EFFECT OF LYSINE IONIZATION ON THE STRUCTURE AND ELECTROCHEMICAL-BEHAVIOR OF THE MET44-]LYS MUTANT OF THE BLUE-COPPER PROTEIN AZURIN FROM PSEUDOMONAS-AERUGINOSA

The structural and spectrochemical effects of the replacement of Met44 in the hydrophobic surface patch of azurin from Pseudomonas aeruginos a by a lysine residue were studied as a function of the ionization sta te of the lysine. In the pH range 5-8, the optical absorption, resonan ce Raman, EPR and electron spin-echo envelope modulation spectroscopic properties of wild-type and Met44-->Lys (M44K) azurin are very simila r, indicating that the Cu-site geometry has been maintained. At higher pH, the deprotonation of Lys44 in M44K azurin (pK(a) 9-10) is accompa nied by changes in the optical-absorption maxima (614 nm and 450 nm in stead of 625 nm and 470 nm) and in the EPR g(parallel-to) 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-excha nge rate of M44K azurin supports the assignment of Lys44 as the ioniza ble group and demonstrates the importance of the hydrophobic patch for electron transfer. The pH dependence of the midpoint potentials of wi ld-type and M44K azurin can be accounted for by the ionizations of His 35 and His83 and by the additional electrostatic effect of the mutatio n.