Effect of lysine ionization on the structure and electrochemical behaviour of the Met44→Lys mutant of the blue-copper protein azurin from Pseudomonas aeruginosa

Authors

  • Mart VAN DE KAMP,

    1. Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, The Netherlands
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  • Gerard W. CANTERS,

    Corresponding author
    1. Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, The Netherlands
      Correspondence to G. W. Canters, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P. O. Box 9502, NL-2300 RA Leiden, The Netherlands
      Fax:+31 71 274537.
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  • Colin R. ANDREW,

    1. Department of Chemical and Biological Sciences, Oregon Graduate Institute of Science and Technology, Portland, USA
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  • Joann SANDERS-LOEHR,

    1. Department of Chemical and Biological Sciences, Oregon Graduate Institute of Science and Technology, Portland, USA
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  • Christopher J. BENDER,

    1. Department of Molecular Pharmacology, Albert Einstein College of Medicine of Yeshiva University, Bronx, USA
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  • Jack PEISACH

    1. Department of Molecular Pharmacology, Albert Einstein College of Medicine of Yeshiva University, Bronx, USA
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Correspondence to G. W. Canters, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P. O. Box 9502, NL-2300 RA Leiden, The Netherlands
Fax:+31 71 274537.

Abstract

The structural and spectrochemical effects of the replacement of Met44 in the hydrophobic surface patch of azurin from Pseudomonas aeruginosa by a lysine residue were studied as a function of the ionization state of the lysine. In the pH range 5–8, the optical absorption, resonance Raman, EPR and electron spin-echo envelope modulation spectroscopic properties of wild-type and Met44-→Lys (M44K) azurin are very similar, indicating that the Cu-site geometry has been maintained. At higher pH, the deprotonation of Lys44 in M44K azurin (pKa 9–10) is accompanied by changes in the optical-absorption maxima (614 nm and 450 nm instead of 625 nm and 470 nm) and in the EPR g value (2.298 instead of 2.241), indicative of a change in the bonding interactions of Cu at high pH. The strong pH dependence of the electron self-exchange rate of M44K azurin supports the assignment of Lys44 as the ionizable group and demonstrates the importance of the hydrophobic patch for electron transfer. The pH dependence of the midpoint potentials of wild-type and M44K azurin can be accounted for by the ionizations of His35 and His83 and by the additional electrostatic effect of the mutation.

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