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Poly-oxo anion chemistry | Bio-inorganic chemistry | Collaborators

Bio-inorganic chemistry

(with Dr. Zhiguang Xiao, Gjoko Buncic, Karrera Djoko, Matthias Zimmermann, Chak Ming Sze and Lee Xin Chong)

The trace metals are essential to life for enzymes but are highly toxic in excess. A balance between deficiency and toxic excess must be maintained. The secrets of their catalytic and structural roles are under intensive scrutiny.

Acquisition of the metals is mediated by molecular membrane pumps and by transport proteins which take the metal to its destinations in biological cells. Figure 1 shows a simplified representation of transport of copper in mammalian cells. Defects in copper metabolism cause Menkes and Wilson diseases in humans and is a component of certain neurodegenerative diseases (Alzheimer, Parkinson, motor neuron, Creutzfeldt-Jakob, mad cow). See, eg, ref 1. Each of the key proteins associated with these diseases is a copper binding protein. Very recently, copper transport proteins have been implicated as critical components of tuberculosis, of cancer cell proliferation and of fungal virulence.

Figure 1. Over-simplified model of copper transport in human cells such as liver hepatocytes which receive much of the copper absorbed from the small intestine. Membrane pumps Ctr1 and ATP7A,B and chaperones Ccs and Atox1 transport Cu^I. Ceruloplasmin (Cp), Cytochrome c oxidase (Cyt ox) and superoxide dismutase (Sod1) are redox enzymes which employ the Cu^II/Cu^I couple. As well as loading Cp with copper, the trans-Golgi network (TGN) inserts copper into many enzymes. The ATP7B pump performs two functions: it transports nutrient copper into the TGN and excess copper out of the cell (by trafficking to the cell membrane via vesicles). It may also supply copper to phagosomes for destruction of invading bacteria. ATP7A is the equivalent pump in most other cells.

The molecular pump Ctr1 (Copper Transporter no. 1) is primarily responsible for import of copper into human cells. It appears to interact with the transport protein (chaperones) via a beautiful Cu₄S₆ cluster (Figure 2).^[2] The chaperone Atox1 carries copper to the Menkes and Wilson disease proteins ATP7A, B. Each of these proteins appears to interact with the enzyme glutathione reductase which may control the copper binding properties of the proteins.

In addition, the cancer drug cis-platin enters certain cells via the Ctr1 pump. Interactions of cis-platin with the above copper proteins appears to be a major cause of loss of drug and side effects. We are studying the pumps and chaperones and their interactions with cis-platin.

Certain bacteria have developed the unusual ability to survive in environments with millimolar concentrations of copper (>1,000 times higher than normal nutrient levels). They have evolved clusters of genes which are induced by copper and whose protein products combine to export excess copper (Figure 3). We have studied the properties of the protein PcoC (CopC) and shown that it has the unique ability to bind copper in either of its oxidation states Cu^I and Cu^II (Figure 4).^[4,5] In addition, the oxidase enzyme PcoA can oxidise Cu^I bound to PcoC catalytically to the less toxic Cu^II form (Figure 5).^[6] This can then be pumped out of the cell.

A new protein, CopK from C. metallidurans also binds two copper ions but with very different structural features (Figure 6). Remarkably, a unique cooperativity means that binding of Cu^I increases the affinity for Cu^II by six orders of magnitude. The Cu^II binding site is assembled upon insertion of Cu^I into its site.^[7]

The proteins are generated via molecular genetics and then purified. The molecular probes needed are provided by techniques such as NMR, ESR, MS, fluorescence, X-ray crystallography, electro-chemistry and quantitative HPLC.

New projects include:-

1. design and synthesis of new chromophoric ligands to act as quantitative probes of bio-metals, both in vivo and in vitro;

2. transport of nutrient metals in normal and in hyper-accumulating plants;^[4]

3. zinc and copper transporters in the simple plant Arabidopsis thaliana.

4. Cell-free expression of membrane proteins (such as CopB, D, R of Figure 4).