NEBRASKA REDOX BIOLOGY CENTER EDUCATIONAL PORTAL

Nebraska Redox Biology Center Educational Portal


Copper

Copper (Cu) is an essential but highly toxic trace element required for survival by all organisms from bacterial cells to mammals [ 1, 2, 3, 4 ]. Both redox states of copper, Cu(II) and Cu(I), are importants for copper chemistry in biological systems. Cu ions serve as essential catalytic redox cofactors in wide range of functionally distinct proteins. Cu dependant proteins are involved in a variety of biological processes and deficiency in these enzymes often cause disease states or pathophysiological conditions including anemia, growth and developmental retardation, cardiac hypertrophy, neurodegenerative diseases, wound healing and angiogenesis. [ 5, 6, 7, 8, 9, 10, 11, 12 ].


In addition ti physiologic function, Cu is also a potent cytotoxin when its content exeeds a cellular needs. Similarly to iron, Cu is involved in reactions that result in the production of highly reactive oxygen species including hydroxyl radical. Thus, Cu content is strictly regulated in Cu-utilizing organisms to avoind toxicity.

Map of soil copper content in the U.S. (red = high copper areas). Courtesy of U.S. Department of the Interior, U.S. Geological Survey, Mineral Resources.

The importance of maintaining this critical balance is underscored by the existence of two well-characterized human genetic diseases in Cu transport, Menkes and Wilson's diseases [ 1, 9, 13 ].

Cu+ + H2O2 → Cu2+ + OH- + . HO;

Cellular Cu transport processes are required by organisms for correct utilization of this element in biochemical processes and to limit the toxicity of excess Cu. Cu import into cells mainly requires the coordinate function of proteins with metal-binding domains. On the other hand, detoxification mechanisms found across species include the binding of Cu to specific proteins (e.g., metallothioneins) and its transfer into cell compartments such as periplasmic space, mitochondria and lysosomes.

There are from one to six known CTR family copper transporters in eukaryotic organisms [ 4, 14 ]. In eukaryotic organisms, Cu ion transport and intracellular distribution is understood in greatest detail in the baker's yeast, Saccharomyces cerevisiae. Copper is first reduced from Cu[II] to Cu[I] by cell surface reductases Fre1/Fre2 prior to uptake. High affinity Cu ion uptake is mediated by the Ctr1 and Ctr3 proteins. Within the cell Cu is bound to the known cytosolic Cu chaperones Atx1, Cox17 and CCS for specific delivery to the secretory pathway, mitochondria and Cu, Zn SOD, respectively. Within the secretory pathway, Ccc2 accepts Cu from Atx1, followed by incorporation of Cu to the multicopper ferroxidase, Fet3 in a manner facilitated by the Gef1 chloride channel. Fet3 forms a complex with the iron permease Ftr1 and both proteins are responsible for high affinity iron uptake at the plasma membrane. In mitochondria Cu delivered by Cox17 is incorporated into cytochrome oxidase, a process that requires the integral inner-membrane protein Sco1 and possibly its homologue, Sco2. CCS delivers Cu specifically to Cu, Zn SOD in the cytosol. Currently it is not known whether specific chaperones are required for incorporation of Cu to the metallothioneins Cup1 and Crs5 in the cytosol, or the nuclear Cu-metalloregulatory transcription factors Ace1 and Mac1 or the vacuole, which is important for proper Cu detoxification. Ctr2 copper transporter is involved in vacuolar copper transport [ 1, 4, 14, 15 ].

Copper transport and distribution in Saccharomyces cerevisiae [ 1 ].

Dietary Cu is primarily absorbed from the stomach and small intestine. hCTR1, a high affinity Cu transporter, may transport Cu into intestinal mucosal cells and MNK is required for Cu transport into the portal circulation. MNK is a P-type ATPase defective in Menkes patients in which Cu is accumulated in intestinal epithelial cells. Once entering the plasma, Cu is bound with albumin and histidine in the portal blood and rapidly deposited in the liver where hCTR1 may play a role in this process. Ceruloplasmin, a major Cu-containing protein in plasma, is synthesized in the liver with the incorporation of Cu by the WND protein in the secretory pathway and has ferroxidase activity that is critical for iron metabolism. WND has high homology with MNK and is defective in Wilson's disease patients who suffer from Cu accumulation in liver. Biliary excretion via the gall bladder is the major route of Cu elimination from the body and a small amount of Cu is found in urine. Heph is a membrane bound ceruloplasmin/Fet3 homologue required for iron egress from the intestine that is defective in sla mice, a model for sex-linked anemia. Tissue uptake of Cu is likely mediated by the hCTR1 Cu transporter. Once transported by hCTR1, the small cytoplasmic Cu chaperones (hCOX17, HAH1, CCS) distribute Cu to specific cellular compartments for the incorporation of Cu into Cu-requiring proteins. hCOX17, HAH1 and CCS deliver Cu to the mitochondria, secretory compartment, and Cu, Zn SOD, respectively. Cu chaperones for metallothionein and the nucleus have not been identified. In tissues other than the liver, MNK transports Cu delivered by HAH1 into the TGN for incorporation of Cu into secretory proteins. Although it is not known how these Cu chaperones take Cu transported from outside, the Atx1 and CCS Cu chaperones directly interact with their specific target molecule to surrender Cu. Cu stimulates the trafficking of MNK from the TGN to the plasma membrane where it may be involved in Cu efflux. In hepatic cells, WND, the Wilson's disease protein is localized in the TGN. Elevated Cu levels stimulates its trafficking from the TGN to an unknown cytosolic vesicular compartment [ 1, 16, 17 ].

Copper metabolism in humans. (A) Model for human Cu absorption and distribution at the organ and tissue level. (B) Model for human Cu uptake and distribution at the cellular level [ 1 ].

The mechanisms involved in Cu transport and homeostasis in prokaryotes are only partially understood. To date, Cu trafficking in bacteria is best described in E. coli and in Enterococcus hirae. In E. hirae, an operon containing five genes, copX, Y, Z, A and B, plays an important role in Cu ion homeostasis. The CopA and CopB proteins are two integral membrane P-type ATPases that are necessary for the transport of Cu into cells under Cu limiting conditions, and Cu efflux under conditions of high Cu ion levels, respectively. CopB is the only Cu transporter to date that has been biochemically demonstrated to drive the accumulation of Cu(I), and the chemically similar metal Ag(I), into reconstituted native inside-out membrane vesicles [ 1, 18, 19, 20, 21, 22 ].

Copper homeostasis mechanisms in E. coli. Several homeostatic mechanisms detoxify the cell of copper. Copper enters the bacterial cell through an unknown importer. CueO, multicopper oxidase; CusF, Cu(I)-binding metallochaperone; CusCBA, RND-driven tripartite complex; CopA, Cu(I)-translocating P-type ATPase; P, phosphate group. CusS, a histidine kinase, senses periplasmic Cu(I) and subsequently phosphorylates CusR, a response regulator, to activate transcription of cusCFBA. CueR senses copper and activates transcription of copA and cueO [ 19 ].


Copper in proteins has been classified into three groups based on their spectroscopic and magnetic properties: type I copper, type II copper, and type III copper. Type I copper shows intense absorption at around 600 nm and narrow hyperfine splittings in the electron paramagnetic resonance (EPR) spectroscopy. Type II copper does not give strong absorption at 600-700 nm and shows hyperfine splittings of the normal magnitude in the EPR spectrum. Unlike type I copper and type II copper, type III copper is not detected in the EPR spectrum [ 23 ]. Copper containing proteins have been identified in all three domains of life [ 23, 24 ].

The biological roles of Cu include electron transfer, oxidation of organic substrates and metal ions, dismutation of superoxide, monooxygenation, transport of dioxygen and iron, reduction of dioxygen, nitrite, and nitrous oxide, etc. Both Cu(I) and Cu(II) are utilized in biological systems [ 1, 2, 3, 4 ].

Plastocyanin, azurin, pseudoazurin, amicyanin, rusticyanin, auracyanin, halocyanin, plantacyanin, umecyanin, mavicyanin, and stellacyanin (also called blue copper proteins) are involved electron transfer a radical scavenger in higher plants. Nitrosocyanin (also called red copper protein) is a variant of the blue Cu protein [ 23, 25 ]. Nitrite reductase is catalyzes the one-electron reduction of nitrite (NO2-) to nitric oxide (NO). Multi-Cu oxidases are involved in intramolecular electron transfer to Cu active sites [ 23, 24 ].

The type 2 Cu-containing enzymes include Cu-Zn superoxide dismutase (Cu-Zn SOD), Cu amine oxidase, peptidylglycine alpha-hydroxylating monooxygenase, and dopamine beta-monooxygenase [ 23, 26 ]. Cu-Zn SOD is involved in superoxide detoxification [ 27 ]. Amine oxidases that catalyze oxidative deamination of amines by reduction of oxygen to hydrogen peroxide [ 28 ]. Peptidylglycine alpha-hydroxylating monooxygenase and dopamine beta-monooxygenase catalyze the hydroxylation of their substrates. They contain two distinct Cu sites that are used to split molecular oxygen to use it as the source of hydroxyl in the hydroxylation reactions [ 26 ].

Cytochrome oxidases are components of respiratory chains. Thera are two subgroups of this family include cytochrome c oxidases and quinol oxidases. [ 29 ].

E. coli Cu(II)-reductase Ndh2 is membrane-bound cupric-reductase that diminishes the susceptibility of the respiratory chain to damaging effects caused by Cu and hydroperoxides and allows cells to survive in extreme Cu conditions. It contributes to antioxidant function and Cu homeostasis [ 30 ].

Tyrosinases are Cu-containing enzymes which are essential for pigmentation and are important factors in wound healing and primary immune response. The copper pair of tyrosinases binds one molecule of atmospheric oxygen to catalyse two different kinds of enzymatic reactions: (1) the ortho-hydroxylation of monophenols (cresolase activity) and (2) the oxidation of o-diphenols to o-diquinones (catecholase activity) [ 31 ].

Galactose oxidase is a secretory fungal copper metalloenzyme that generates hydrogen peroxide in the extracellular space by oxidizing simple alcohols and subsequently reducing dioxygen to hydrogen peroxide [ 32 ].

Copper binding proteins with Cu donor set and redox potential.

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