Metal binding to Pseudomonas aeruginosa azurin: a kinetic investigation

The interaction between azurin from Pseudomonas aeruginosa and Ag(I), Cu(II ), Hg(II), was investigated as a function of protein state, i.e. apo-, redu ced and oxidised azurin. Two different metal binding sites, characterized b y two different spectroscopic absorbancies, were detected: one is accessibl e to Ag(I) and Cu(II) but not to Hg(II); the other one binds Ag(I) and Hg(I I) but not copper. When added in stoichiometric amount, Ag(I) shows high af finity for the redox center of apo-azurin, to which it probably binds by th e -SH group of Cys112; it can displace Cu(I) from reducedazurin, while it d oes not bind to the redox center of oxidizedazurin. Kinetic experiments sho w that Ag(I) binding to the reduced form is four times faster than binding to the ape-form. This result suggests that metal binding requires a conform ational rearrangement of the active site of the azurin. Interaction of Ag(I) or Hg(II) ions to the second metal binding site, induc es typical changes of UV spectrum and quenching of fluorescence emission.