Yeast Contain a Non-proteinaceous Pool of Copper in the Mitochondrial Matrix

The yeast mitochondrion is shown to contain a pool of copper that is distinct from that associated with the two known mitochondrial cuproenzymes, superoxide dismutase (Sod1) and cytochrome c oxidase (CcO) and the copper-binding CcO assembly proteins Cox11, Cox17, and Sco1. Only a small fraction of mitochondrial copper is associated with these cuproproteins. The bulk of the remainder is localized within the matrix as a soluble, anionic, low molecular weight complex. The identity of the matrix copper ligand is unknown, but the bulk of the matrix copper fraction is not protein-bound. The mitochondrial copper pool is dynamic, responding to changes in the cytosolic copper level. The addition of copper salts to the growth medium leads to an increase in mitochondrial copper, yet the expansion of this matrix pool does not induce any respiration defects. The matrix copper pool is accessible to a heterologous cuproenzyme. Co-localization of human Sod1 and the metallochaperone CCS within the mitochondrial matrix results in suppression of growth defects of sod2Δ cells. However, in the absence of CCS within the matrix, the activation of human Sod1 can be achieved by the addition of copper salts to the growth medium. The yeast mitochondrion is shown to contain a pool of copper that is distinct from that associated with the two known mitochondrial cuproenzymes, superoxide dismutase (Sod1) and cytochrome c oxidase (CcO) and the copper-binding CcO assembly proteins Cox11, Cox17, and Sco1. Only a small fraction of mitochondrial copper is associated with these cuproproteins. The bulk of the remainder is localized within the matrix as a soluble, anionic, low molecular weight complex. The identity of the matrix copper ligand is unknown, but the bulk of the matrix copper fraction is not protein-bound. The mitochondrial copper pool is dynamic, responding to changes in the cytosolic copper level. The addition of copper salts to the growth medium leads to an increase in mitochondrial copper, yet the expansion of this matrix pool does not induce any respiration defects. The matrix copper pool is accessible to a heterologous cuproenzyme. Co-localization of human Sod1 and the metallochaperone CCS within the mitochondrial matrix results in suppression of growth defects of sod2Δ cells. However, in the absence of CCS within the matrix, the activation of human Sod1 can be achieved by the addition of copper salts to the growth medium. Copper is an essential cell nutrient acting as a cofactor in nearly 20 enzymes (1Linder M.C. Hazegh-Azam M. Am. J. Clin. Nutr. 1996; 63: 797S-811SGoogle Scholar). However, excess accumulation of copper ions results in toxicity. Evidence for the effectiveness of copper ions as a toxin comes from its historic use as a fungicide, molluscide, and algicide. Homeostatic mechanisms exist in cells to regulate the cellular concentration of copper ions, thus maintaining copper balance and minimizing deleterious effects. Cells appear to maintain a quota for essential metal ions; this quota is primarily the quantity necessary to metallate the various copper proteins (2Outten C.E. O'Halloran T.V. Science. 2001; 292: 2488-2492Google Scholar, 3Finney L.A. O'Halloran T.V. Science. 2003; 300: 931-936Google Scholar). Copper ions are required for at least three key enzymes in Saccharomyces cerevisiae. The cuproenzymes include the cytosolic superoxide dismutase Sod1, 1The abbreviations used are: Sod, superoxide dismutase; CcO, cytochrome c oxidase; IMS, mitochondrial intermembrane space; IM, inner membrane; OM, outer membrane; h-, human; y-, yeast; ER, endoplasmic reticulum; MS, mass spectroscopy; ICP-OES, inductively coupled plasma-optical emission spectroscopy. the plasma membrane ferroxidase Fet3, and the mitochondrial inner membrane enzyme cytochrome c oxidase (CcO). The copper quota of the yeast S. cerevisiae is about 5 × 105 atoms per cell (3Finney L.A. O'Halloran T.V. Science. 2003; 300: 931-936Google Scholar, 4Rae R.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-807Google Scholar). It is unclear what fraction of the 5 × 105 copper atoms per cell is from copper in Sod1, Fet3, and CcO. Expression of these copper-binding proteins varies with growth conditions, suggesting that the copper may be distributed differently depending on growth conditions. Clearly, a significant fraction of the cellular copper is associated with Sod1; however, not all Sod1 molecules are metallated (4Rae R.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-807Google Scholar, 5Culotta V.C. Joh H.D. Lin S.J. Slekar K.H. Strain J. J. Biol. Chem. 1995; 270: 29991-29997Google Scholar). Fet3 requires four copper ions for activity, but levels of this protein are dependent on iron status of the medium (6Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Google Scholar). CcO levels vary depending on whether the cells are grown by fermentation or respiration. In addition, a varying quantity of cellular copper exists bound to two metallothioneins, Cup1 and Crs5 (7Karin M. Najarian R. Haslinger A. Valenzuela P. Welch J. Fogel S. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 337-341Google Scholar, 8Jensen L.T. Howard W.R. Strain J.J. Winge D.R. Culotta V.C. J. Biol. Chem. 1996; 271: 18514-18519Google Scholar). Expression of CUP1 and CRS5 is regulated by copper levels through the copper-responsive transcription factor Ace1 (9Furst P. Hu S. Hackett R. Hamer D. Cell. 1988; 55: 705-717Google Scholar, 10Thiele D.J. Mol. Cell. Biol. 1988; 8: 2745-2752Google Scholar). An increase in the free Cu(I) ion pool activates Ace1 through formation of a polycopper cluster (11Dameron C.T. Winge D.R. George G.N. Sansone M. Hu S. Hamer D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6127-6131Google Scholar). Activated Ace1 induces transcription of CUP1, CRS5, and SOD1. Each protein is capable of buffering the copper ion concentration in the cytoplasm. Copper buffering regulated by Ace1 must be highly efficient because yeast cells are predicted to lack a pool of free copper ions in the cytoplasm (4Rae R.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-807Google Scholar). Cells concentrate copper by several orders of magnitude from the culture medium to achieve the copper quota (2Outten C.E. O'Halloran T.V. Science. 2001; 292: 2488-2492Google Scholar). This gradient is generated by multiple metal ion permeases on the plasma membrane. Copper ion uptake is mediated by high affinity Ctr1 and Ctr3 and low affinity Smf1 and Fet4 permeases (12Puig S. Thiele D.J. Curr. Opin. Chem. Biol. 2002; 6: 171-180Google Scholar). Within the cell other transporters are necessary for transmembrane movement of copper ions. Translocation of copper ions into the lumen of trans-Golgi vesicles is achieved by P-type ATPase transporters (Ccc2 in yeast, ATP7A and ATP7B in animal cells) (13Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Google Scholar). Translocation of copper ions across the vacuolar membrane is mediated by Ctr2 (14Portnoy M.E. Schmidt P.J. Rogers R.S. Culotta V.C. Mol. Genet. Genomics. 2001; 265: 873-882Google Scholar). Copper ions are shuttled to sites of utilization by protein-mediated transfer O'Halloran T.V. 2001; Scholar, Culotta V.C. J. 2002; Scholar). the of copper ions to sites through a and the of copper ions with other Copper into Sod1 in yeast requires the of the metallochaperone (4Rae R.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-807Google Scholar, V.C. Strain J. J. Biol. Chem. Scholar, J. Scholar, D. J. Biol. Chem. Scholar). In the absence of CCS copper ions are not into yeast Sod1 cells are in medium high levels of copper and this is by a in levels of the by a of (4Rae R.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-807Google Scholar). This is an of the cytosolic of Copper into Fet3 within The copper is across the membrane by the P-type ATPase (13Yuan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Google Scholar). Cu(I) ions are shuttled to the by the metallochaperone Pufahl R.A. Dancis A. O'Halloran Culotta V.C. J. Biol. Chem. Scholar, O'Halloran T.V. J. Biol. Chem. Scholar). of or results in and an to on medium Pufahl R.A. Dancis A. O'Halloran Culotta V.C. J. Biol. Chem. Scholar). Cu(I) ions by the can be used to metallate heterologous molecules within trans-Golgi vesicles R. Culotta V.C. J. Biol. Chem. 2003; Scholar). The of copper ion to the mitochondrion for assembly of cytochrome c oxidase is copper ion to Sod1 and is the is that copper to the mitochondrion be proteins in copper ion to the mitochondrion are and A. A. J. Biol. Chem. 1996; 271: Scholar, J. A. J. Biol. Chem. Scholar, J. A. J. Biol. Chem. 2002; Scholar). proteins are in cells and a in the and the mitochondrial intermembrane J. A. J. Biol. Chem. Scholar, J. A. J. Biol. Chem. 2002; Scholar). The is an the mitochondrial inner membrane and outer membrane and is by in the and are in 2002; Scholar). in copper ion to the on its and the that the of cells is by high levels of copper in the growth medium A. A. J. Biol. Chem. 1996; 271: Scholar). copper for is because Cu(I) through three in a D. T. Winge D.R. J. Biol. Chem. Scholar). to in the of three J. A. J. Biol. Chem. 2002; however, these in are not of the Cu(I) D. T. Winge D.R. J. Biol. Chem. Scholar). cells or are and of cytochrome c oxidase of these by the addition of copper is in cells. Within the mitochondrion two inner membrane and Cox11, are in copper ion into cytochrome c oxidase M. Mol. Genet. 1988; Scholar, Curr. Genet. Scholar, A. A. J. Biol. Chem. 1996; 271: Scholar, A. A. Curr. Genet. 1999; Scholar, T. George G.N. Winge D.R. J. Biol. Chem. 2001; Scholar, George G.N. Winge D.R. J. Biol. Chem. 2002; Scholar). and are in of copper transfer to the and The two copper exist within of cytochrome c copper ions must be shuttled to the mitochondrion for into within the inner membrane. may Cu(I) to and for to the and proteins may exist for and in the of Cu(I) ions to the may Cu(I) ions to a mitochondrial in to the of Cu(I) ions to the trans-Golgi may Cu(I) ions across the mitochondrial proteins are across the through the of the outer membrane but these proteins are as proteins M. EMBO J. Scholar, Curr. Opin. Biol. 2002; 14: Scholar). of Cu(I) across the by of the to its copper The other known mitochondrial copper protein is Sod1, a within the and the L.A. L.T. R. Culotta V.C. J. Biol. Chem. 2001; Scholar). of Sod1 is localized within the L.A. L.T. R. Culotta V.C. J. Biol. Chem. 2001; Scholar). The fraction of Sod1 is dependent on the CCS metallochaperone Y. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2003; Scholar). of CCS to the a heterologous mitochondrial accumulation of Sod1 within the IMS, Sod1 can to the in the absence of CCS L.A. L.T. R. Culotta V.C. J. Biol. Chem. 2001; Scholar, Y. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2003; Scholar). Copper ion of Sod1 within the because mitochondrial Sod1 is dependent on the Y. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2003; Scholar). The of copper ions for the of Sod1 in the is unknown, but does not appear to formation of Sod1 within the L.A. L.T. R. Culotta V.C. J. Biol. Chem. 2001; Scholar). CCS does not CcO V.C. Strain J. J. Biol. Chem. Scholar). that is required for the or accumulation of copper in the suggesting that as for the bulk of mitochondrial for the that a significant pool of copper exists within the mitochondrion that is not associated with CcO or This pool with matrix and does not appear to be This pool of copper can be to metallate a heterologous human Sod1, to the mitochondrial from and its used for all for and from Cells from Cells by R. and T. The by in a Expression from P. J. into and to the of the yeast that the mitochondrial The the of the with the of this in the of to This the mitochondrial of and cytosolic by and by contain CCS to of in a of the to Y. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2003; a of of to that CCS to the into yeast by the of as L.A. 1995; Scholar). used to that in a the by in the of at × and at × the the The a gradient on and at × for The from the gradient in and at × protein the into and at × to the The as and into and membrane by or in of protein from the mitochondrial or fraction on a and a membrane. in and protein to the vacuolar and from to cytochrome by R. to and by Culotta and to from in 5 of and and and 5 of in at in of The to into for of metal used to a of or in the for and to c oxidase in by the of cytochrome c at by of by three of at of a The fraction from the fraction by at × The fraction into and with a The the a in of A. The protein with 5 of A. gradient of and by in The remainder of the a or in 20 the and by into The remainder of the used for or for mass Copper to the and as mitochondrial copper ion for assembly of cytochrome c oxidase on in the cytoplasm and the of J. A. J. Biol. Chem. Scholar). In addition, proteins are capable of copper ions. that Cu(I) within a cluster George G.N. Winge D.R. 2001; Scholar). is a copper-binding are copper ion for assembly of CcO, the is that cells or mitochondrial copper The copper levels in from a of cells of the CcO assembly The of copper within the mitochondrion of the of or and a by a shown in the mitochondrial copper not by this In the mitochondrial copper concentration not in yeast is the of CcO that the or in are of The of mitochondrial copper of the used for yeast not The lack of an in mitochondrial copper in cells a CcO that contain a pool of copper not associated with CcO or CcO assembly of the fraction of mitochondrial copper associated with the CcO the pool within CcO is the protein to use and all is associated with the CcO complex. defects in assembly of CcO and are of within the mitochondrion A. A. J. Biol. Chem. 1996; 271: Scholar, A. A. J. Biol. Chem. 1996; 271: Scholar, A. D. EMBO J. Scholar). an for the of CcO can be from the of M. L.A. 1995; Scholar). the CcO three copper ions per the fraction of mitochondrial copper associated with CcO can be This that of the mitochondrial copper associated with CcO This is on the that all CcO molecules are This is because is known that that to copper into CcO in an that does not contain A. A. J. Biol. Chem. 1996; 271: Scholar, A. A. J. Biol. Chem. 1996; 271: Scholar). The of copper to protein or by growth in a This is with the in that of a CcO or several assembly significant on the copper The known assembly in copper ion of CcO are not to to the mitochondrial copper pool because are low mitochondrion to be a low protein in levels of the CcO S. U. J. Biol. Chem. Scholar). The copper pool is associated with the mitochondrion and not a mitochondrial may contain vacuolar and L.A. 1995; Scholar). on and to and in the mitochondrial but the proteins in the mitochondrial fraction a in vacuolar and in the copper of of not The in gradient with multiple a copper in the to the that vacuolar is not the of the mitochondrial copper, from cells that small Mol. Biol. Cell. Scholar). The mitochondrial copper as the of copper to that the copper not from vacuolar that not a from cells is the P-type ATPase copper that is for of Cu(I) into The mitochondrial copper in cells to that of cells The Copper with Copper two known copper enzymes within are CcO and fraction of the cytosolic Sod1 in the mitochondrial L.A. L.T. R. Culotta V.C. J. Biol. Chem. 2001; Scholar). The CCS metallochaperone for copper into Sod1 is in the of Sod1 to the L.A. L.T. R. Culotta V.C. J. Biol. Chem. 2001; Scholar). Cells CCS levels of Sod1 within the that of CCS does not the mitochondrial copper pool other cytosolic copper-binding proteins include the Cup1 and Crs5 proteins multiple Cu(I) ions within polycopper L.T. Howard W.R. Strain J.J. Winge D.R. Culotta V.C. J. Biol. Chem. 1996; 271: 18514-18519Google Scholar). of these molecules within the mitochondrion may a significant quantity of mitochondrial Expression of CUP1 and CRS5 is regulated by the copper-responsive of results in a in CUP1 and CRS5 in yeast V.C. Howard W.R. J. Biol. Chem. Scholar). Cup1 and Crs5 not to the mitochondrial copper pool because cells levels of mitochondrial copper cells at least levels of mitochondrial The other known cuproenzymes in yeast are the plasma membrane Fet3 and vacuolar D. A. S. J. Cell. Scholar, J. Biol. Chem. 1999; Scholar). of these is to the copper pool in In addition, the mitochondrial copper pool is in cells in high medium in Fet3 and are The mitochondrial copper pool is not by copper ion Cells CUP1 from the on a high not the of mitochondrial copper not of human the copper Sod1 levels in the not the mitochondrial copper pool not of the Copper three distinct the IMS, the and the of with results in of the and of proteins localized within the and are with the of yeast in results in of the bulk of mitochondrial copper with the fraction that Sod1 by the with its within the the known matrix protein with the The of Sod1 in yet of the bulk of the copper with is with the with cells that Sod1 does not to the mitochondrial copper whether copper associated with the membrane fraction or matrix to to of the membrane fraction and fraction that the fraction CcO but a of the copper and the protein and The membrane fraction the bulk of the CcO and the protein results that the bulk of the mitochondrial copper exists within the matrix This matrix of copper is with known in the the a to ions, the matrix copper pool must across the Copper the mitochondrial pool of copper to changes in copper levels in the growth cells in medium It known that yeast in medium copper the copper CUP1 is in to the cellular copper and a significant fraction of the cellular copper is in the cytoplasm as Hamer J. Winge D.R. J. Biol. Chem. Scholar). that a fraction of the cellular The mitochondrial copper pool is in cells with The mitochondrial copper on respiration as by culture with of the copper is associated with the Cup1 and Crs5 L.T. Howard W.R. Strain J.J. Winge D.R. Culotta V.C. J. Biol. Chem. 1996; 271: 18514-18519Google Scholar). The of these molecules can be by cells with and of CUP1 and cells are to copper D.J. Mol. Cell. Biol. 1988; 8: 2745-2752Google cells with for 5 these from cells a increase in copper The bulk of the cellular copper is associated with Cup1 within the However, an increase in mitochondrial copper accumulation in cells. The accumulation in cells that the mitochondrion copper, and this may to the of the The addition of to the growth medium of cells not the mitochondrial copper pool but the mitochondrial and the addition of but not to the culture medium the mitochondrial copper The in mitochondrial copper by the addition of in cellular that and may with copper for mitochondrial concentration in of of of of in a of uptake of copper and the lack of known copper enzymes in the matrix that may of the fraction a The bulk of the copper and with of a gradient The of the copper pool significant from mitochondrial as matrix proteins are The fraction by by The copper pool in to the of a protein of This fraction however, of at not to proteins in the fraction by or for in In a fraction and to contain by for a and for the the copper pool to any protein of The of is the of a copper-binding the from to the by The of the copper fraction in the gradient by with within the fraction The of within this fraction yet the from that copper is by small molecules or a protein of does not to the of this copper as the by with and the at not In addition the does not the fraction is by or are with from cells and cells with The of the copper pool in not from cells with copper a increase in the quantity of the copper The of this pool from to that of that the pool is and are not of this pool on in by In fraction of copper at be the not a sod2Δ of copper into the mitochondrial matrix and by a small may this pool to metallate a heterologous copper-binding the yeast Sod1 be in the absence of CCS in the culture conditions, copper of Sod1 can in cells the cytosolic copper is (4Rae R.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-807Google Scholar). yeast Sod1, human Sod1 can be metallated into an in the absence of whether the matrix copper pool is accessible to heterologous human Sod1 to the mitochondrial matrix in an sod2Δ the mitochondrial matrix of yeast to human in the of into the and The sod2Δ is to of Proc. Natl. Acad. Sci. U. S. A. Scholar, Scholar). The for matrix in to the growth of sod2Δ cells cells on The sod2Δ cells are in This by but growth with the addition of that the of the sod2Δ cell to matrix and not levels of cytosolic the yeast but the mitochondrial The localized in the matrix growth of sod2Δ cells the for cytosolic Sod1 in or on of the of copper to the and The lack of suppression of sod2Δ cells by is with the that the iron superoxide dismutase in the growth defects of yeast sod2Δ cells localized to the but localized within the matrix R. D.R. 1999; Scholar). the in suppression of growth of sod2Δ the as in suppression of growth of cells not of growth defects of sod2Δ cells by matrix localized human growth of sod2Δ in with medium or copper in a with for growth or in for cells human Sod1 to the matrix and cells with and CCS as as cells with human Sod1 localized in the growth of sod2Δ with as the Cells on or in the or absence of The for copper salts in the growth medium for efficient suppression of sod2Δ cell CCS within the of CCS to a of of results in of CCS into the mitochondrial matrix S. EMBO J. Scholar). Cells matrix and CCS in to in copper salts of these cells with the addition of to the growth medium. cells and in copper to the growth medium The of the sod2Δ by that the copper in the matrix can be used for of The of matrix of is by the of CCS within the The does not a on respiration in cells. are shown to contain a pool of copper that is distinct from the two known mitochondrial cuproenzymes, Sod1 and CcO. Only a small fraction of mitochondrial copper is associated with these two The bulk of the remainder is localized within the matrix as a soluble, anionic, low molecular weight complex. The mitochondrial copper pool is dynamic, responding to changes in the cytosolic copper level. The addition of copper salts to the growth medium leads to an increase in mitochondrial This increase in mitochondrial copper is Cup1 is by of The mitochondrial copper pool is not by of CUP1 or suggesting that a for copper ion uptake into the mitochondrion of copper ions within the mitochondrion may be a of cellular copper The identity of the matrix copper ligand is of that the matrix copper fraction is not protein-bound. protein in the fraction by and proteins by mass the on not with The that the matrix pool is on the that for the matrix fraction not copper ions from a protein complex. to may copper from an This is copper are the It is that the pool is copper because cells in medium to the mitochondrial copper level. the of this pool from cells not of exist within the matrix and may be that the mitochondrial copper pool is and can as a the is that are not within the and are to the matrix copper The matrix copper pool is Co-localization of human Sod1 and CCS within the mitochondrial matrix results in suppression of growth defects of sod2Δ cells. In the absence of CCS within the matrix, the activation of requires the addition of copper salts to the growth medium. mitochondrial of proteins into the matrix as the copper of must within the The CCS for efficient activation of Sod1 may from its as a copper metallochaperone in copper ion in (4Rae R.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-807Google Scholar). CCS may be in the formation of the essential in Sod1 Y. O'Halloran T.V. Culotta V.C. J. Biol. Chem. 2003; Scholar). In the absence of CCS within the matrix, the copper necessary for may to an necessary for of the growth defects of sod2Δ cell can from in of the as are J. Biol. Chem. Scholar). The suppression of sod2Δ cell by and CCS molecules from because the on the The cytoplasm is predicted to contain free ions (4Rae R.D. Schmidt P.J. Pufahl R.A. Culotta V.C. O'Halloran T.V. Science. 1999; 284: 805-807Google Scholar). Copper ion buffering within the cytoplasm by Sod1 and the two metallothioneins, Cup1 and of copper ions by may a however, the of Cup1 must any copper The that mitochondrial copper uptake may to the cytosolic copper The for mitochondrial copper ion uptake is It is that is for copper to this mitochondrial with the results that mitochondrial copper levels are in cells or that is localized to the Winge D.R. J. Biol. Chem. Scholar). The addition of salts to the growth medium mitochondrial copper levels but not cellular copper copper ions may be into the mitochondrial matrix through a that and ions. the mitochondrial copper multiple metal ions, the mitochondrion may a buffering for metal ions. Cells activity, but mitochondrial levels are M. M. Culotta V.C. Proc. Natl. Acad. Sci. U. S. A. 2003; Scholar). cells are to contain a pool of may contain a pool for The pool of in with cytosolic D. 2002; Scholar, D. Proc. Natl. Acad. Sci. U. S. A. 2003; Scholar). iron levels increase within the of yeast in cluster formation and L.T. Culotta V.C. Mol. Cell. Biol. Scholar, U. M. R. Mol. Genet. 2002; Scholar). The increase in the mitochondrial iron pool deleterious The in iron in of cells for or results in an increase in formation A. R. J. Biol. Chem. Scholar, S. J. Proc. Natl. Acad. Sci. U. S. A. 2002; Scholar). in the iron pool in are a increase in the matrix copper pool is not associated with mitochondrial The of the matrix copper must the of this The of the matrix copper pool is Copper ions are for assembly of and sites in CcO as as of Sod1 within the of copper ion to the may exist to copper ions for CcO assembly and of Cu(I) to the may through a within the or through a copper may be to the from the The matrix copper pool may a pool of copper to an for to the are to a from the of to the for at the of with