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Introduction of a π-π interaction at the active site of a cupredoxin: Characterization of the met16Phe pseudoazurin mutant

Lookup NU author(s): Sachiko Yanagisawa, Dr Katsuko Sato, Professor Christopher Dennison

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Abstract

The Met16Phe mutant of the type 1 copper protein pseudoazurin (PACu), in which a phenyl ring is introduced close to the imidazole moiety of the His81 ligand, has been characterized. NMR studies indicate that the introduced phenyl ring is parallel to the imidazole group of His81. The mutation has a subtle effect on the position of the two S(Cys)→Cu(II) ligand-to-metal charge transfer bands in the visible spectrum of PACu(II) and a more significant influence on their intensities resulting in a A459/A598 ratio of 0.31 for Met16Phe as compared to a A453/A594 ratio of 0.43 for wild-type PACu(II) at pH 8. The electron paramagnetic resonance spectrum of the Met16Phe variant is more axial than that of the wild-type protein, and the resonance Raman spectrum of the mutant exhibits subtle differences. A CγH proton of Met86 exhibits a much smaller hyperfine shift in the paramagnetic 1H NMR spectrum of Met16Phe PACu(II) as compared to its position in the wild-type protein, which indicates a weaker axial Cu-S(Met86) interaction in the mutant. The Met16Phe mutation results in an ∼60 mV increase in the reduction potential of PACu. The pKa value of the ligand His81 decreases from 4.9 in wild-type PACu(I) to 4.5 in Met16Phe PACu(I) indicating that the π-π contact with Phe16 stabilizes the Cu-N(His81) interaction. The Met16Phe variant of PACu has a self-exchange rate constant at pH* 7.6 (25 °C) of 9.8 × 103 M-1 s-1 as compared to the considerably smaller value of 3.7 × 103 M-1 s-1 for the wild-type protein under identical conditions. The enhanced electron transfer reactivity of Met16Phe PACu is a consequence of a lower reorganization energy due to additional active site rigidity caused by the π-π interaction between His81 and the introduced phenyl ring.


Publication metadata

Author(s): Yanagisawa S, Sato K, Kikuchi M, Kohzuma T, Dennison C

Publication type: Article

Publication status: Published

Journal: Biochemistry

Year: 2003

Volume: 42

Issue: 22

Pages: 6853-6862

ISSN (print): 0006-2960

ISSN (electronic): 1943-295X

Publisher: American Chemical Society

URL: http://dx.doi.org/10.1021/bi030030p

DOI: 10.1021/bi030030p

PubMed id: 12779340


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