Alkaline transition of pseudoazurin from Achromobacter cycloclastes studied by paramagnetic NMR and its effect on electron transfer

Paramagnetic NMR studies on the Cu(II) form of pseudoazurin have been used to demonstrate that the alkaline transition of this protein results in an a ctive site in which the axial Cu-S(Met) interaction is considerably decreas ed. This observation confirms the conclusion made from various other spectr oscopic methods. Furthermore, we show that the alkaline transition of pseud oazurin coincides with a dramatic increase in the electron self-exchange ra te constant of the protein. The self-exchange rate constant (25 degrees C) at pH 8.2 is 3.5 x 10(3) M-1 s(-1) (I = 0.10 M), consistent with a previous ly determined value (25 degrees C) of 2.9 x 10(3) M-1 s(-1) (I = 0.10 M) at pH 7.5. Upon increasing the pH value to 10.9 the self-exchange rate consta nt (25 degrees C) increases to 1.7 x 10(4) M-1 s(-1) (I = 0.10 M). The incr eased self-exchange reactivity at high pH is due to the deprotonation of a number of lysine residues that surround the hydrophobic patch of the protei n, the most likely docking surface for the Self-exchange process. The conco mitant active site changes indicate that the deprotonation of one or more s urface lysine residues is responsible for the alkaline transition in pseudo azurin.