Binuclear Sites and Multi-Centred Copper Proteins

Several site-directed mutations were carried out with a new member of the blue oxidases , the Fet3p protein from Saccharomyces cerevisiae which is involved in iron trafficking. The protein oxidizes Fe2+ to Fe3+ as first step in the iron uptake chain. The mutations were targeted to block this ferroxidase activity and thus to identify the residues involved in iron binding. The respective mutants showed no perturbed type 1 sites.91 

A new putative member of the blue multi-copper oxidases has been isolated using the Escherichia coli yacK gene. Six copper ions per polypeptide chain were determined and assigned to two type 1 copper centers and further one type 2 and one type 3 copper. Phenoloxidase and ferroxidase properties were ascertained98 A new copper containing nitrite reductase was purified from a halophilic archaeon and the ligands to type 1 and type 2 coppers in the sequence were 

Ceruloplasmin is involved in copper storage and transport as well as in iron mobilisation and oxidation. Among the blue oxidases it is unique since it contains, in addition to the usual motif of a type 1 combined with the trinuclear cluster, two other type 1 coppers. Electron transfer occurs, however, only between five of the six copper ions since one of the type 1 centres is not catalytically relevant due to its too high redox potential. The redox potentials of the centres were determined and possible electron transfer pathways among the copper sites were discussed.101 

The other copper-only binuclear centre to be considered is the CuA or purple copper complex. It is part of the terminal oxidase in mitochondrial respiration, cytochrome c oxidase (COX). Its EPR signature, a seven-line spectrum, has since long been known to be different from the classes type 1 to 3 and arises from two copper ions in a 1.5 valence (or mixed valence) state, first proposed from EPR-analysis of a similar center in nitrous oxide (N20) reductase. There is a close correspondence between the blue and purple states of copper since each of the two copper ions in CuA can be considered as being structurally related to the mononuclear blue site coordination. 

With this rationale, mutations of the axial ligand to one of the coppers were performed in a water soluble recombinant CuA containing protein from the 

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