Characterization of the Cytochrome c Oxidase Assembly Factor Cox19 of Saccharomyces cerevisiae

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Sco1 is implicated in the copper metallation of the Cu A site in Cox2 of cytochrome oxidase. The structure of Sco1 in the metallated and apo-conformers revealed structural dynamics primarily in an exposed region designated loop 8. The structural dynamics of loop 8 in Sco1 suggests it may be an interface for interactions with Cox17, the Cu(I) donor and/or Cox2. A series of conserved residues in the sequence motif 217 KKYRVYF 223 on the leading edge of this loop are shown presently to be important for yeast Sco1 function. Cells harboring Y219D, R220D, V221D, and Y222D mutant Sco1 proteins failed to restore respiratory growth or cytochrome oxidase activity in sco1⌬ cells. The mutant proteins are stably expressed and are competent to bind Cu(I) and Cu(II) normally. Specific Cu(I) transfer from Cox17 to the mutant apo-Sco1 proteins proceeds normally. In contrast, using two in vivo assays that permit monitoring of the transient Sco1-Cox2 interaction, the mutant Sco1 molecules appear compromised in a function with Cox2. The mutants failed to suppress the respiratory defect of cox17-1 cells unlike wild-type SCO1. In addition, the mutants failed to suppress the hydrogen peroxide sensitivity of sco1⌬ cells. These studies implicate different surfaces on Sco1 for interaction or function with Cox17 and Cox2.Cytochrome c oxidase (CcO) 2 is the terminal enzyme of the energy-transducing, electron transfer chain within the mitochondrial inner membrane (IM). The enzyme contains two copper centers important for its function (1). One center is the binuclear copper site (Cu A ) residing in the Cox2 subunit. The second center is the Cu B site in the Cox1 subunit that forms a heterobimetallic site with heme a 3 . The formation of the copper sites in the two mitochondrially encoded subunits occurs within the mitochondrial intermembrane space (IMS) by a series of accessory proteins Cox11, Cox17, and Sco1 (2). Cox17 is a soluble Cu(I)-binding protein largely localized within the IMS. In yeast, CuCox17 appears to specifically transfer Cu(I) to the Cu A site via the IM-tethered Sco1 and to the Cu B site via the IM-anchored Cox11 (3).The role of Cox17 in formation of the Cu A site was initially implicated from the isolation of SCO1 as a high copy suppressor of cox17-1 respiratory deficient cells (4). Yeast lacking Sco1 are devoid of CcO activity and show greatly attenuated Cox2 protein levels (5, 6). Cu(I) bound to Cox17 can be transferred to Sco1 through an apparent transient protein-mediated complex. The C57Y mutation in the cox17-1 strain precludes Cu(I) transfer to Sco1, but not Cox11, consistent with each transfer occurring through a specific protein complex (3). Although attempts to isolate the Cox17⅐Sco1 complex have failed, a mass spectrometry study with human Sco1 and Cox17 revealed a low abundance ion consistent with a complex (7). Copper metallation of Sco1 and Cox11 is an intermediate step in the transfer of Cu(I) to Cox2 and Cox1, respectively. One preliminary study reported an interaction of Sco1 with Cox2 (8), but no direct...

Section: Methodsmentioning

confidence: 99%

Mapping the Functional Interaction of Sco1 and Cox2 in Cytochrome Oxidase Biogenesis

Rigby

Cobine

Khalimonchuk

et al. 2008

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“…Cox19, is a member of the CX 9 C family and an important accessory protein in the assembly of cytochrome c oxidase (CcO) in yeast. Cells lacking Cox19 are respiratory deficient and have reduced CcO activity (10). Cox19 was recently proposed to be involved also in copper efflux to mitochondria (11).…”

mentioning

confidence: 99%

“…Cox19 was recently proposed to be involved also in copper efflux to mitochondria (11). So far, little structural information exists for this protein, except that it contains two disulfides bonds between its external (Cys30-Cys62) and internal (Cys40-Cys52) Cys in the native state (10,12), a topological linkage thought to be conserved in the CX 9 C family (13).…”

mentioning

confidence: 99%

The Mitochondrial Intermembrane Space Oxireductase Mia40 Funnels the Oxidative Folding Pathway of the Cytochrome c Oxidase Assembly Protein Cox19

Fraga

Bech-Serra

Canals

et al. 2014

Background: Mia40 accelerates Cox19 folding through the specific recognition of the third Cys in the second CX 9 C motif. Results: The chaperone catalysis renders a native-like intermediate that oxidizes in a slow uncatalyzed reaction into native Cox19. Conclusion:The role of Mia40 is funnelling an already sequentially encoded, but rough, substrate folding landscape. Significance: These results provide a rationale for the substrate promiscuity of the chaperone Mia40.

“…Cox17 contains a twin Cx 9 C structural motif that is also present in a growing number of IMS proteins required for COX assembly. These proteins include the soluble Cox19 (17) and Cox23 (42) and the peripherally bound to the inner membrane Pet191 (18) and Cmc1 (19). A recent system-atic analysis of the twin Cx 9 C protein family identified a total of 14 mitochondrial proteins, which absence produce heterogeneous effects not only in cytochrome c oxidase activity but also in cytochrome c reductase activity (20).…”

mentioning

confidence: 99%

“…The respiratory deficiency of a cox23 null mutant is rescued by copper supplementation, albeit only with concomitant overexpression of COX17 (21). Recombinant Cox19 binds copper (17), but the COX assembly defect of a cox19 null mutant strain is not rescued by copper supplementation (22). Cox19 was proposed to participate in a different part of the Cox23-Cox17 pathway (21).…”

mentioning

confidence: 99%

The Conserved Mitochondrial Twin Cx9C Protein Cmc2 Is a Cmc1 Homologue Essential for Cytochrome c Oxidase Biogenesis

Horn

Zhou

Trevisson

et al. 2010

Mitochondrial copper metabolism and delivery to cytochrome c oxidase and mitochondrially localized CuZn-superoxide dismutase (Sod1) requires a growing number of intermembrane space proteins containing a twin Cx 9 C motif. Among them, Cmc1 was recently identified by our group. Here we describe another conserved mitochondrial metallochaperonelike protein, Cmc2, a close homologue of Cmc1, whose function affects both cytochrome c oxidase and Sod1. In the yeast Saccharomyces cerevisiae, Cmc2 localizes to the mitochondrial inner membrane facing the intermembrane space. In the absence of Cmc2, cytochrome c oxidase activity measured spectrophotometrically and cellular respiration measured polarographically are undetectable. Additionally, mutant cmc2 cells display 2-fold increased mitochondrial Sod1 activity, whereas CMC2 overexpression results in Sod1 activity decreased to 60% of wild-type levels. CMC1 overexpression does not rescue the respiratory defect of cmc2 mutants or vice versa. However, Cmc2 physically interacts with Cmc1 and the absence of Cmc2 induces a 5-fold increase in Cmc1 accumulation in the mitochondrial membranes. Cmc2 function is conserved from yeast to humans. Human CMC2 localizes to the mitochondria and CMC2 expression knockdown produces cytochrome c oxidase deficiency in Caenorhabditis elegans. We conclude that Cmc1 and Cmc2 have cooperative but nonoverlapping functions in cytochrome c oxidase biogenesis.