title: Role of the axial ligand in type 1 Cu centers studied by point mutations of met148 in rusticyanin.
abstract: 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.
authors.affiliation: CCLRC Daresbury Laboratory, Warrington, Cheshire WA4 4AD, and Faculty of Applied Sciences, De Montfort University, The Gateway, Leicester LE1 9BH, U.K.
authors.forename: J F
authors.initials: JF
authors.lastname: Hall
authors.forename: L D
authors.initials: LD
authors.lastname: Kanbi
authors.forename: R W
authors.initials: RW
authors.lastname: Strange
authors.forename: S S
authors.initials: SS
authors.lastname: Hasnain
mesh_terms: D001400:Azurin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D003300:Copper / Q000737:chemistry*; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D008715:Methionine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; C012543:rusticyanin; D001400:Azurin; D003300:Copper; D008715:Methionine