Cytochrome c Oxidase (CcO), the terminal enzyme in the electron transfer chain of eukaryotes and prokaryotes, is responsible for over 90% of the oxygen utilization in the biosphere. The enzyme serves a dual role of (i) maintaining electron flow for oxidative phosphorylation, by catalyzing the four- electron reduction of O2 to H2O and (ii) creating a proton gradient for ATP production, by coupling the oxygen reduction chemistry to proton translocation. Although the oxygen reduction chemistry is relatively well understood, the mechanism by which the energy of the redox-linked oxygen reduction reaction is harnessed for proton translocation is unresolved. It remains as one of the major unsolved issues in bioenergetics. This knowledge gap is in part a result of the difficulty in detecting protons in the vast protein matrix of the enzyme. It is our hypothesis that the vibrational modes of the heme peripheral groups can serve as reporters of proton occupancy and movement in the enzyme. Based on this hypothesis, as supported by our preliminary data, a new methodology, hydrogen/deuterium exchange resonance Raman spectroscopy, will be developed and used to investigate the critical driving elements for proton translocation in CcO. The objective of this project is to improve our understanding of how the electron transfer and oxygen reduction chemistry regulates proton translocation. To achieve this objective three Specific Aims are proposed: (i) Define the resonance Raman markers of the peripheral heme groups that are sensitive to solvent H/D exchange;(ii) Determine how the solvent H/D sensitive resonance Raman modes are modulated by the redox processes;and (iii) Identify the roles of critical residues involved in coupling oxygen chemistry to proton translocation. To accomplish these Aims, the new technology will be combined with fast kinetic techniques and mutagenesis methods to investigate a mammalian enzyme, as well as its bacterial analogs with differing heme types. The experimental results will be complemented by computational modeling to advance our understanding of the proton pumping mechanism in CcO at the molecular level, as well as to shed new light on the evolutionary conservation of the structure and function of the oxidase superfamily of enzymes. The information derived from this multifaceted approach, which is unattainable by other techniques, will provide a foundation for the rational design of therapeutics targeting CcO related diseases.

Public Health Relevance

The proposed line of research will provide the mechanistic details underlying the coupling between the oxygen reduction chemistry and proton translocation in cytochrome c oxidase, one of the most important enzymes in physiology. It is relevant to the part of the NIH's mission that pertains to developing fundamental knowledge that will ultimately help reduce the burden of human disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM098799-03
Application #
8542873
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2011-09-20
Project End
2015-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
3
Fiscal Year
2013
Total Cost
$419,003
Indirect Cost
$168,103
Name
Albert Einstein College of Medicine
Department
Physiology
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
Ishigami, Izumi; Zatsepin, Nadia A; Hikita, Masahide et al. (2017) Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature. Proc Natl Acad Sci U S A 114:8011-8016
Nelson, Garrett; Kirian, Richard A; Weierstall, Uwe et al. (2016) Three-dimensional-printed gas dynamic virtual nozzles for x-ray laser sample delivery. Opt Express 24:11515-30
Ahn, Young O; Lee, Hyun Ju; Kaluka, Daniel et al. (2015) The two transmembrane helices of CcoP are sufficient for assembly of the cbb3-type heme-copper oxygen reductase from Vibrio cholerae. Biochim Biophys Acta 1847:1231-9
Ishigami, Izumi; Hikita, Masahide; Egawa, Tsuyoshi et al. (2015) Proton translocation in cytochrome c oxidase: insights from proton exchange kinetics and vibrational spectroscopy. Biochim Biophys Acta 1847:98-108
Egawa, Tsuyoshi; Haber, Jonah; Fee, James A et al. (2015) Interactions of Cu(B) with Carbon Monoxide in Cytochrome c Oxidase: Origin of the Anomalous Correlation between the Fe-CO and C-O Stretching Frequencies. J Phys Chem B 119:8509-20
Hayden, Eric Y; Kaur, Prerna; Williams, Thomas L et al. (2015) Heme Stabilization of ?-Synuclein Oligomers during Amyloid Fibril Formation. Biochemistry 54:4599-610
Ahn, Young O; Mahinthichaichan, Paween; Lee, Hyun Ju et al. (2014) Conformational coupling between the active site and residues within the K(C)-channel of the Vibrio cholerae cbb3-type (C-family) oxygen reductase. Proc Natl Acad Sci U S A 111:E4419-28
Sabat, Joseph; Egawa, Tsuyoshi; Lu, Changyuan et al. (2013) Catalytic intermediates of inducible nitric-oxide synthase stabilized by the W188H mutation. J Biol Chem 288:6095-106
Egawa, Tsuyoshi; Yeh, Syun-Ru; Rousseau, Denis L (2013) Redox-controlled proton gating in bovine cytochrome c oxidase. PLoS One 8:e63669
Li, Wenbing; Chen, Li; Lu, Changyuan et al. (2013) Regulatory role of Glu546 in flavin mononucleotide-heme electron transfer in human inducible nitric oxide synthase. Inorg Chem 52:4795-801

Showing the most recent 10 out of 12 publications