An outer-sphere hydrogen-bond network constrains copper coordination in blue proteins.

In azurins and other blue copper proteins with relatively low reduction potentials (E(0) [Cu(II)/Cu(I)]<400 mV vs. normal hydrogen electrode), the folded polypeptide framework constrains both copper(II) and copper(I) in such a way as to tune the reduction potentials to values that differ greatly from those for most copper complexes. Largely conserved networks of hydrogen bonds organize and lock the rest of the folded protein structure to a loop that contains three of the ligands to copper. Changes in hydrogen bonds that allow copper(I) to revert more closely to its preferred geometry [relative to the copper(II) geometry] accordingly lead to an increase in E(0). This paper reports mutations in the ligand loop of amicyanin from P. denitrificans that relax the constraints on ligation for copper(I) and significantly raise E(0) for these mutants (for example 415+/-4 mV) relative to that of the native amicyanin (265+/-4 mV). These mutations also shift the pK(a) of a ligand histidine to below 5 relative to 7.0 in the wild type.