The effect of pH and ligand exchange on the redox properties of blue copper proteins.

A study of the structure and redox properties of the copper site in azurins by means of EXAFS, NMR, redox titrations, potentiometry, equilibrium cyclic voltammetry and rapid scan voltammetry on protein films is reported. The results are discussed in light of existing theories on structure and function of type-1 copper sites. The exit and entry of electrons take place through the C-terminal histidine ligand of the copper. The hydrophobic patch through which this residue penetrates the protein surface plays an important role in partner docking (cf. The rim of the porphyrin ring sticking through the surface of the cytochromes-c). We find no experimental evidence for strain around the metal site. The active centre is able to maintain ET activity even in the presence of fairly gross disturbances of the site structure. The analysis of the thermodynamics of the redox reaction shows that the protein matrix and the solvent play an important role in 'tuning' the redox potential around a "design" value of around 300 mV at room temperature. The metal site appears "designed" to stabilise the Cu(II) instead of the Cu(I) form. The remarkable evolutionary success of the blue copper proteins is ascribed to the sturdy overall beta-sandwich structure of the protein in combination with a metal site that is structurally adaptable because three of its four ligands are located on a loop. The electronic "gate" that occurs in the middle of a hydrophobic patch allows for fine tuning of the docking patch for recognition purposes.