Lennart G. Lundberg

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.

Flash-photolysis studies of the electron transfer from genetically modified spinach plastocyanin to photosystem I

Plastocyanin (Pc) has been modified by site‐directed mutagenesis at two separate electron‐transfer (ET) sites: Leu‐12‐Glu at a hydrophobic patch, and Tyr‐83‐His at an acidic patch. The reduction potential at pH 7.5 is decreased by 26 mV in Pc(Leu‐12‐Glu) and increased by 35 mV in Pc(Tyr‐83‐His). The latter mutant shows a 2‐fold slower intracomplex ET to photosystem I (PSI) as expected from the decreased driving force. The affinity for PSI is unaffected for this mutant but is drastically decreased for Pc(Leu‐12‐Glu). It is concluded that the hydrophobic patch is more important for the ET to PSI.

Rack-induced bonding in blue copper proteins: spectroscopic properties and reduction potential of the azurin mutant Met-121 Leu

Site‐directed mutagenesis has been used to prepare azurin in which the methionine‐121 residue has been replaced by leucine. The oxidized mutant protein displays the strong blue color and characteristic EPR signal of a type 1 Cu(II) ion, showing that methionine is not an obligatory component of a blue copper site. The optical absorption maximum is shifted 5 nm towards longer wavelength and the extinction coefficient increased by about 10% compared to the wild‐type protein. In addition, there are small changes in the EPR parameters, in particular the copper hyperfine splitting. The reduction potential is increased by 70 mV. The results show that a small change in primary structure without any alteration in the three strong ligands can perturb the Cu(II) site and shift the reduction potential, in accord with the concept of rack‐induced bonding in blue copper proteins.

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.

Expression of spinach plastocyanin in E. coli.

An expression vector designed for overexpression of plastocyanin in the periplasmic space of E. coli has been developed. The vector contains the signal peptide sequence of Pseudomonas aeruginosa azurin and the mature sequence of spinach plastocyanin. The precursor is efficiently translocated to the periplasmic space and correctly processed to mature plastocyanin. No detectable amount of plastocyanin was present in the cytoplasmic or in the membrane fraction. A large scale preparation of the recombinant plastocyanin in a 20 litre fennentor yielded approximately 30 mg of pure plastocyanin. The recombinant protein obtained from E. coli shows CD, EPR and optical properties identical to plastocyanin isolated from spinach.

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.

The structural gene for cytochrome c551 from Pseudomonas aeruginosa: The nucleotide sequence shows a location downstream of the nitrite reductase gene

The gene coding for Pseudomonas aeruginosa cytochrome c 551 has been cloned and its nucleotide sequence determined. Cytochrome c 551 is expressed as a 104 amino acid pre‐proteinfrom which a signal peptide of 22 amino acids is cleaved off during the translocation across the cytoplasmic membrane. The gene is located just downstream of the gene coding for nitrite reductase on the Pseudomonas aeruginosa chromosome, suggesting that these genes form an operon.The gene coding for Pseudomonas aeruginosa cytochrome c551 has been cloned and its nucleotide sequence determined. Cytochrome c551 is expressed as a 104 amino acid pre-protein from which a signal peptide of 22 amino acids is cleaved off during the translocation across the cytoplasmic membrane. The gene is located just downstream of the gene coding for nitrite reductase on the Pseudomonas aeruginosa chromosome, suggesting that these genes form an operon.

The reactivity of spinach plastocyanin mutants with inorganic oxidants [Fe(CN)6]3– and [Co(phen)3]3+

Stopped-flow rate constants for the oxidation of spinach plastocyanin PCuI mutants Leu12Glu, Leu12Asn, Asp42Asn and Tyr83Phe have been determined, and are discussed in terms of two-site reactivity for electron transfer.

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.

Crystallization and preliminary crystallographic data for the azurin mutant End-121 from Pseudomonas aeruginosa

Pseudomonas aeruginosa azurin has been crystallized from a mutant where residues from Met 121 to Lys128 have been deleted from the protein. The crystals form pale-blue well formed prisms in the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 60.79 (5), b = 123.47 (5), c = 187.77 (5) A. The crystals diffract to 3.0 A and there are eight molecules in the asymmetric unit.