COX deficiency in human presents a plethora of phenotypes which is not surprising given the complexity of the enzyme structure and the multiple factors and many steps required for its assembly. A functional COX requires three mitochondrially encoded subunits (Cox1p, Cox2p and Cox3p), at least 10 nuclearly encoded subunits, some of which are tissue specific, and a yet unknown number of assembly factors. Mutations in four of these factors, hSco1p, hSco2p, hCox10p and hSurf1p, have been associated with lethal COX deficiency in patients. Sco proteins, conserved from prokaryotes to eukaryotes, are probably involved in the insertion of copper in COX. The role of hSco1p and hSco2p in this process was investigated in this work. Moreover the importance of some hSco mutations found in patients was analysed. Both in vitro and in vivo analyses show that the hSco proteins are localised in the mitochondria. Both proteins are per se unable to substitute for ySco1p. However, a chimeric construct consisting of the N-terminal portion, the TM and a part of the C-terminal portion of ySco1p and the remaining C-terminal part derived from hSco1p was able to complement a ysco1 null mutant strain. This construct was used to define the role of a point mutation (P174L) found in the hSCO1 gene of infants suffering from ketoacidotic coma. These mutation was shown to affect the COX activity and the levels of Cox1p and Cox2p. The fact that copper was able to suppress this mutation, strongly outlined the importance of Sco proteins in the copper insertion in COX. The C-terminal portions of recombinant hSco1p and hSco2p were purified from E. coli by affinity chromatography. The purified proteins were subjected to atomic emission and absorption analyses and were shown to specifically bind copper. A stoichiometry of 1:1 for hSco2p and of 0,6:1 for hSco1p was determined. To identify the Aa residues involved in copper binding, in vitro mutagenesis was performed. hSco1p and hSco2p, lacking the cysteines of the predicted metal binding site CxxxC, show a dramatic decrease in the ability to bind copper. A model for the structure of the metal binding site in hSco proteins is proposed. hSco proteins could bind copper with trigonal coordination, involving the two cysteines of the CxxxC motif and a conserved histidine. The purified recombinant proteins were also used in an enzymatic assay to test their ability to reduce disulfide bridges, similar to thioredoxin-like proteins involved in the assembly of bacterial COX. Both hSco proteins were not able to act as thioredoxins suggesting a role for the hSco proteins as copper chaperones. To define the pathway of the copper transfer to COX, hSco proteins were tested for their ability to interact with hCox17p, a mitochondrial copper chaperone, and with Cox2p, which contains two copper ions. An interaction between hSco1p and Cox2p was detected. Both hSco proteins were shown to homomerise and to form heterodimers one with each other. Two mutations found in hSCO2 patients suffering from hypertrophic cardiomyopathy, (E140K and S225F) were shown not to affect the copper binding properties, the intracellular localisation and the ability to form homomers. In accordance to these data, a model is proposed in which hSco2p dimers transfer copper to hSco1p dimers. hSco1p dimers interact with COX and insert copper in the binuclear centre of Cox2p.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:24109 |
| Date | 17 December 2001 |
| Creators | Paret, Claudia |
| Contributors | Rödel, Gerhard, Göttfert, Michael, Jaksch, Michaela |
| Publisher | Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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