Mitochondrial oxidative phosphorylation is capable of supplying more than 95% of the total ATP requirement in respiring eucaryotic cells. It is driven by a respiratory chain composed of a number of multimeric membrane proteins that act in series to affect the transfer of electrons from reduced substrates to oxygen. Previous studies have emphasized the importance of the respiratory chain itself in regulating oxidative phosphorylation and have identified cytochrome c oxidase, its terminal member, as a key enzyme in the overall regulation of cellular energy production. At present, it is unclear how eucaryotic cells alter their cytochrome c oxidase activity levels in response to energy demand. However, the recent discovery of isoforms to the nuclearcoded subunits of cytochrome c oxidase in many eucaryotes, including humans, has led to the hypothesis that these polypeptides play a role in the modulation of cytochrome c oxidase activity. In this grant we will address this hypothesis. Initially, we will use the two subunit V isoforms, Va and Vb, of yeast cytochrome c oxidase as a model. Previous studies have shown that these isoforms affect some catalytic properties of holocytochrome c oxidase in vivo and that the expression of their genes, COX5a and COX5b, is differentially regulated by oxygen. Here, we propose to: 1) examine the structural-functional basis for the differential effects of Va and Vb on the electron transport activities of the holoenzyme; 2) determine if Va and Vb alter the proton pumping activity of the holoenzyme; 3) identify the domain(s) in Va and Vb that modulate holoenzyme activities; 4) determine if COX5a and COX5b are oxygen sensors that regulate the number of holocytochrome c oxidase molecules that are assembled in vivo; and 5) develop and use a heterologous complementation system to determine if human cytochrome c oxidase has subunit isoforms that function like yeast Va and Vb. These studies should enhance our understanding of cellular energetics and cytochrome c oxidase structure-function, and may provide an assay as well as a molecular basis for understanding the growing number of human diseases (i.e., tissue specific myopathies, cardiopathies, and hepatopathies) that are being linked to defects in cytochrome c oxidase. In addition, they should provide new opportunities to examine, and possibly modify, the mechanism of cytochrome c oxidase catalysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM039324-04
Application #
2179747
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1991-04-01
Project End
1995-11-30
Budget Start
1994-04-01
Budget End
1995-11-30
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Colorado at Boulder
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Boulder
State
CO
Country
United States
Zip Code
80309
Kwast, K E; Burke, P V; Poyton, R O (1998) Oxygen sensing and the transcriptional regulation of oxygen-responsive genes in yeast. J Exp Biol 201:1177-95
Burke, P V; Raitt, D C; Allen, L A et al. (1997) Effects of oxygen concentration on the expression of cytochrome c and cytochrome c oxidase genes in yeast. J Biol Chem 272:14705-12
Kwast, K E; Burke, P V; Brown, K et al. (1997) REO1 and ROX1 are alleles of the same gene which encodes a transcriptional repressor of hypoxic genes in Saccharomyces cerevisiae. Curr Genet 32:377-83
Poyton, R O; Bellus, G; McKee, E E et al. (1996) In organello mitochondrial protein and RNA synthesis systems from Saccharomyces cerevisiae. Methods Enzymol 264:36-42
Poyton, R O; McEwen, J E (1996) Crosstalk between nuclear and mitochondrial genomes. Annu Rev Biochem 65:563-607
Bunn, H F; Poyton, R O (1996) Oxygen sensing and molecular adaptation to hypoxia. Physiol Rev 76:839-85
Zhao, X J; Raitt, D; V Burke, P et al. (1996) Function and expression of flavohemoglobin in Saccharomyces cerevisiae. Evidence for a role in the oxidative stress response. J Biol Chem 271:25131-8
Poyton, R O; Goehring, B; Droste, M et al. (1995) Cytochrome-c oxidase from Saccharomyces cerevisiae. Methods Enzymol 260:97-116
Allen, L A; Zhao, X J; Caughey, W et al. (1995) Isoforms of yeast cytochrome c oxidase subunit V affect the binuclear reaction center and alter the kinetics of interaction with the isoforms of yeast cytochrome c. J Biol Chem 270:110-8
Zhao, X J; Caughey, W S; Poyton, R O (1995) Fourier transform infrared analysis of carbonyl and nitrosyl complexes of cytochrome-c oxidase from Saccharomyces cerevisiae. Methods Enzymol 260:399-406

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