Gene Synthesis, High-Level Expression, and Mutagenesis of Thiobacillus ferrooxidans Rusticyanin: His 85 Is a Ligand to the Blue Copper Center

Abstract

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/lacOR 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).(ABSTRACT TRUNCATED AT 250 WORDS)

The HiPIP from the acidophilic Acidithiobacillus ferrooxidans is correctly processed and translocated in Escherichia coli, in spite of the periplasm pH difference between these two micro-organisms

“…Rusticyanin, a blue-copper protein, has been produced in the cytoplasm (Casimiro et al, 1995) or in the periplasm (Bengrine et al, 1998) but, in both cases, the copper was not incorporated and reconstitution in vitro was required. Similarly, copper insertion in subunit II of the aa 3 -type cytochrome oxidase from A. ferrooxidans did not occur when produced in the periplasm of E. coli (J.…”

Cloning, expression, and spectroscopic characterization of Cucumis sativus stellacyanin in its nonglycosylated form

“…Subsequently, the nucleotide and deduced amino acid sequences of the genes for various mononuclear blue copper proteins from different microorganisms became available, notably for azurin, pseudoazurin, amicyanin, and more recently for halocyanin (Canters, 1987;Yamamoto et al, 1987;Arvidsson et al, 1989;Chistoserdov et al, 1991;Ubbink et al, 1991;Mattar et al, 1994). In addition, an artificial gene for rusticyanin has been constructed and amplified using PCR based on the amino acid sequence (Casimiro et al, 1995). The availability of these genes has stimulated research in this area significantly, particularly in the case of plastocyanin and azurin, where various mutant proteins have been engineered.…”

Structural Analysis of Respirasomes in Electron Transfer Pathway of Acidithiobacillus ferrooxidans: A Computer-Aided Molecular Designing Study

“…Further, previous reports have indicated that Asp73 might serve as one of the Cu coordinating ligands [56]. In contrast, subsequent site-specific mutants and gene expression results revealed that His85 acts as the Cu coordinating ligand [57]. In our present docking model, we observed that Asp73 is actually situated at the opposite surface of Cu site in RcY and thus is unlikely a coordinate Cu atom.…”