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 abs orption bands at 459 (epsilon greater-than-or-equal-to 1460) and 595 n m (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 yeas t copper-zinc superoxide dismutase (CuZnSOD) using site-directed mutag enesis. It was expressed in a T7 polymerase expression system in E. co li, purified to homogeneity by ion-exchange and gel-filtration chromat ography, 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 envelop e modulation (ESEEM) spectroscopies demonstrates that this green coppe r-containing protein is a new member of the blue copper protein family . It has UV-vis, MCD, and resonance Raman (RR) spectra that are simila r to those of the blue copper center in Achromobacter cycloclastes nit rite reductase and EPR spectra that are similar to those of the blue c opper protein stellacyanin. The UV-vis spectrum of the Co(II) derivati ve is also similar to those of cobalt-substituted blue copper proteins . The two strong absorption bands at 459 and 595 nm in the copper deri vative are assigned to cysteine sulfur-to-Cu(II) charge-transfer trans itions. An examination of the published visible spectral parameters of many blue copper protein centers leads to the conclusion that all hav e absorption bands of variable intensity at or near 460 nm and that th e 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 i ncrease in the bonding interactions with a fourth ligand which further displaces the copper from the Cu-N2S plane formed by the conserved cy steinyl sulfur atom and the two histidine nitrogen atoms. We also find that the presence of the thiolate-Cu(II) bond greatly increases the r edox reactivity of this site relative to that of the Cu2Cu2 wild type protein. This result is consistent with the thiolate providing an effi cient superexchange pathway for electron transfer.