Loop-directed mutagenesis of the blue copper protein amicyanin from Paracoccus versutus and its effect on the structure and the activity of the type-1 copper site

Four loop-mutants of the blue copper protein amicyanin from Paracoccus vers utus have been constructed and characterized. The mutations replaced the lo op containing three (Cys93, His96, Met99) of the four copper ligands in ami cyanin by the "ligand loops" of P. aeruginosa azurin (AmiAzu), A. fuecalis pseudoazurin (AmiPaz), P. nigra plastocyanin (AmiPcy), and P. aureofaciens nitrite reductase (AmiNiR). The copper centers of all variants appear to be perfect type-1 Cu sites although the AmiNiR variant exhibits diminished st ability. The optical spectra of the AmiAzu and AmiPaz variants display a si gnificant dependence on temperature. Excitation at 457 nm as well as 647 nm results in similar resonance Raman spectra. The reduction potentials of th e three stable variants are all higher than that of wt amicyanin. The reduc ed forms of the loop-mutants protonate at the C-terminal histidine, with pK (a), values of 5.6 (AmiAzu), 5.4 (AmiPaz), and 5.7 (AmiPcy) (6.8 for wt ami cyanin). AmiAzu is the first known cupredoxin with mon than two amino acids between the cysteine and histidine ligands that undergoes this protonation . Tnc electron self-exchange rate constants at 25 degrees C are 5.7-7.5 tim es lower for the loop mutants than for wt amicyanin (1.2 x 10(5) M-1 s(-1)) . The results are interpreted by taking into consideration that three metal ligands are intimately connected with the stable beta-sandwich structure o f the cupredoxin. This leaves considerable freedom in positioning the fourt h one, the C-terminal His, on the "ligand loop". This explains the ease by which the C-terminal histidine ligand can be excised and replaced by extern al ligands without losing the metal binding property of the protein. The re sults also help understand the remarkable evolutionary success of the combi nation of cupredoxin fold and Cu site for mediating biological electron tra nsfer.