Type 1 Cu centers in cupredoxins, nitrite reductases, and multi-copper oxidases utilize the same trigonal core ligation to His-Cys-His, with a weak axial ligand generally provided by a Met sulfur. In azurin, an additional axial ligand, a carbonyl oxygen from a Gly, is present. The importance of these axial ligands and in particular the Met has been debated extensively in terms of their role in fine-tuning the redox potential, spectroscopic properties, and rack-induced or entatic state properties of the copper sites. Extensive site-directed mutagenesis of the Met ligand has been carried out in azurin, but the presence of an additional carbonyl oxygen axial ligand has made it difficult to interpret the effects of these substitutions. Here, the axial methionine ligand (Met148) in rusticyanin is replaced with Leu, Gln, Lys, and Glu to examine the effect on the redox potential, acid stability, and copper site geometry. The midpoint redox potential varies from 363 (Met148Lys) to 798 mV (Met148Leu). The acid stability of the oxidized proteins is reduced except for the Met148Gln mutant. The Gln mutant remains blue at all pH values between 2.8 and 8, and has a redox potential of 563 mV at pH 3.2. The optical and rhombic EPR properties of this mutant closely resemble those of stellacyanin, which has the lowest redox potential among single-type 1 copper proteins (185 mV). The Met148Lys mutant exhibits type 2 Cu EPR and optical spectra in this pH range. The Met148Glu mutant exhibits a type 2 Cu EPR spectrum above pH 3 and a mixture of type 1 and type 2 Cu spectra at lower pH. The Met148Leu mutant exhibits the highest redox potential ( approximately 800 mV at pH 3.2) which is similar to the values in fungal laccase and in the type 1 Cu site of ceruloplasmin where this axial ligand is also a Leu.
The expression of rusticyanin in Escherichia coli and a number of mutants for Ser86 is reported. Mutations of Ser86 to Asn, Asp, Gln, and Leu were undertaken as this is an Asn residue in other structurally characterized cupredoxins, and it has been suggested that this may be partly responsible for the high redox potential (680 mV) and extreme acid stability of rusticyanin. N-Terminal sequence analysis, together with other biochemical and spectrochemical characterization, shows that the recombinant wild-type protein is indistinguishable from native rusticyanin. All four mutants retain the rhombic nature of the EPR spectra and a significant absorption maximum at approximately 450 nm, thus confirming that the overall geometry of the Cu ligands is essentially maintained. The oxidized form of all four mutants is less acid stable than the wild-type protein, although the detailed mechanism of lability varies. Ser86Leu readily loses copper as the pH is reduced from 4.0, but the protein does not denature. A significant proportion (approximately 30%) of Ser86Gln is denatured at lower pH values, whereas Ser86Asn and Ser86Asp are stable as the reduced (CuI) protein. The redox potential also varies by approximately 110 mV (590-702 mV) upon these single point mutations, thus providing direct experimental support to the idea that this residue is at least in part responsible for the acid stability and the highest redox potential of rusticyanin in the cupredoxin family.
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