Amino acid sequence of the blue copper protein rusticyanin from Thiobacillus ferrooxidans

Abstract

Rusticyanin is a small blue copper protein isolated from Thiobacillus ferrooxidans. The amino acid sequence of the rusticyanin has been determined by the structural characterization of tryptic and endoproteinase Asp-N peptides with use of amino terminal microsequencing, fast atom bombardment mass spectrometry, and electrospray triple-quadrupole mass spectrometry techniques. Amino acid analysis, carboxy-terminal sequence analysis, and circular dichroism spectroscopy were also performed on the protein. Amino acid sequence identity among rusticyanin and six other small blue copper proteins is apparent only in the limited C-terminal region of each protein bearing three of the four putative copper ligands. A structural model of the rusticyanin is proposed where the protein is principally a beta-barrel comprised of six strands. This model is consistent with the circular dichroism data and computational predictions of the secondary structure of rusticyanin. A feature of the model is the hypothesis that Asp 73 may serve as a fourth copper ligand.

Engineering type 1 copper sites in proteins

“…1). For rusticyanin one of the coordinating histidines could not be located in the primary sequence [22,23]. It has been suggested that its position might be taken up by an aspartate [23].…”

Respiratory Isozyme, Two Types of Rusticyanin ofAcidithiobacillus ferrooxidans

“…1) from this known sequence of strain ATCC 23270. 21) These primers were used to amplify and clone rus from several strains of the bacterium. This strategy was successful for cloning rus of several strains.…”

“…The rusticyanin open reading frame was cloned by PCR using genomic DNA with the primers. The primers for PCR were based on the previously identiˆed rus gene, 21,22) or on the new gene identiˆed here. The primers are summarized in Table 1 and Fig.…”

“…5,[17][18][19][20] The gene for rusticyanin (rus ) is well known, havingˆrst been sequenced from A. ferrooxidans strain ATCC 23270. 21) Later, the complete gene was cloned from strain ATCC 33020 22) and found to be comprised of 564 bp, encoding 155 amino acids, with additional codons encoding a 32-amino acid signal peptide. [21][22][23][24] The primary structure of the rus gene was found to be the same for both genes.…”

“…21) Later, the complete gene was cloned from strain ATCC 33020 22) and found to be comprised of 564 bp, encoding 155 amino acids, with additional codons encoding a 32-amino acid signal peptide. [21][22][23][24] The primary structure of the rus gene was found to be the same for both genes. 22) Furthermore, rus was shown to be situated downstream of genes for C-type cytochrome and cytochrome oxidase (COX ) 22) and these three have been thought to represent an operon present as a single copy within a cluster of genes encoding electron transfer proteins.…”