Gene synthesis, expression, and mutagenesis of the blue copper proteins azurin and plastocyanin.

Abstract

Genes for the blue copper proteins Populus nigra var. italica plastocyanin and Pseudomonas aeruginosa azurin have been constructed by a stepwise procedure. The leader sequence for azurin has been placed before the genes directing plastocyanin and azurin transport to the periplasmic space when the genes are expressed in Escherichia coli. Sitesaturation mutagenesis has been used to alter two copperbinding residues of azurin and Met-92 of plastocyanin. While the plastocyanin mutants do not appear to bind copper, the azurin variants all bind copper and show characteristic type I blue copper centers. In particular, the electronic spectra reflect the dominance of the charge transfer interaction between copper and the thiolate of Cys-112, being relatively insensitive to changes in Met-121 or His-46. In contrast, removal of Met-121 appreciably alters the EPR spectra of the mutants, although, to a first order, the spectra of all mutants are themselves similar, suggesting a more distorted geometry around copper in the mutants than in the wild type.The family of blue copper proteins includes plastocyanins (1) from green plants and some algae and azurin (2) from bacteria. These proteins perform essential roles as electron carriers (3, 4) in such important processes as photosynthesis and bacterial respiration. They provide a unique ligand environment (5-8) to their single type I copper atom that endows them with a rich blue color as well as an unusually high potential for the Cu(II)-Cu(I) couple (9). Furthermore, the ligation geometries are essentially identical for both Cu(II) and Cu(I) forms near neutral pH, giving these proteins the ability to transfer electrons very rapidly (10,11). Lastly, the three-dimensional structures for a representative azurin at 1.8 A (12) and plastocyanin at 1.6 A (13) have been determined. For these reasons, the blue copper proteins provide attractive candidates for mutagenic structurefunction studies (14) aimed at gaining insights into such diverse aspects of their behaviors as electronic spectra, paramagnetic properties, redox potentials, rates of electron transfer, transmission of electrons through the interior of the protein for subsequent transfer to redox partners, and surface sites involved in such protein-protein interactions. The close relationship in both structure and function of the plastocyanins and azurins provides an additional attraction for the concurrent study of these questions against these similar, but nevertheless significantly different, protein backgrounds.We have approached this problem by the total synthesis of genes for poplar (Populus nigra var. italica) leaf plastocyanin (because of its highly refined three-dimensional structure; ref. 13) and Pseudomonas aeruginosa azurin (15). The synthetic genes were introduced adjacent to synthetic leader sequences for plastocyanin and azurin and the resulting constructs were expressed in Escherichia coli. When grown in the presence of 1 mM Cu(II), the properly processed and folded native proteins can be isolated from ...

The role of His117 in the redox reactions of azurin from Pseudomonas aeruginosa

“…[9], that the presence of a histidine-imidazole, or even of a nitrogen atom, at the fourth ligand position of the copper is no prerequisite for the typical spectral properties of blue copper proteins. Absorbance spectra resembling that of wild-type azurin have also been reported for mutants lacking either histidine 46 or methionine 121 [10,11] but not for a mutant lacking cysteine 112 [12], underscoring the fact that it is primarily the cysteine-sulphur that determines the spectral signature of type-1 copper.…”

Electron‐Transfer Properties and Active‐Site Structure of the Type 1 (Blue) Copper Protein Umecyanin

“…["I Site-directed mutagenesis studies of azurin have demonstrated that the axial methionine is not essential for the creation of a Type 1 blue copper site.[". 131 Similar studies of plastocyanin [13] and the Type 1 copper site of nitrite reductase [14] have so far failed to produce a Cu" binding active site. The fact that the Metl21Gln azurin mutant appears to possess many of the properties of oxidised ~tellacyanin['~~ also indicates the presence of an axial glutamine ligand in stellacyanin.…”

Engineering type 1 copper sites in proteins

“…The information for crossing more than one membrane is hidden in the plastocyanin signal sequence. Apart from the cloning of naturally occurring genes there is also a report of a complete in vitro synthesis of the genes of an azurin and a plastocyanin [41].…”