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 active site in which the axial Cu-S(Met) interaction is considerably decreased. This observation confirms the conclusion made from various other spectroscopic methods. Furthermore, we show that the alkaline transition of pseudoazurin coincides with a dramatic increase in the electron self-exchange rate 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 previously 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 constant (25 degrees C) increases to 1.7 x 10(4) M-1 s(-1) (I = 0.10 M). The increased self-exchange reactivity at high pH is due to the deprotonation of a number of lysine residues that surround the hydrophobic patch of the protein, the most likely docking surface for the Self-exchange process. The concomitant active site changes indicate that the deprotonation of one or more surface lysine residues is responsible for the alkaline transition in pseudoazurin.