Mechanisms in Respiration James A. Fee GM35342 Cytochrome c oxidase is an enzyme that provides energy for the cell. It does so by coupling dioxygen reduction to proton pumping leading to oxidative phosphorylation. The cytochrome ba3 from Thermus thermophilus is a distant homolog of the mammalian enzymes that contains a storehouse of natural mutations that, while retaining an almost identical three-dimesional structure, confer on the enzyme novel chemical behaviours that provide unique insight on function. During past funding periods we developed a homologous expression system, learned how to make mutant forms, demonstrated the presence of a single pathway for access of protons into the active site, developed efficient methods to obtain X-ray diffracting crystals, determined the structures of several novel forms of the enzyme, introduced two very powerful forms of spectroscopy to examine the enzyme in single crystals, and published, as hypothesis, a detailed chemical mechanism for coupling proton pumping with dioxygen reduction. Here we propose four Specific Aims: [1] to explore the mechanism of the enzyme using rapid measurement of O- isotope shifted resonance Raman spectra. [2] Combine structure-directed, site-directed mutation to regulate the several structural pathways whereby substrates (electrons, dioxygen, water and protons) enter and leave the active site. Our hope to trap new intermediates for detailed chemical study.
In Aims [3&4] we focus on obtaining the structures of oxygen-intermediates that occur in the overall mechanism. The significance of this work to human health lies in providing fundamental information about the conservation of energy in biological systems.

Public Health Relevance

Mechanisms in Respiration James A. Fee GM35342 Cytochrome c oxidase is an enzyme that helps power the eukaryotic cell. It combines electrons from the food we eat, oxygen from the air we breath, and protons from the fluid of which we are composed and creates an extreme proton gradient across the mitochondrial membrane. The most common use of this gradient is its capture and dissipation by the ATP synthase that provides the ATP to drive our metabolism. The chemistry whereby cytochrome c oxidase generates the proton gradient remains one of the deepest and most mysterious of biological chemistry. The purpose of our research is to understand this mechanism in respiration at the molecular level.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035342-29
Application #
8282829
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1984-12-01
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
29
Fiscal Year
2012
Total Cost
$428,078
Indirect Cost
$202,655
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
McDonald, William; Funatogawa, Chie; Li, Yang et al. (2014) Conserved glycine 232 in the ligand channel of ba3 cytochrome oxidase from Thermus thermophilus. Biochemistry 53:4467-75
McDonald, William; Funatogawa, Chie; Li, Yang et al. (2013) Ligand access to the active site in Thermus thermophilus ba(3) and bovine heart aa(3) cytochrome oxidases. Biochemistry 52:640-52
Liu, Bin; Zhang, Yang; Sage, J Timothy et al. (2012) Structural changes that occur upon photolysis of the Fe(II)(a3)-CO complex in the cytochrome ba(3)-oxidase of Thermus thermophilus: a combined X-ray crystallographic and infrared spectral study demonstrates CO binding to Cu(B). Biochim Biophys Acta 1817:658-65
Chang, Hsin-Yang; Choi, Sylvia K; Vakkasoglu, Ahmet Selim et al. (2012) Exploring the proton pump and exit pathway for pumped protons in cytochrome ba3 from Thermus thermophilus. Proc Natl Acad Sci U S A 109:5259-64
Neehaul, Yashvin; Chen, Ying; Werner, Carolin et al. (2012) Electrochemical and infrared spectroscopic analysis of the interaction of the Cu(A) domain and cytochrome c(552) from Thermus thermophilus. Biochim Biophys Acta 1817:1950-4
Egawa, Tsuyoshi; Chen, Ying; Fee, James A et al. (2012) The rate-limiting step in O(2) reduction by cytochrome ba(3) from Thermus thermophilus. Biochim Biophys Acta 1817:666-71
Tiefenbrunn, Theresa; Liu, Wei; Chen, Ying et al. (2011) High resolution structure of the ba3 cytochrome c oxidase from Thermus thermophilus in a lipidic environment. PLoS One 6:e22348
Smirnova, Irina; Reimann, Joachim; von Ballmoos, Christoph et al. (2010) Functional role of Thr-312 and Thr-315 in the proton-transfer pathway in ba3 Cytochrome c oxidase from Thermus thermophilus. Biochemistry 49:7033-9
Szundi, Istvan; Funatogawa, Chie; Fee, James A et al. (2010) CO impedes superfast O2 binding in ba3 cytochrome oxidase from Thermus thermophilus. Proc Natl Acad Sci U S A 107:21010-5
Hayashi, Takahiro; Lin, Myat T; Ganesan, Krithika et al. (2009) Accommodation of two diatomic molecules in cytochrome bo: insights into NO reductase activity in terminal oxidases. Biochemistry 48:883-90

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