CONSTRUCTION OF A BLUE COPPER SITE AT THE NATIVE ZINC SITE OF YEAST COPPER-ZINC SUPEROXIDE-DISMUTASE

A stable, green, copper-containing protein with two strong visible absorption bands at 459 (epsilon greater-than-or-equal-to 1460) and 595 nm (epsilon greater-than-or-equal-to 1420 M-1 cm-1) was constructed by replacing a histidine (His80) with a cysteine in the zinc site of yeast copper-zinc superoxide dismutase (CuZnSOD) using site-directed mutagenesis. It was expressed in a T7 polymerase expression system in E. coli, purified to homogeneity by ion-exchange and gel-filtration chromatography, converted to the apoprotein, and reconstituted by addition of Cu2+ to both the copper and the zinc sites. Spectral characterization by electronic absorption (UV-vis), magnetic circular dichroism (MCD), electron paramagnetic resonance (EPR), and electron spin echo envelope modulation (ESEEM) spectroscopies demonstrates that this green copper-containing protein is a new member of the blue copper protein family. It has UV-vis, MCD, and resonance Raman (RR) spectra that are similar to those of the blue copper center in Achromobacter cycloclastes nitrite reductase and EPR spectra that are similar to those of the blue copper protein stellacyanin. The UV-vis spectrum of the Co(II) derivative is also similar to those of cobalt-substituted blue copper proteins. The two strong absorption bands at 459 and 595 nm in the copper derivative are assigned to cysteine sulfur-to-Cu(II) charge-transfer transitions. An examination of the published visible spectral parameters of many blue copper protein centers leads to the conclusion that all have absorption bands of variable intensity at or near 460 nm and that the ratio of the intensity of the 460-nm band to that of the 600-nm band correlates with the rhombicity of the EPR signal. A likely structural explanation for the difference between these spectral parameters and those of the metal center of plastocyanin is that they are due to an increase in the bonding interactions with a fourth ligand which further displaces the copper from the Cu-N2S plane formed by the conserved cysteinyl sulfur atom and the two histidine nitrogen atoms. We also find that the presence of the thiolate-Cu(II) bond greatly increases the redox reactivity of this site relative to that of the Cu2Cu2 wild type protein. This result is consistent with the thiolate providing an efficient superexchange pathway for electron transfer.