Type I blue copper proteins as enantioselective quenchers of the photoluminescence of Delta,Lambda-Eu(pyridine-2,6-dicarboxylate)(3)(3-): azurin fromPseudomonas aeruginosa and its Met44 -> Lys mutant, amicyanin from Paracoccus versutus and parsley plastocyanin

The dynamic quenching of the luminescence of racemic Eu(III)(pyridine-2,6-d icarboxylate = dpa)(3)(3-) by the title proteins is investigated and the en antioselectivity of the proteins in the quenching of the Delta and Lambda e nantiomers of Eu(dpa)(3)(3-) is determined. The two diastereomeric quenchin g rate constants pertaining to azurin (k(q)(Delta) = 3.3 x 10(6), k(q)(Lamb da) = 2.7 x 10(6) M-1 s(-1), pH 7.2, ionic strength I = 22 mM) are lower th an for its Met-->44Lys mutant (k(q)(Delta) = 1.9 x 10(7), k(q)(Lambda) = 1. 4 x 10(7) M-1 s(-1), same pH and I), indicating that energy transfer occurs from Eu(dpa)33- to the Cu(II) centre when the luminophore is bound to the hydrophobic patch of the protein near residue 44. The enantioselectivity re mains unaltered by the mutation: k(q)(Delta)/k(q)(Lambda) = 1.27 +/- 0.04, so Lys44 is probably not in direct contact with the Eu chelate. The I and p H dependence of k(q) indicate that the lysine residue interacts electrostat ically with Eu(dpa)(3)(3-). For plastocyanin the quenching rates are of the order of 10(6) M-1 s(-1); for amicyanin they are two orders of magnitude l arger (k(q)(Delta) =12 x 10(7), k(q)(Lambda) = 11 x 10(7) M-1 s(-1), pH 7.2 , I = 22 mM). The variation of k(q) is attributed to differences in the cha rge distribution on the proteins, which influences the binding of the lumin ophore to the protein surface. For amicyanin the anion binding site near Ly s59 and Lys60 may be involved in the energy transfer.