EXAFS ANALYSIS OF THE PH-DEPENDENCE OF THE BLUE-COPPER SITE IN AMICYANIN FROM THIOBACILLUS-VERSUTUS

The room temperature Cu K-edge EXAFS (extended X-ray absorption fine structure) spectrum of reduced and oxidized amicyanin, the blue copper protein from Thiobacillus versutus, was measured at low and high pH. The data interpretation was partly based on independent NMR evidence for the occurrence of a ligand histidine protonation at low pH (pK(a) = 6.9) in the reduced protein. In the oxidized protein two nitrogen-donors (from two histidines; Cu-N distances 1.95-2.01 angstrom and 1.86-1.89 angstrom) and a sulfur-donor (from a cysteine; Cu-S distance 2.11-2.13 angstrom) were identified and the coordination appears independent of pH. Upon reduction at high pH the Cu-S bond and one of the Cu-N bonds lengthen slightly (from 2.11 to 2.19 angstrom and from 2.01 to 2.18 angstrom, respectively). Upon lowering of the pH one of the N-donors of the Cu in reduced amicyanin disappears from the Cu EXAFS and a second S-donor (from a methionine) becomes visible at 2.41 angstrom from the Cu. The Debye-Waller factors are compatible with a Cu-N vibrational stretch frequency in the range of 150-250 cm-1 and one > 285 cm-1, and a Cu-S vibrational stretch frequency of about 150 cm-1 (Cu-Smet; reduced amicyanin at low pH) and one in the range of 230-800 cm-1 (Cu-Scys).