Funds are being requested to study the molecular mechanism of respiratory oxidases. Virtually all aerobic organisms on the planet utilize a respiratory oxidase from either the heme-copper superfamily or from the bd-family of enzymes. We are studying both groups of enzymes as part of the proposed research. All of the oxidases reduce dioxygen to water, and use the free energy to generate a voltage across the membrane. Most of the charge separation that is driven by these enzymes comes from protons moving across the membrane within the oxidase protein, either to the enzyme active site, to be consumed to form water, or to the bulk solution on the opposite side of the membrane from which protons are taken up, i.e., proton pumping. We are primarily interested in the pathways through which protons move within the proteins, and in the driving forces that result in proton translocation. Which groups are protonated and when? The catalytic cycle consists of a sequence of proton-coupled electron transfer reactions, and we are interested in how the electron transfer is coupled to moving protons, including the proton pump. We use rapid kinetics techniques to examine individual steps in the reaction, monitoring charge movements across the membrane by time- resolved voltage measurements, and correlating this to the electron transfer events, monitored by UV/vis spectroscopy. FTIR spectroscopy is also utilized to monitor changes in protein structure and the protonation of individual residues. Several X-ray structures serve as guides for site-directed mutagenesis, and we can characterize each mutant by measuring the uptake and release of protons from the enzymes, as well as intra-protein proton translocation. In the next grant period, the emphasis will be on trying to characterize intermediates in the reaction chemistry, and to expand our studies to include """"""""non-canonical"""""""" oxidases, which lack what we have assumed to be essential residues, although these """"""""non-canonical oxidases function perfectly well. The human mitochondrial aa3-type cytochrome c oxidase is a member of the heme-copper superfamily. One aspect of medical interest concerns the many mitochondrial genetic defects which influence the function of the mitochondrial oxidase, leading to insufficient ability to make ATP and a variety of symptoms depending on the nature of the lesion. A second point of medical relevance concerns the possible vulnerability of pathogenic bacteria to agents that compromise their aerobic respiration. The need for pathogens to survive under conditions of low oxygen, as in intracellular regions, often requires specialized oxidases, such as the bd-type of cbb3-type enzymes.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
Project #
Application #
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Hasan, Ahmed AK
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Illinois Urbana-Champaign
Schools of Arts and Sciences
United States
Zip Code
Padayatti, Pius S; Leung, Josephine H; Mahinthichaichan, Paween et al. (2017) Critical Role of Water Molecules in Proton Translocation by the Membrane-Bound Transhydrogenase. Structure 25:1111-1119.e3
Hammer, Neal D; Schurig-Briccio, Lici A; Gerdes, Svetlana Y et al. (2016) CtaM Is Required for Menaquinol Oxidase aa3 Function in Staphylococcus aureus. MBio 7:
Mahinthichaichan, Paween; Gennis, Robert B; Tajkhorshid, Emad (2016) All the O2 Consumed by Thermus thermophilus Cytochrome ba3 Is Delivered to the Active Site through a Long, Open Hydrophobic Tunnel with Entrances within the Lipid Bilayer. Biochemistry 55:1265-78
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
von Ballmoos, Christoph; Gonska, Nathalie; Lachmann, Peter et al. (2015) Mutation of a single residue in the ba3 oxidase specifically impairs protonation of the pump site. Proc Natl Acad Sci U S A 112:3397-402
Lin, Myat T; Fukazawa, Risako; Miyajima-Nakano, Yoshiharu et al. (2015) Escherichia coli auxotroph host strains for amino acid-selective isotope labeling of recombinant proteins. Methods Enzymol 565:45-66
Schurig-Briccio, Lici A; Yano, Takahiro; Rubin, Harvey et al. (2014) Characterization of the type 2 NADH:menaquinone oxidoreductases from Staphylococcus aureus and the bactericidal action of phenothiazines. Biochim Biophys Acta 1837:954-63
Y?ld?z, Gülgez Gökçe; Gennis, Robert B; Daldal, Fevzi et al. (2014) The K(C) channel in the cbb3-type respiratory oxygen reductase from Rhodobacter capsulatus is required for both chemical and pumped protons. J Bacteriol 196:1825-32
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
Hoeser, Jo; Hong, Sangjin; Gehmann, Gerfried et al. (2014) Subunit CydX of Escherichia coli cytochrome bd ubiquinol oxidase is essential for assembly and stability of the di-heme active site. FEBS Lett 588:1537-41

Showing the most recent 10 out of 168 publications