Let's Sco1, Oxidase! Let's Sco!

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    Figure 1. Mechanism of Pore Formation byActinoporins

    (A) Toxin monomers bind to the membranesurface by an aromatic rich surface (blue).(B) The N-terminal segment with amphipathichelix (red) dislocates from the sandwich coreand anchors into the membrane-water interface,exposing polar residues toward solution.

    (C) After aggregation of four monomers, a transmembrane funnel-shaped pathway is set up by lipid headgroups and helices, exposing sidechains of acidic amino acids (yellow) in the lumen of the toroidal pore. Residues 110 are not shown.

    de los Rios, V., Mancheno, J.M., Lanio, M.E., Onaderra, M., and Podlesek, Z., Macek, P., Turk, D., Gonzales-Manas, J.M., Lakey,Gavilanes, J.G. (1998). Eur. J. Biochem. 252, 284289. J.H., et al. (2002). J. Biol. Chem. 277, 4191641924.

    Heuck, A.P., Tweten, R., and Johnson, A.E. (2001). -barrel pore- Lakey, J.H., and Slatin, S.L. (2001). Curr. Top. Microbiol. Immunol.forming toxins: intriguing dimorphic proteins. Biochemistry 40, 257, 131161.90659073. Malovrh, P., Barlic, A., Dalla Sera, M., Podlesek, Z., Lakey, J.H.,Hermoso, J.A., Mancheno, J.M., Martn-Benito, J., Martnez-Ripoll, Macek, P., Menestrina, G., and Anderluh, G. (2003). J. Biol. Chem.M., and Gavilanes, J.G. (2003). Structure, 11, this issue, 13191328. 278, 2267822685.Hinds, M.G., Zhang, W., Anderluh, G., Hansen, P.E., and Norton, Valcarcel, C.A., Dalla Sera, M., Potrich, C., Bernhart, I., Tejuca, M.,R.S. (2002). J. Mol. Biol. 315, 12191229. Martinez, D., Pazos, F., Lanio, M.E., and Menestrina, G. (2001). Bio-

    phys. J. 80, 27612774.Hong, Q., Gutierrez-Aguirre, I., Barlic, A., Malovrh, P., Kristan, K.,

    Structure, Vol. 11, November, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/j .str .2003.10.006

    drion for assembly of CcO is unknown. As Cu deliveryLets Sco1, Oxidase! Lets Sco!to Sod1 and the P-type ATPase transporters is proteinmediated, the prediction is that Cu insertion into nascentCcO chains will also be protein mediated. Although Sco1

    The solution structure of Sco1 from Bacillus subtilis is not a copper shuttle analogous to Atx1 and CCS, itis the first structure of a protein important in the as- is postulated to function as a cometallochaperone insembly of cytochrome c oxidase (CcO). The assembly the insertion of Cu into CcO. Mammalian CcO consistsof CcO requires the insertion of multiple cofactors. of a 13 subunit complex embedded within the IM. ThreeSco1 is a conserved protein implicated in formation core subunits of the complex, Cox1-3, are encoded byof the binuclear CuA center. the mitochondrial genome, whereas the remaining 10

    subunits are nuclear encoded. Copper ions exist in twoCopper is an essential nutrient serving as a cofactor CcO centers designated CuA and CuB enfolded by twoin numerous redox enzymes and enzymes involved in mitochondrial-encoded subunits (Tsukihara et al., 1995).oxidative reactions. Copper ions are shuttled to sites of The CuA center in Cox2 is a binuclear center within autilization by protein-mediated transfer reactions involv- domain that protrudes into the soluble intermitochon-ing metallochaperones (Huffman and OHalloran, 2001; drial membrane space (IMS). This domain is the dockingRosenzweig, 2001). Copper transfer is mediated by pro- site for cytochrome c for electron transfer to the CuAtein:protein interactions between the metallochaperone center. The CuB center is buried within Cox1 forming aand the respective target molecule. Copper insertion binuclear site with one of the two heme A cofactors.into the Cu,Zn-superoxide dismutase (Sod1) requires Copper metallation of CcO involves at least three pro-the function of the CCS metallochaperone. Secretory teins, Sco1, Cox11, and Cox17. Cox17 is implicated incupro-enzymes are metallated within post-Golgi vesi- the delivery of copper ions to the mitochondrion,cles by copper ions translocated across the membrane whereas Sco1 and Cox11 are believed to be cometallo-by P-type ATPases. Cu(I) ions are presented to the chaperones assisting Cox17 in the metallation of CcOATPase translocase by the Atx1 metallochaperone. Atx1 (Carr and Winge, 2003). Cox11 appears to be specificand CCS share a conserved structural motif that is the for CuB site formation. Sco1 was first implicated in CuCu(I) binding domain. The Cu(I) binding site is near the delivery to CcO by the observation that the respiratory-surface of the protein being partially solvent shielded, deficient phenotype of a cox17 yeast mutant can beallowing access for the target molecule yet protection suppressed by overexpression of SCO1 or a relatedfor the cell (Arnesano et al., 2002). The metallochaperone gene designated SCO2 (Glerum et al., 1996). Sco1 anddata suggest that individual molecules exist for the load- Sco2 are highly similar yeast proteins associated withing of cupro-enzymes. the mitochondrial IM. Both proteins contain a single

    A major cupro-enzyme is cytochrome c oxidase (CcO), transmembrane helix anchoring a soluble domain thatwhich resides within the mitochondrial inner membrane projects into the IMS. Yeast cells lacking Sco1, but not

    Sco2, are respiratory deficient and show diminished(IM). The mechanism of Cu(I) routing to the mitochon-

  • Structure1314

    CcO activity. Two SCO genes, HsSCO1 and HsSCO2, the Atx1:Ccc2 ATPase interaction. Complex formationare essential in humans. The defective CcO activity in between the two proteins may bring the Cu(I) ion in anHsSCO2 patient myoblasts was rescued by the addition exposed loop of Sco1 in close juxtaposition for ligandof exogenous copper to the culture medium (Jaksch et exchange reactions to drive the Cu(I) ion to Cox2. CuAal., 2001). site formation requires two Cu ions in the center. Since

    The working model of copper ion insertion in eukary- Sco1 binds only a single Cu(I) ion, two successive dock-otic CcO is that Sco1 mediates the transfer of Cu(I) ions ing and exchange reactions may be required for forma-from Cox17 to Cox2 forming CuA, and Cox11 mediates tion of the binuclear center. Within the CuA center, thethe transfer of Cu(I) to Cox1 forming CuB. In support two Cu ions are bridged by two Cys residues within aof this model, both Sco1 and Cox11 are Cu(I) binding CxxxC motif, similar to part of the Cu binding site inproteins (Carr and Winge, 2003). A conserved histidine Sco1. These Cys residues in Cox2 may mediate theand two conserved cysteine residues in CxxxC motif are ligand exchange reactions.essential for Sco1 function and Cu(I) ligation. In addition, As shown in the paper, Sco1 can bind Cu(II) in additionSco1 is known to interact with Cox2 in vitro (Lode et al., to Cu(I). However, the identity of the additional ligands2000). and significance of Cu(II) binding are unknown. Interest-

    Data supporting a role for Sco1 in CuA site formation ingly, the structurally related peroxiredoxins have thiol-in CcO comes from Bacillus subtilis. This organism con- disulfide oxidoreductase activity, so oxidation of Cystains two related terminal oxidases, one of which is a residues in Sco1 may promote copper ion transfer tocytochrome c oxidase with the same heme and copper Cox2.cofactor requirements as eukaryotic CcO. The secondoxidase is the menaquinol oxidase that contains the

    Dennis R. WingeCuB center but not the CuA center and does not useDepartments of Biochemistry and Medicinecytochrome c as a reductant. Deletion of the Sco1 ho-University of Utah Health Sciences Centermolog BsSco1 (ypmQ) results in loss of CcO activity butSalt Lake City, Utah 8413not menaquinol oxidase activity (Mattatall et al., 2000).

    CcO activity is also restored in the null mutant by theaddition of exogenous copper to cultures.

    Selected ReadingThe solution structure of the apo-conformer of BsSco1reveals that the soluble domain adopts a thioredoxin- Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffoni, S., Molteni, E., Huff-type fold characterized by a central sheet flanked man, D.L., and OHalloran, T.V. (2002). Genome Res. 12, 255271.by three helices (Balatri et al., 2003, this issue). Two Balatri, E., Banci, L., Bertini, I., Canttini, F., and Ciofi-Baffoni, S.important aspects of the BsSco1 structure emerge. (2003). Structure 11, this issue, 14311443.First, two loops projecting off central strands contain Carr, H.S., and Winge, D.R. (2003). Acc. Chem. Res. 36, 309316.the candidate Cu(I) binding ligands. The two conserved Glerum, D.M., Shtanko, A., and Tzagoloff, A. (1996). J. Biol. Chem.Cys residues in the CxxxC motif exist in the chain turn 271, 2053120535.of one loop and the conserved His residue exists in the Huffman, D.L., and OHalloran, T.V. (2001). Annu. Rev. Biochem. 70,second loop. These loops exhibited the highest disorder 677701.in the structure consistent with the absence of bound Jaksch, M., Paret, C., Stucka, R., Horn, N., Muller-Hocker, J., Hor-Cu(I). Cu(I) binding to these candidate residues in the vath, R., Trepesch, N., Stucker, G., Freisinger, P., Thirion, C., et al.protruding loops would likely generate a partially solvent (2001). Hum. Mol. Genet. 10, 30253035.accessible site as is the case with CuAtx1. Second, a Lode, A., Kuschel, M., Paret, C., and Rodel, G. (2000). FEBS Lett.negative electrostatic patch exists near the putative 448, 16.Cu(I) site and residues contributing to this electronega- Mattatall, N.R., Jazairi, J., and Hill, B.C. (2000). J. Biol. Chem. 275,tive surface are conserved. Modeling suggests that 2880228809.Cox2 has a positive electrostatic surface potential con- Rosenzweig, A.C. (2001). Acc. Chem. Res. 34, 119128.sistent with complimentary electrostatic interactions be- Tsukihara, T., Aoyama, H., Yamashita, E., Tomizaki, T., Yamaguchi,ing important for the docking of Sco1 on Cox2. Compli- H., Shinzawa-Itoh, K., Hakashima, R., Yaono, R., and Yoshikawa, S.

    (1995). Science 269, 10691074.mentary electrostatic interactions are also important in