Electron transfer ceruloplasmin

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... 

Copper has an essential role in a number of enzymes, notably those involved in the catalysis of electron transfer and in the transport of dioxygen and the catalysis of its reactions. The latter topic is discussed in Section 62.1.12. Hemocyanin, the copper-containing dioxygen carrier, is considered in Section 62.1.12.3.8, while the important role of copper in oxidases is exemplified in cytochrome oxidase, the terminal member of the mitochondrial electron-transfer chain (62.1.12.4), the multicopper blue oxidases such as laccase, ascorbate oxidase and ceruloplasmin (62.1.12.6) and the non-blue oxidases (62.12.7). Copper is also involved in the Cu/Zn-superoxide dismutases (62.1.12.8.1) and a number of hydroxylases, such as tyrosinase (62.1.12.11.2) and dopamine-jS-hydroxylase (62.1.12.11.3). Tyrosinase and hemocyanin have similar binuclear copper centres. 

The several functions of ceruloplasmin cannot be explained at present. It seems reasonable that this diversity is related to the activity of the copper centres. The general pattern of oxidase activity is probably similar to that of the other blue oxidases, with a type 3 binuclear site serving to bind and reduce dioxygen, with electrons transferred from the type 1 site. The type 2 copper may represent a substrate-binding site. 

Wilson s disease is a pathological accumulation of copper in tissue which is later released into the bloodstream, leading to anaemia, and final accumulation of copper in liver and brain. It is the result of a mutation in the Wilson s disease gene in chromosome 13 which ordinarily codes for a cation transporting ATPase so that copper can be incorporated into ceruloplasmin prior to excretion. Also known as ferroxi-dase, in acknowledgement of its primary function as an oxidoreductase responsible for electron transfer, this enzyme contains iron and, more importantly, six copper atoms. It accounts for the transport of 90% of copper in the plasma so any impairment in its production or efficacy has a major impact on copper homeostasis. The greatly reduced concentration of ceruloplasmin in the blood of Wilson s disease sufferers correlates with their inability to metabolize copper effectively. It leads to chronic liver disease, for which the only real cure is a liver transplant,... 

Machonkin, T. E. and Solomon, E. T. (2000) The thermodynamics, kinetics, end molecular mechanism of intramolecular electron transfer in human ceruloplasmin, J. Am. Chem. Soc. 122,12547-12560. 

Ceruloplasmin is an intensely blue glycoprotein of the a2-globulin fraction of mammalian blood, which acts as a copper transfer protein and probably has a role in iron storage. The structure is known it contains three Type 1 (Tl) sites (one of which seems to be inactive) and a Type 2/Type 3 (T2/T3) trinuclear cluster. It is believed to be part of the process of oxidizing Fe(II) to Fe(III) in the transfer of iron from ferritin to transferrin. Reduction of two Tl sites and the T3 pair is fast, but reduction of the T2 Cu site is slow the pathways of electron transfer between the sites have been investigated, but the complete mechanism is still unknown. 

The reduction potential is central for the function of electron-transfer proteins, since it determines the driving force of the reaction. In particular, it must be poised between the reduction potentials of the donor and acceptor species. Therefore, electron-transfer proteins normally have to modulate the reduction potential of the redox-active group. This is very evident for the blue copper proteins, which show reduction potentials ranging from 184 mV for stellacyanin to 1000 mV for the type 1 copper site in domain 2 of ceruloplasmin [1,110,111]. 

Cu 72 Electron transfer systems (blue copper proteins) O2 storage and transport (haemocyanin) Cu transport proteins (ceruloplasmin)... 

Studies of intramolecular ET in oxidases provide interesting examples of how pulse radiolysis is employed to obtain insights into both (1) these enzymes respective mechanisms of action and (2) electron transfer along protein polypeptide matrices that were most probably selected by evolution (9,10, 30-32). Thus, early attempts to study the electron uptake mechanism by the blue oxidase, ceruloplasmin, showed that a diffusion-controlled decay process of the eaq in solutions of this protein is paralleled by the formation of transient optical absorptions due to electron adducts of protein residues, primarily of cystine disulfide bonds (30). The monomolecular decay of the latter absorption was found to have the same rate constant as that at which the type 1 Cu(II) absorption band was reduced. These results were interpreted as being the combined result of the high reactivity of the e q and the relatively inaccessible type 1 Cu(II) site, yielding an indirect, intramolecular electron transfer pathway from surface-exposed residues (30). 

The blue oxidases like ascorbate oxidase, laccase, and ceruloplasmin, and the terminal oxidases of aerobic respiratory chains like cytochrome oxidases and quinol oxidases are the only enzymes so far known that catalyze the direct four-electron reduction of molecular oxygen to water. Thereby, the reducing substrates like ascorbate, quinol, Fe " ", and cytochrome c are oxidized in one-electron transfer steps. The substrates of quinol oxidases, ubiquinol, or menaquinol, may be oxidized in two-electron transfer steps. For the two cases the following general reaction formulae can be defined ... 

Type 1 copper proteins are the class of proteins for which cupredoxins were originally named. Type 1 copper proteins include both proteins with known electron transfer function (e.g., plastocyanin and rusticyanin), and proteins whose biological functions have not been determined conclusively (e.g., stellacyanin and plantacyanin). Although these proteins with unknown function cannot be called cupredoxins by the strict functional definition, they have been classified as cupredoxins because they share the same overall structural fold and metal-binding sites as cupredoxins. In addition, many multidomain proteins, such as laccase, ascorbate oxidase, and ceruloplasmin, contain multiple metal centers, one of which is a type 1 copper. Those cupredoxin centers are also included here. Finally, both the Cua center in cytochrome c oxidase (CcO) and nitrous oxide reductase (N2OR), and the red copper center in nitrocyanin will be discussed in this chapter because their metal centers are structurally related to the type 1 copper center and the protein domain that contains both centers share the same overall structural motif as those of cupredoxins. The Cua center also functions as an electron transfer agent. Like ferredoxins, which contain either dinuclear or tetranuclear iron-sulfur centers, cupredoxins may include either the mononuclear or the dinuclear copper center in their metal-binding sites. 

The inhibition of ceruloplasmin is also quite comphcated, and a part of this may be associated with the suggested conformational sensitivity of the molecule. Azide, at least, inhibits by decreasing the rate of decomposition of the 420 nm absorbing intermediate, possibly by inhibiting intramolecular electron transfer reactions necessary for the reduction of O2 to H2O. 

As for iron, differences in redox potentials are relatively small between the major oxidation states of copper, that is, Cu(I) and Cu(II). This gives copper its main function as a cofactor in enzymatic reactions involving electron-transfer processes. In the human body, most of the copper (about 40%) is present in muscle tissue with significant amounts also present in the liver, brain, and skeleton. About 5% of the copper can be found in serum, of which 80-90% is present as ceruloplasmin. Ceruloplasmin in serum and hephaestin at the basolateral side of the mucosa ensure oxidation of circulating Fe to Fe for iron binding to transferrin. Unbound Fe is a major source of oxidative stress through Fenton/Haber-Weiss chemistry. Copper together with zinc is also a cofactor for superoxide dismutase, a key molecule in the anti-oxidant defense system of the body ]74]. 

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