Blue copper proteins oxidation site

H.B. Gray, California Institute of Technology The rate of Co(phen) 3 oxidation of the chromium derivatives of the reduced blue copper protein is surprisingly close to the rate for the native protein, in my view. Could you comment further on what you think this result means in terms of the proposed interaction sites in the various mechanisms ... 

Copper-containing amine oxidases (non-blue copper proteins) catalyze the oxidative deamination of primary amines to the corresponding aldehydes with the release of ammonia and concomitant reduction of oxygen to hydrogen peroxide. They typically use a quinone redox cofactor [topaquinone (TPQ)], which is bound covalently in the active site, and are thought to form a Cu(I)-TPQ semi-quinone radical intermediate during the redox reaction [13]. 

Blue copper proteins in their oxidized form contain a Cu2+ ion in the active site. The copper atom has a rather unusual tetra-hedral/trigonal pyramidal coordination formed by two histidine residues, a cysteine and a methionine residue. One of the models of plastocyanin used in our computational studies (160) is pictured in Fig. 7. Among the four proteins, the active sites differ in the distance of the sulfur atoms from the Cu center and the distortion from an approximately trigonal pyramidal to a more tetrahedral structure in the order azurin, plastocyanin, and NiR. This unusual geometrical arrangement of the active site leads to it having a number of novel electronic properties (26). 

The other way to study the "conductivity of protein molecules towards electron tunneling is to investigate the quenching of luminescence of electron-excited simple molecules by redox sites of proteins [95,96]. Experiments of this sort on reduced blue copper proteins have involved electron-excited Ru(II)(bpy)3, Cr(III)(phen)3, and Co(III)(phen)3 as oxidants. The kinetics of these reactions exhibit saturation at protein concentrations of 10 3 M, suggesting that, at high protein concentrations, the excited reagent is bound to reduced protein in an electron transfer precursor complex. Extensive data have been obtained for the reaction of reduced bean plastocyanin Pl(Cu(I)) with Cr(III)(phen)3. To analyze quenching experimental data, a mechanistic model that includes both 1 1 and 2 1 [Pl(Cu(I))/ Cr(III)(phen)3] complexes was considered [96]... 

The ability to exist in more than one oxidation state allows transition-metal complexes to serve as the active site of enzymes whose function is to transfer electrons (39). A great deal of effort has been directed at understanding the mechanisms of electron transfer in metalloproteins, such as cytochromes and blue copper proteins (40). Of particular interest is the mechanism by which an electron can tunnel from a metal center that is imbedded in a protein matrix to a site on the outer surface of the protein (7). A discussion of current theories is given in this volume. 

Type I copper is present at the active site of blue copper proteins (BCP see chapter by Nersissian and Shipp, this volume) where it is involved in the transfer of a single electron, as well as in multicopper enzymes (Gray et al., 2000 Malmstrdm, 1994 Randall et al., 2000 Sykes, 1991) (see Section V). BCP are single-domain proteins with a (3-barrel fold defined by two (3-sheets that can contain 6 to 13 strands following a Greek-key motif (Fig. 1) (Adman, 1991 Messerschmidt, 1998 Murphy ei a/., 1997 Sykes, 1991). These proteins are stable in both the reduced, Cu(I), and the oxidized, Cu(II), forms. 

The active sites of oxidized blue copper proteins are characterized by unique features relative to those of normal Cu(II) complexes. These features include an intense absorption... 

L. M. Murphy, S. S. Hasnain, R. W. Strange, I. Harvey, and W. J. Ingledew, Xafs Studies on Blue Copper Proteins The Effect of ph and Oxidation State Changes on the Copper Site, in X-ray Absorption Fine Structure , ed. S. S. Hasnain, EUis Horwood, London, 1991, p. 152. 

Metalloproteins, where the active site includes one or more metals, represent a very different class of proteins than those discussed above. The particular kinds of metalloproteins discussed here are those where the metal is redox active and represents a functional and not structural component of the system. Many mechanistic studies of metalloproteins have been carried out using radiation chemistry in the past 50 years. Two different ways of using radiation chemistry to query mechanisms will be illustrated here. The first, as described in the earliest of these studies using blue copper proteins such as azurin, involves using pulse radiolysis to change an oxidation state and thus... 

This topic has been reviewed by Ingledew (55). The major components of the respiratory chain for T. ferrooxidans are a cytochrome oxidase of the Ci type, cytochromes c, and the blue copper protein rusticyanin. Initial electron transfer from Fe(II) to a cellular component takes place at the outer surface of the plasma membrane in the periplasmic space. The rate of electron transfer from Fe(II) to rusticyanin is too slow for rusticyanin to serve as the initial electron acceptor. Several proposals have been made for the primary site of iron oxidation. Ingledew (56) has suggested that the Fe(II) is oxidized by Fe(III) boimd to the cell wall the electron then moves rapidly through the polynuclear Fe(III) complex to rusticyanin or an alternative electron acceptor. Other proposals for the initial electron acceptor include a three-iron-sulfur cluster present in a membrane-bound Fe(II) oxidoreductase (39, 88), a 63,000 molecular weight Fe(II)-oxidizing enzyme isolated from T. ferrooxidans (40), and an acid-stable cytochrome c present in crude extracts of T. ferrooxidans (14). 

ABSTRACT, The cyclic voltanraietric behaviour of four mononuclear copper(II) complexes and one binuclear copper(I) complex with the ligand 1,6-bis(3,5-dimethyl-l-pyrazolyl)-2,5-dithiahexane (bddh) was studied. Their oxidation-reduction properties will be discussed in terms of the possible relevance of these complexes as models of the Type 1 site of the blue-copper proteins. 

Redox inert complexes [Cr(phen)3] and [CitCN) ] have been shown by nmr to bind at different points on the surface of the blue copper protein plastocyanin. Both sites are close to electron channels to the copper center and are the likely sites occupied by the oxidants [Co(phen)3] and [Fe(CN)6], which have been shown to bind to the protein.The reaction of [Co(phen)3] in inhibited by [Cr(phen)3]. ... 

---