Modulating the Redox Potential and Acid Stability of Rusticyanin by Site-Directed Mutagenesis of Ser86

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• 作者：John F. Hall ; Lalji D. Kanbi ; Ian Harvey ; Loretta M. Murphy ; and S. Samar Hasnain
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：August 18, 1998
• 年：1998
• 卷：37
• 期：33
• 页码：11451 - 11458
• 全文大小：118K
• 年卷期：v.37,no.33(August 18, 1998)
• ISSN：1520-4995

文摘

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 structurallycharacterized cupredoxins, and it has been suggested that this may be partly responsible for the highredox potential (680 mV) and extreme acid stability of rusticyanin. N-Terminal sequence analysis, togetherwith other biochemical and spectrochemical characterization, shows that the recombinant wild-type proteinis indistinguishable from native rusticyanin. All four mutants retain the rhombic nature of the EPR spectraand a significant absorption maximum at ~450 nm, thus confirming that the overall geometry of the Culigands 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 pHis reduced from 4.0, but the protein does not denature. A significant proportion (~30%) of Ser86Gln isdenatured at lower pH values, whereas Ser86Asn and Ser86Asp are stable as the reduced (Cu^I) protein.The redox potential also varies by ~110 mV (590-702 mV) upon these single point mutations, thusproviding direct experimental support to the idea that this residue is at least in part responsible for theacid stability and the highest redox potential of rusticyanin in the cupredoxin family.