Copper
The chemical properties of copper that make it useful for biochemistry are the stability of its redox-interchangeable prevalent ionic forms, its ability to bind oxygen and its affinity for functional groups that occur in proteins. Accordingly, copper enzymes are found in electron transfer pathways, such as respiration (cytochrome oxidase) and photosynthesis (plastocyanin), and in enzymes/proteins that bind oxygen, such as oxidases (ferroxidase) and oxygen transport proteins (hemocyanin). Because of its role in fundamental biochemical pathways, copper is an essential micronutrient for organisms as diverse as bacteria, fungi, plants and mammals. But the same properties that make copper a useful cofactor in biochemical redox reactions or oxygen chemistry can wreak havoc within the cell when intracellular copper content is excessive, and accordingly, organisms have developed methods for avoiding toxicity. The mechanism of intracellular copper homeostasis – the balance between uptake, utilization and detoxification pathways – has been a subject of considerable interest and research activity in the past 15 years.
Copper deficiency
Studies of biological responses to copper have historically focused more heavily on toxicity. Deficiency is much more difficult to control in the laboratory, since copper is required only at trace levels: for instance, the adult human contains only 1% the amount of copper compared to iron (Frieden, 1985). Nevertheless, deficiency in humans does occur in premature or malnourished infants, in individuals with excessive consumption of zinc, or as a consequence of genetic disease, and is not uncommon in animals in the natural environment. The consequences of copper-deficiency are most easily studied at the molecular level in microorganisms, many of which have developed adaptive mechanisms to survive transient, or even sustained, deficiency (Merchant, 1998). A first response is the induction of uptake pathways to avoid deficiency, but there are situations where intracellular copper is still insufficient despite the operation of assimilatory mechanisms. How are metabolic pathways with copper-containing enzymes affected in this situation? And how is metabolic flux through these pathways modified in response to copper-deficiency? These are the questions being addressed in our project.
Chlamydomonas: My group developed the popular photosynthetic eukaryote, Chlamydomonas reinhardtii, (insert a picture of chlamy here) as an experimental model for the dissection of the molecular mechanisms underlying copper-responsive signal transduction in the context of deficiency (Merchant, 1998). Besides the advantage of genetic amenability including a draft genome sequence, Chlamydomonas offers the additional advantage of growth in a simple, well-defined salts medium, which facilitates nutritional studies of trace elements (Quinn and Merchant, 1998). The organism displays extraordinary capacity for copper assimilation: in a deficient medium, the cells deplete the copper so that it is undetectable in the medium (Hill et al., 1996). At the same time, the organism displays exquisite control over copper assimilation: all the intracellular copper can be entirely and exactly accounted for by the stoichiometry of known copper proteins, consistent with a non-existent or very small non-enzyme bound pool (Merchant et al., 1991). Studies of metabolic adaptations to copper-deficiency are complicated for most organisms because of the now well-established molecular connection between copper and iron metabolism (Askwith and Kaplan, 1998). Specifically, because high affinity iron uptake requires a multicopper oxidase (Fet3p in Saccharomyces cerevisiae or ceruloplasmin and hephaestin in mammals), most situations of copper-deficiency in eukaryotes generate secondary iron deficiency. This is distinctly not the situation in Chlamydomonas where a back up mechanism for iron uptake operates in the copper-deficient situation (La Fontaine et al., 2002; Eriksson et al., 2004). This feature is useful for distinguishing copper-specific metabolic adaptations, and it adds great interest to the study of the copper-deficiency targets because of the possibility for discovery of a novel iron assimilation pathway.
Plastocyanin and cytochrome c6 abundance is reciprocally dependent on Crr1: The best example of metabolic adaptation to copper-deficiency is found in cyanobacteria and eukaryotic green algae. Plastocyanin, a photosynthetic electron transfer catalyst, is the most abundant copper protein in these organisms. When the environmental availability of copper cannot sustain plastocyanin accumulation at the stoichiometry required for photosynthesis, an iron protein – cytochrome c6– substitutes. In Chlamydomonas, the reciprocal accumulation of plastocyanin vs. cytochrome c6 is effected by separate mechanisms but controlled by the same master regulator of copper homeostasis, called Crr1 (for copper response regulator). Cytochrome c6 synthesis in copper-deficient cells occurs by transcriptional activation of the CYC6 gene through copper response elements containing an absolutely essential GTAC core and Crr1, which is a novel DNA binding protein (Eriksson et al., 1995; Quinn and Merchant, 1995). In copper-replete cells, the CYC6 gene is turned off. Transcriptional regulation of CYC6 expression is paralleled by a loss of plastocyanin, resulting from induced degradation of a thermodynamically destabilized apoprotein (Li and Merchant, 1995; Li et al., 1996). The degradation of plastocyanin, which is the most abundant copper protein in the Chlamydomonas cell, is important in the copper-deficient cell for re-allocation of copper to cytochrome oxidase, for which there are no effective “back up” mechanisms. The degradation of plastocyanin is also regulated by Crr1, presumably by transcriptional activation of a lumen-targeted protease, the net effect being that cytochrome c6 and plastocyanin show a reciprocal copper nutrition- and Crr1-dependent pattern of accumulation (Merchant, 1998; Eriksson et al., 2004). The two proteins therefore represent the prototypical copper-deficiency targets and serve as diagnostic markers of copper nutrition in Chlamydomonas (Quinn and Merchant, 1998).
Other adaptations to Cu deficiency in Chlamydomonas: Besides plastocyanin loss and cytochrome c6 gain, there are other adaptive responses known: copper uptake, modification of iron assimilation, and altered flux through the chloroplast branch of the tetrapyrrole pathway, and each of these responses is dependent on Crr1 (Eriksson et al., 2004). The activation of copper uptake is an obvious adaptation and involves up-regulation of an extracellular cupric reductase and a CuI transporter, but these molecules have not yet been identified (Hill et al., 1996). Modification of iron metabolism is another important adaptation because it allows iron assimilation in the situation where ferroxidase (a multicopper oxidase) function is compromised by copper-deficiency. This adaptive response is genetically defined by the CRD2 locus (Eriksson et al., 2004): a loss of function crd2 strain displays a copper-deficiency-conditional iron-deficiency chlorosis and can be rescued for growth by excess iron (insert figure). The connection between copper and tetrapyrroles is less understood but it recalls a similar connection noted decades earlier in copper deficient animals. While the copper requirement for iron uptake accounts for many of the observations in those studies, there remain some less well-understood aspects. Our previous studies with Chlamydomonas point to two key oxygen-dependent steps in the tetrapyrrole pathway that are regulated by copper: 1) CPX1, encoding a chloroplast isoform of coproporphyrinogen III oxidase, is 10-fold up-regulated in copper-deficiency and 2) CHL27A and CHL27B, encoding differently localized isoforms of the MgProtoporphyrin IX monomethylester cyclase, are reciprocally regulated by copper nutrition (Hill and Merchant, 1995; Moseley et al., 2000). We have proposed that the adjustment of flux through compartmentalized parallel branches of the tetrapyrrole pathway allows re-modelling of the electron transfer chain in the chloroplast to accommodate the reduced efficiency of cytochrome c 6 in –Cu cells (the Fe-containing back up protein) relative to plastocyanin in +Cu cells.
New copper enzymes of Chlamydomonas: Analysis of a draft genome of Chlamydomonas (www.jgi.doe.gov) indicates the occurrence and expression of several other copper enzymes or novel proteins based on sequence similarities and the presence of copper binding site motifs. Of particular interest are candidate (mono- and di-) amine oxidases related to fungal peroxisomal enzymes and mammalian amiloride-sensitive enzymes, multicopper oxidases related to ascorbate oxidases and laccases, and a new family of matrix metalloproteinases (MMPs) with an atypical QExxH metal binding site that show 10-fold selectivity for copper vs. zinc for activity. The physiological functions of these enzymes are not established definitively for any organism yet, but candidate pathways include nitrogen utilization, metabolism of biogenic amines, and re-modelling of the extracellular matrix. The extracellular matrix is a dynamic cell structure and a critical mediator of growth, development and response to wounding. The role of copper in contributing to this structure is well established but the mechanistic details are not understood, nor is the consequence of copper-deficiency on the biochemistry of the extracellular matrix explored.
Generality of trace element-dependent metabolic changes: Although copper-dependent modification of metabolic pathways is best established in the Chlamydomonas system, the phenomenon is not unique to this organism. For example, in methanotrophic bacteria, particulate methane monooxygenase, which is a multicopper oxidase, is expressed in a copper-replete environment, but under copper-deficiency, a soluble di-iron enzyme substitutes. In the copper-deficient rat, the expression of MnSOD is increased to compensate for loss of function of CuZnSOD, while in crustaceans that need copper for maintenance of the oxygen carrier hemocyanin, a novel cytosolic MnSOD functions in place of cytosolic CuZnSOD.
References
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Eriksson, M., Gardestrom, P. and Samuelsson, G. (1995) Isolation, purification and characterization of mitochondria from Chlamydomonas reinhardtii. Plant Physiol, 107, 479-483.
Eriksson, M., Moseley, J.L., Tottey, S., del Campo, J.A., Quinn, J.M., Kim, Y. and Merchant, S. (2004) Genetic dissection of nutritional copper signaling in Chlamydomonas distinguishes regulatory and target genes. Genetics, 168, 795-807.
Frieden, E. (1985) New perspectives on the essential trace elements. J Chem Ed, 62, 917-923.
Hill, K.L., Hassett, R., Kosman, D. and Merchant, S. (1996) Regulated copper uptake in Chlamydomonas reinhardtii in response to copper availability. Plant Physiol, 112, 697-704.
Hill, K.L. and Merchant, S. (1995) Coordinate expression of coproporphyrinogen oxidase and cytochrome c6 in the green alga Chlamydomonas reinhardtii in response to changes in copper availability. EMBO J, 14, 857-865.
La Fontaine, S., Quinn, J.M., Nakamoto, S.S., Page, M.D., Göhre, V., Moseley, J.L., Kropat, J. and Merchant, S. (2002) Copper-dependent iron assimilation pathway in the model photosynthetic eukaryote Chlamydomonas reinhardtii. Eukaryot Cell, 1, 736-757.
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Quinn, J.M. and Merchant, S. (1998) Copper-responsive gene expression during adaptation to copper deficiency. Methods Enzymol, 297, 263-279.