Torbjörn Pascher

Expression of the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli

The structural gene for the blue copper protein azurin from Pseudomonas aeruginosa has been subcloned in different expression plasmid vectors. The highest yield of expression was obtained when the gene with its native ribosome‐binding site was placed downstream of the lac promoter in plasmid pUC18. The protein is exported to the periplasmic space in Escherichia coli and the amount corresponds to 27% of the total protein content in the periplasmic space. The preprotein is cleaved correctly according to N‐terminal sequencing of the purified protein. Azurin has been purified in large amounts and is spectroscopically indistinguishable from the protein purified from P. aeruginosa.

Electronic absorption spectra of M(II)(Met121X) azurins (MCo, Ni, Cu; XLeu, Gly, Asp, Glu): charge-transfer energies and reduction potentials

Abstract Electronic absorption spectra of the Co(II) and Ni(II) derivatives of Met121X (XLeu, Gly, Asp, Glu) azurin mutants have been measured. Coordination of carboxylate to the metal ion is indicated by LF and LMCT band shifts in the Met121Glu proteins. The relatively low reduction potentials of the Cu(II)(Met121X) (XAsp, Glu) azurins accord with the LMCT energies of the corresponding Co(II) derivatives.

Modification of the electron-transfer sites of Pseudomonas aeruginosa azurin by site-directed mutagenesis

Site‐directed mutagenesis of the structural gene for azurin from Pseudomonas aeruginosa has been used to prepare azurins in which amino acid residues in two separate electron‐transfer sites have been changed: His‐35‐Lys and Glu‐91‐Gln at one site and Phe‐114‐Ala at the other. The chargetransfer band and the EPR spectrum are the same as in the wild‐type protein in the first two mutants, whereas in the Phe‐114‐Ala azurin, the optical band is shifted downwards by 7 nm and the copper hyperfine splitting is decreased by 4·10−4/cm. This protein also shows an increase of 20–40 mV in the reduction potential compared to the other azurins. The potentials of all four azurins decrease with increasing pH in phosphate but not in zwitterionic buffers with high ionic strength. The rate constant for electron exchange with cytochrome c 551 is unchanged compared to the wild‐type protein in the Phe‐114‐Ala azurin, but is increased in the other two mutant proteins. The results suggest that Glu‐91 is not important for the interaction with cytochrome c 551 and that His‐35 plays no critical role in the electron transfer to the copper site.

Structural Characterisation of Azurin from Pseudomonas aeruginosa and some of Its Methionine-121 Mutants

Azurin from Pseudomonas aeruginosa and two mutants where the methionine ligand has been mutated have been studied in order to directly investigate the functional and structural significance of this ligand in the blue copper proteins. Redox potentials, x-ray absorption fine structure (XAFS), electron paramagnetic resonance (EPR) and optical spectra are obtained in an attempt to provide a direct correlation between the spectrochemical properties and the immediate structure of this redox centre.