Effect of lysine ionization on the structure and electrochemical behaviour of the Met44Lys mutant of the blue-copper protein azurin from Pseudomonas aeruginosa (original) (raw)

1993, European Journal of Biochemistry

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 (pK, 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 gll 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. Fax: +31 71 274537. Abbreviations. Ches, 2-(N-cyclohexylamino)ethmesulfonic acid; Cu(1) azurin, reduced azurin; Cu(I1) azurin, oxidized azurin; Em, midpoint potential ; ESE, electron self-exchange ; ESEEM, electron spin-echo envelope modulation; M44K, Met44+Lys ; RR, resonance Raman; wt, wild type; FT, Fourier transform. due His117 is located in the centre of this patch. The replacement of Met44 by the protonated lysine residue hardly affects the spectroscopic properties of the Cu site, but causes a considerable decrease of the k,,, value of azurin. At pH >8, deprotonation of Lys44 produces a new type-] Cu site, whereas the magnitude of the k,,, value of the protein is largely restored. At pH 5 and pH 8, the midpoint potential, Em, of Met44+Lys (M44K) azurin is higher than the wildtype (wt) azurin Em by 60 mV. At pH > 8, deprotonation of Lys44 in M44K azurin reduces the En, difference from 60 mV to 40 mV. The pH dependence of the wt and M44K azurin En, values is analyzed in terms of the titration of His35, His83 and Lys44. In addition, the effects of the M44K mutation and the Cu-ion oxidation state (I or TI) on the pK, values of the titratable His35 and His83 of azurin are reported and analyzed. The structural and functional implications of the findings are discussed.