Four loop-mutants of the blue copper protein amicyanin from Paracoccus versutus have been constructed and characterized. The mutations replaced the loop containing three (Cys93, His96, Met99) of the four copper ligands in amicyanin 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 stability. The optical spectra of the AmiAzu and AmiPaz variants display a significant dependence on temperature. Excitation at 457 nm as well as 647 nm results in similar resonance Raman spectra. The reduction potentials of the three stable variants are all higher than that of wt amicyanin. The reduced 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 amicyanin). 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 times 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 of the cupredoxin. This leaves considerable freedom in positioning the fourth 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 external ligands without losing the metal binding property of the protein. The results also help understand the remarkable evolutionary success of the combination of cupredoxin fold and Cu site for mediating biological electron transfer.