The effect of pH and temperature on the structure of the active site of azurin from Pseudomonas aeruginosa

Abstract

1. INTRODUCTION The unusual spectroscopic properties of the blue copper proteins, such as azurin and plastocyanin, have attracted much attention [ 1,2]. The success of spectroscopic methods in predicting the coordina- tion environment of the copper could be assessed on publication of the crystal structures of a plastocya- nin [3] and an azurin [4,5]. It appears that in poplar plastocyanin, the Cu-atom is coordinated by two histidines (residues 37 and 87), a cysteine (Cys 84) and a methionine (Met 92) in a distorted tetrahedral configuration 131. Although the resolution of the crystal structure of azurin is somewhat less, the data suggest that the Cu is coordinated by two histidines (His 46 and 117) a cysteine (Cys 112) .and a methionine (Met 121), again in a distorted tetra- hedral configuration. Possible contributions from other ligands, e.g., from the peptide backbone, could not be completely excluded [5]. of magnitude slower in exchanging electrons cyto- chrome c-555 than the former. The redox potential of the ‘inactive’ form is 60 mV lower than that of the ‘active’ form, indicating a stabilization of the Cu(I1) state towards high pH [6-81. The transition from active to inactive state involves deprotonation of the histidine [6] which was shown in NMR studies [9- 121 to participate in a slow proton-exchange process. In a recent NMR study [ 131 this residue has been as- signed to His 35, which is adjacent to the ligand His 46 [4,5]. We wish to report the results of some NMR ex- periments on