Expression and characterization of Met92Gln mutant plastocyanin from Silene pratensis.
To investigate the role of the copper-ligand Met92 in the structural and functional properties of silene plastocyanin (PC), Met92 was replaced with Gln, which is the purposed fourth copper-ligand in another blue copper protein, stellacyanin. By use of the recently developed expression system [Hibino et al. (1994) J. Biochem. 116, 826-832], the Met92Gln mutant of intermediate precursor plastocyanin was successfully expressed in Escherichia coli and accumulated in the periplasmic space as a mature protein. In contrast to the wild type, most of the Met92Gln mutant PC accumulated as an apoprotein. After purification, mutant apoprotein could incorporate copper ions, although less efficiently than the wild-type apoprotein. The absorption peak of Met92Gln mutant PC was blue-shifted from 597 nm in the wild type to 591 nm. The rhombic type EPR spectrum was obtained for the mutant in place of the axial spectrum in the wild type. Compared with that of the wild-type PC, the oxidation-reduction potentials of the Met92Gln mutant PC were lower by about 35 mV over the whole pH range examined. These results indicate that the Met92Gln mutant exhibited "stellacyanin-like" spectroscopic properties. Interestingly, the electron-transfer activities of the mutant PC with the physiological electron donor (cytochrome f) and acceptor (Photosystem I) were similar to that of the wild-type PC. Since Met92 is conserved in all the plastocyanins whose primary structures are known, we propose that the primarily function of Met as a copper-ligand is in the uptake of copper ions during folding rather than in electron-transfer activities.
Hibino
T
T
Department of Chemistry, Faculty of Science & Technology, Meijo University, Aichi.
Lee
B H
BH
Takabe
T
T
Takabe
T
T
D001483:Base Sequence; D003580:Cytochromes / Q000737:chemistry; D045348:Cytochromes f; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics*; D005973:Glutamine / Q000235:genetics*; D007700:Kinetics; D008715:Methionine / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010970:Plastocyanin / Q000096:biosynthesis / Q000235:genetics*; D013054:Spectrophotometry, Atomic
D003580:Cytochromes; D005973:Glutamine; D010970:Plastocyanin; D045348:Cytochromes f; D008715:Methionine