Rack‐induced bonding in blue‐copper proteins

Abstract

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 preformed 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 π back bonding, and this is supported by a linear increase in ΔLF (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. Copyright © 1994, Wiley Blackwell. All rights reserved