observed. At higher pH (8.5±0.1), the protein binds upto two copper atoms per protein molecule, and copper titrations exhibit a blue transition at 595nm in the UV–Vis spectrum. The EPR data are consistent with two monomeric sites very similar to one another having hyperfines A ∥ =182 and 150G, g ∥ =2.24 and 2.22 and a similar g ⊥ value of 2.01. These results indicate that both bridging cysteines play a critical role in the CuA center, and replacing them with serines is not enough to maintain the symmetrical diamond core structure or the characteristic electronic and functional properties of the CuA center. [Copyright &y& Elsevier]