An artificial gene of the blue copper protein rusticyanin from Thiobacillus ferrooxidans was constructed from eight overlapping oligonucleotides in a recursive ''one-pot'' polymerase chain reaction. The gene was placed behind the T7/lacO(R) promoter of pET24a and expressed in Escherichia coli as a soluble protein. A purification scheme involving a pH titration step, cation-exchange chromatography, and reverse-phase HPLC separation provided yields of the apoprotein ranging from 70 to 100 mg/L of cell culture; reconstitution with Cu(II) is quantitative at pH 3.4-5.5. The redox reactions and the electronic absorption and EPR spectra of the recombinant Cu(II)-rusticyanin and NMR spectra of the reduced holoprotein are indistinguishable from those of the protein derived from T. ferrooxidans, Rusticyanin possesses the phylogenetically conserved carboxy-terminal loop of three copper ligands (Cys 138, His 143, and Met 148), but the identity of the fourth ligand was not clear from sequence homology to other blue copper proteins. To address this question directly, we have prepared two site-specific mutants where two of the proposed ligands, Asp 73 and His 85, have been replaced with alanine. The Asp73Ala mutant retained the electronic properties of the wild-type blue copper center (absorption maxima at 452, 597, and 750 nm), whereas the His85Ala variant gave rise to a green type 1 copper protein (absorption maxima at 455 and 618 nm), The ability of the His85Ala mutant to bind exogenous ligands such as chloride ions is consistent with the idea that a cavity has been introduced at the copper site; the N-15 HSQC spectrum of the chloro derivative of the mutant indicates that the mutation at position 85 does not perturb the overall fold of the protein. These results, together with those of a recent NMR study [Hunt, A. H., Toy-Palmer, A., Assa-Munt, N., Cavanagh, J., Blake, R. C., II, and Dyson, H. J. (1993) J. Mel. Biol. 244, 370-384], firmly establish that rusticyanin has the classical ''blue-copper'' coordination set of CysHis(2)Met. The availability of a high-level bacterial expression system will allow us to address the source of the unusual acid stability and redox properties of the protein through combination of site-directed mutagenesis experiments and structural analyses.
