Rack-induced bonding in blue-copper proteins.

The unique spectroscopic properties of blue-copper centers, i.e. the strong charge-transfer band at approximately 600 nm and the narrow hyperfine coupling in the EPR spectrum, are reviewed. The concept of rack-induced bonding is summarized. The tertiary structure of the protein creates a performed chelating site with very little flexibility, the geometry of which is in conflict with that preferred by Cu2+. The structure of the metal site in azurin is discussed. It is shown that the three strong ligands, one thiolate S and two imidazole N, are in a configuration intermediate between those preferred by Cu2+ and Cu+. It is emphasized that cysteine is an obligatory component of a blue site, whereas the weak interaction with a methionine S is not necessary. The minimum rack energy is estimated to be 70 kJ.mol-1. It is pointed out that the high reduction potentials of blue-copper centers are a result of the protein-forced ligand-field-destabilized site structure. It is suggested that the potentials are tuned by variations in pi back bonding, and this is supported by a linear increase in delta LF (ligand field) with decreasing electron-transfer enthalpy. Site-directed mutagenesis has shown that large hydrophobic residues in the site increase the potential, whereas negative groups or water decrease it. It is also shown that the fine-tuning of the properties of the metal site by rack-induced bonding can alter the electron-transfer reorganization energy. Kinetic results with azurin mutants support a through-bond tunneling mechanism for intramolecular electron transfer in proteins. Finally, it is pointed out that the concept of rack-induced bonding is a universal principle of macromolecular structure/function relationships, which should be applied also to other systems.