An amicyanin C-terminal loop mutant where the active-site histidine donor cannot be protonated

A novel blue copper protein was constructed by replacing the C-terminal loo p of amicyanin (Paracoccus versutus) by the homologous loop of rusticyanin. The C-terminal loop of both amicyanin and rusticyanin contains three (His, Cys, Met) of the four copper ligands. The amicyanin mutant exhibits all sp ectroscopic properties normally encountered for blue copper sites. The midp oint potential (369 mV) is the highest reported value for an amicyanin muta nt. Cyclic voltammetry and NMR studies of the reduced form indicate that, i n contrast to wild-type amicyanin and all amicyanin mutants described so fa r, the C-terminal histidine ligand does not protonate in the accessible pH range (pK(a)<4.5).