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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES003817-32
Application #
8416902
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Reinlib, Leslie J
Project Start
1985-05-01
Project End
2015-01-31
Budget Start
2013-02-01
Budget End
2015-01-31
Support Year
32
Fiscal Year
2013
Total Cost
$435,324
Indirect Cost
$146,072
Name
University of Utah
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
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Bestwick, Megan; Jeong, Mi-Young; Khalimonchuk, Oleh et al. (2010) Analysis of Leigh syndrome mutations in the yeast SURF1 homolog reveals a new member of the cytochrome oxidase assembly factor family. Mol Cell Biol 30:4480-91
Robinson, Nigel J; Winge, Dennis R (2010) Copper metallochaperones. Annu Rev Biochem 79:537-62
Bestwick, Megan; Khalimonchuk, Oleh; Pierrel, Fabien et al. (2010) The role of Coa2 in hemylation of yeast Cox1 revealed by its genetic interaction with Cox10. Mol Cell Biol 30:172-85

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