This proposal focuses on the assembly of metal centers cytochrome c oxidase (CcO) using yeast as an experimental system. CcO deficiency is an important cause of respiratory chain disorders in infants that can present as Leigh Syndrome, cardiomyopathy, encephalocardiomyopathy or hepatic failure. The Mendelian- inherited CcO deficiency disorders represent mutations in CcO assembly factors that act either in the synthesis or insertion of copper or heme redox cofactors for CcO. These factors are conserved in eukaryotes and insights into their function were first gleaned from studies in yeast. Thus, yeast studies on the assembly of redox cofactor centers of CcO have direct relevance to human biology and CcO deficiencies disorders. We will elucidate the assembly pathway of the two heme a and two copper centers in Cox1 and Cox2. We postulate that heme insertion into Cox1 is a sequential process with the heme a site forming early in Cox1 maturation followed by heme a3 insertion involving Shy1. The human Shy1 ortholog SURF1 is the most frequently mutated gene in Leigh Syndrome patients. Our data indicate that concurrent to heme a3 insertion, Cox11-dependent copper metallation of the CuB site occurs. We will define the mechanistic roles of Cox11 in CuB site formation in Cox1. We hypothesize that cofactor insertion into Cox1 is completed prior to the addition of Cox2 containing the CuA cofactor site. We will define the role of Sco1 in CuA site formation in Cox2 and Cox17 as the copper donor to both Sco1 and Cox11. Arrest of CcO assembly at steps downstream of heme a3 insertion generates a pro- oxidant intermediate that leads to growth impairment in cells stressed with hydrogen peroxide. We used the H2O2 sensitivity assay to establish the role of Shy1 in heme a3 insertion and to characterize the function of two new CcO assembly factors Coa1 and Coa2. The presence of the pro-oxidant heme a3:Cox1 stalled intermediate in patients with CcO deficiencies may add oxidative stress to the observed pathology. The Cu- transfer steps occur in the intermembrane space of mitochondria that is susceptible to oxidative stress. Thus, CcO assembly will be likely sensitive to environmental agents that induce oxidative stress. The unifying theme in this grant is that defining the CcO assembly pathway in yeast will open up strategies to dissect the pathway in human cells and perhaps identify human specific assembly factors.

Public Health Relevance

Assembly of the metal cofactor centers in cytochrome c oxidase is a conserved process in yeast and human cells. Mutations in cytochrome oxidase assembly factors that act in the synthesis or insertion of copper and heme redox cofactors leads to cytochrome oxidase deficiencies that can present as Leigh Syndrome, cardiomyopathy, encephalocardiomyopathy or hepatic failure. The most frequently mutated gene in Leigh Syndrome patients is SURF1 that encodes a protein that acts in heme a insertion in cytochrome oxidase.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Reinlib, Leslie J
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University of Utah
Internal Medicine/Medicine
Schools of Medicine
Salt Lake City
United States
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Cui, Tie-Zhong; Conte, Annalea; Fox, Jennifer L et al. (2014) Modulation of the respiratory supercomplexes in yeast: enhanced formation of cytochrome oxidase increases the stability and abundance of respiratory supercomplexes. J Biol Chem 289:6133-41
Na, Un; Yu, Wendou; Cox, James et al. (2014) The LYR factors SDHAF1 and SDHAF3 mediate maturation of the iron-sulfur subunit of succinate dehydrogenase. Cell Metab 20:253-66
Bohovych, Iryna; Donaldson, Garrett; Christianson, Sara et al. (2014) Stress-triggered activation of the metalloprotease Oma1 involves its C-terminal region and is important for mitochondrial stress protection in yeast. J Biol Chem 289:13259-72
Patil, Vinay A; Fox, Jennifer L; Gohil, Vishal M et al. (2013) Loss of cardiolipin leads to perturbation of mitochondrial and cellular iron homeostasis. J Biol Chem 288:1696-705
Kim, Hyung J; Winge, Dennis R (2013) Emerging concepts in the flavinylation of succinate dehydrogenase. Biochim Biophys Acta 1827:627-36
Kim, Hyung J; Khalimonchuk, Oleh; Smith, Pamela M et al. (2012) Structure, function, and assembly of heme centers in mitochondrial respiratory complexes. Biochim Biophys Acta 1823:1604-16
Khalimonchuk, Oleh; Kim, Hyung; Watts, Talina et al. (2012) Oligomerization of heme o synthase in cytochrome oxidase biogenesis is mediated by cytochrome oxidase assembly factor Coa2. J Biol Chem 287:26715-26
Khalimonchuk, Oleh; Jeong, Mi-Young; Watts, Talina et al. (2012) Selective Oma1 protease-mediated proteolysis of Cox1 subunit of cytochrome oxidase in assembly mutants. J Biol Chem 287:7289-300
Eletsky, Alexander; Jeong, Mi-Young; Kim, Hyung et al. (2012) Solution NMR structure of yeast succinate dehydrogenase flavinylation factor Sdh5 reveals a putative Sdh1 binding site. Biochemistry 51:8475-7
Kim, Hyung J; Jeong, Mi-Young; Na, Un et al. (2012) Flavinylation and assembly of succinate dehydrogenase are dependent on the C-terminal tail of the flavoprotein subunit. J Biol Chem 287:40670-9

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