The proposed research for the next grant period is focused on prokaryotic respiratory oxygen reductases. Oxygenic respiration is responsible for providing virtually all the energy required by aerobic organisms. There are two families of enzymes which catalyze the reduction of O2 to water and conserve the free energy liberated by this reaction by generating a proton motive force- (1) The heme-copper oxygen reductases;(2) the bd-type oxygen reductases. The heme-copper oxygen reductases are themselves subdivided into 3 major subfamilies that are highly divergent- the A-, B- and C-families. In the last grant period we definitively showed that members of each of the 3 families of heme-copper oxygen reductases pump protons. We will pursue two major thrusts in the next grant period. Of the greatest medical relevance is the fact than many human pathogens contain either a C-family oxygen reductase (cytochrome cbb3) and/or cytochrome bd. The reason is that these enzymes are adapted to function best under conditions of low [O2], such as inside macrophages or in granulomas or in the intestine. Under conditions in which O2 is essential for the bioenergetic needs of the pathogen, inhibiting cytochrome cbb3 or cytochrome bd will be lethal. We have designed a high-throughput drug screening protocol that will allow us to identify inhibitors of aerobic respiration in live bacteria. We will focus on 1) Mycobacterium tuberculosis, which requires cytochrome bd in the persistent (dormant) state;2) Haemophilus influenzae, for which cytochrome bd has been recently shown to be essential for growth;and 3) Neisseria gonorrhoeae, which relies on cytochrome cbb3 for aerobic growth. Respiratory oxygen reductases are a new class of drug targets and we plan to demonstrate their importance in the next grant period. At the same time, we will continue our work on structure/function relationships in the heme-copper oxygen reductases. An important open question is what group or groups are directly involved in proton pumping. We have proposed that the propionate-A group of the active-site heme is the most likely """"""""proton loading site"""""""" of the pump. This will be examined by time-resolved FTIR to directly observe protonation of the proton loading site. We also plan to address the question about how O2 diffuses through the protein to the active site, and how the pathways for this differ in the A-, B- and C-families of oxygen reductases.
All aerobic organisms derive a substantial portion of their energy from the reduction of oxygen to water, accomplished by membrane-bound enzymes called oxygen reductases. Several different kinds of enzymes can do this same reaction and we are interested in the mechanism of how the chemical energy from the reaction is harnessed in a usable form. Many human pathogens, including the bacterium causing tuberculosis, have unusual enzymes that are used for this purpose, and we propose to find drugs that will inhibit these enzymes from functioning and, therefore, kill the pathogenic organism with no harm to the person.
|Lencina, Andrea M; Franza, Thierry; Sullivan, Matthew J et al. (2018) Type 2 NADH Dehydrogenase Is the Only Point of Entry for Electrons into the Streptococcus agalactiae Respiratory Chain and Is a Potential Drug Target. MBio 9:|
|Sun, Chang; Benlekbir, Samir; Venkatakrishnan, Padmaja et al. (2018) Structure of the alternative complex III in a supercomplex with cytochrome oxidase. Nature 557:123-126|
|Mahinthichaichan, Paween; Gennis, Robert B; Tajkhorshid, Emad (2018) Cytochrome aa3 Oxygen Reductase Utilizes the Tunnel Observed in the Crystal Structures To Deliver O2 for Catalysis. Biochemistry 57:2150-2161|
|Ahn, Young O; Albertsson, Ingrid; Gennis, Robert B et al. (2018) Mechanism of proton transfer through the KC proton pathway in the Vibrio cholerae cbb3 terminal oxidase. Biochim Biophys Acta Bioenerg 1859:1191-1198|
|Mahinthichaichan, Paween; Gennis, Robert B; Tajkhorshid, Emad (2018) Bacterial denitrifying nitric oxide reductases and aerobic respiratory terminal oxidases use similar delivery pathways for their molecular substrates. Biochim Biophys Acta Bioenerg 1859:712-724|
|Murali, Ranjani; Gennis, Robert B (2018) Functional importance of Glutamate-445 and Glutamate-99 in proton-coupled electron transfer during oxygen reduction by cytochrome bd from Escherichia coli. Biochim Biophys Acta Bioenerg 1859:577-590|
|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|
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