Modulating the redox potential and acid stability of rusticyanin by site-directed mutagenesis of Ser86.
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.
School of Applied Sciences, De Montfort University, Leicester, U.K.
J F
JF
Hall
L D
LD
Kanbi
I
I
Harvey
L M
LM
Murphy
S S
SS
Hasnain
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001400:Azurin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006851:Hydrochloric Acid; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012694:Serine / Q000235:genetics*
D001426:Bacterial Proteins; C012543:rusticyanin; D001400:Azurin; D012694:Serine; D006851:Hydrochloric Acid