Solution Structure of Cox11, a Novel Type of β-Immunoglobulin-like Fold Involved in CuB Site Formation of Cytochrome c Oxidase

Abstract

Cytochrome c oxidase assembly process involves many accessory proteins including Cox11, which is a copper-binding protein required for Cu incorporation into the Cu B site of cytochrome c oxidase. In a genome wide search, a number of Cox11 homologs are found in all of the eukaryotes with complete genomes and in several Gram-negative bacteria. All of them possess a highly homologous soluble domain and contain an Nterminal fragment that anchors the protein to the membrane. An anchor-free construct of 164 amino acids was obtained from Sinorhizobium meliloti, and the first structure of this class of proteins is reported here. The apoform has an immunoglobulin-like fold with a novel type of ␤-strand organization. The copper binding motif composed of two highly conserved cysteines is located on one side of the ␤-barrel structure. The apoprotein is monomeric in the presence of dithiothreitol, whereas it dimerizes in the absence of the reductant. When copper(I) binds, NMR and extended x-ray absorption fine structure (EXAFS) data indicate a dimeric protein state with two thiolates bridging two copper(I) ions. The present results advance the knowledge on the poorly understood molecular aspects of cytochrome c oxidase assembly.Cytochrome c oxidase (CcO) 1 is the terminal enzyme in the electron transport system, reducing oxygen to water and generating the proton gradient that drives ATP synthesis. Multiple subunits and several cofactors are necessary for catalytic activity including two hemes a, a magnesium ion, a zinc ion, and three copper ions. In particular, the copper ions are located in subunits COX I and COX II, which contain the Cu B and Cu A centers, respectively. The insertion of these cofactors and assembly of the CcO complex in the inner mitochondrial membrane requires accessory proteins (for a general review, see Ref.1). It has recently become clear that the required metal ions cannot simply diffuse to the requisite compartment of the cell for insertion into the desired protein, but a complex machinery of metal importers and chaperones is required (2). In eukaryotes, two metallochaperones, Cox17 and Cox19, were proposed to shuttle copper in the mitochondrion (3, 4). Other nuclear genes also are required for the proper insertion of copper into CcO (5-8). In particular, it is well established that a mitochondrial inner membrane protein, Sco1, interacting with COX II subunit (9), is important for copper insertion into the binuclear Cu A site (10, 11). On the contrary, the process through which copper is provided to COX I subunit, which contains a copper ion buried 13 Å below the membrane surface, still remains essentially obscure, even if it is known that it requires another mitochondrial inner membrane protein, Cox11. Cox11 was first shown to be implicated in the CcO maturation process from the observation that ⌬cox11 yeast lacked CcO activity and was deficient in heme a (5). It was also observed that, for cox11 mutants lacking CcO activity, RNA and protein synthesis of the core subunits I and II are normal, sugg...

Yeast Cox17 Solution Structure and Copper(I) Binding

“…It could be that this apo protein is binding less than the full complement of Cu(I), but this possibility is unlikely because thiol quantitation clearly indicates that all seven cysteines are reduced and available for metal binding. Another [23][24][25][26][27] in addition to the helical residues (residues 28 -39 and 48 -58). explanation is that Cox17 isolated from cells grown with CuSO 4 has an artificially high number of Cu(I) ions.…”

“…To understand the function of Cox17, knowledge of its three-dimensional structure and clarification of its copper binding stoichiometry are required. In particular, it is not known how Cox17 can recognize and interact with both Sco1 and Cox11, which have very different structures from one another (24,25). It is also unclear how copper might be transferred from a polynuclear cluster in Cox17 to the mononuclear site in Sco1 or to the dinuclear site in Cox11 and subsequently to the dinuclear Cu A center or the mononuclear Cu B center.…”

“…The C-terminal soluble domain of Cox11 is a dimer and binds one Cu(I) ion/monomer. EXAFS data indicate the presence of a dinuclear cluster with each Cu(I) ion ligated by three sulfurs (10,25). The solution structure of dimeric Cu(I)-loaded Cox11 from Sinorhizobium meliloti suggests that two ligands derive from a CXC motif located on the side of an immunoglobulin-like ␤ barrel structure, with the third ligand donated by the same CXC motif in the second monomer (25).…”

Functional Characterization of Mia40p, the Central Component of the Disulfide Relay System of the Mitochondrial Intermembrane Space

“…Mia40p might not be the only substrate of Erv1p in the IMS of mitochondria, since there are, in addition to proteins with twin CX n C motifs, other proteins containing disulfide bonds. Examples are the Rieske Fe/S protein of the respiratory chain, the assembly factor Cox11p of the cytochrome oxidase, and the superoxide dismutase Sod1p and its copper chaperone Ccs1p (47)(48)(49)(50).…”

Crystallography with X-ray and optical spectroscopies for metalloproteins structural studies

“…(Elam et al 2002;Rosenzweig 2001) At CERM we have focussed our work on the study of copper transport proteins in different organisms by x-ray crystallography and by coupling NMR and x-ray absorption (XAS) spectroscopic techniques that, combined, offer the possibility to achieve the complete structure determination of a metalloprotein in solution and provide unique information on the electronic structure of the metal ion and on how it influences its binding to the protein (Arnesano et al 2003;Banci et al 2003;Banci et al 2004;Banci et al 2005a,b;Banci et al 2006). The most recent applications of the NMR-XAS approach to the structure determination of copper proteins involved in the assembly of bacterial and human cytochrome C oxidase will be presented and discussed as well as the comparison with crystallographic results.…”