Bacteria frequently encounter microoxic environments, particularly in dense communities associated with biofilms, sites of infection, and in symbiotic life. Microbes have evolved to respire O2 even when its concentrations are low but the mechanisms that underlie microoxic respiration are not well understood. In many bacteria, including Pseudomonas aeruginosa (Pa), O2 reduction to water is catalyzed by cbb3 terminal oxidases that have several features distinct from those of mammalian oxidases. Success of four-electron O2 reduction depends on the coordinated play of multiple redox centers and, under microoxic conditions, also on regulatory mechanisms to deal with the limited supply of O2. We have identified two metalloproteins critical for the microoxic function of cbb3 oxidases in Pa: the electron carrier cytochrome c4 (cyt c4) and the O2 carrier hemerythrin (Hr); both of them are required for robust Pa growth at low O2. Cbb3 oxidases in Pa and many other proteobacteria have an extended chain of six metal redox centers and their electron carrier has two. We propose that diheme architecture of cyt c4 and the extended electron-transfer (ET) chain in cbb3 oxidases, together with unique conformational dynamics, are used to modulate ET through these respiratory proteins providing opportunities for regulation of the redox flow and enabling efficient energy conversion during catalysis. Furthermore, we hypothesize that Hr aids in directing O2 for use in respiration by membrane-bound Pa cbb3 oxidases. In this project, the spectroscopic and electrochemistry studies of protein components and their interactions in vitro are combined with genetics and phenotypic analyses in vivo to (1) characterize the role of diheme moieties in regulating ET properties of cyt c4 and of the CcoP subunit of cbb3 oxidase and establish the mechanism of the electron injection to the enzyme; (2) elucidate the mechanism of electron flow within the extended ET chain in cbb3 oxidase and coordination of conformational changes with ET steps; and (3) establish the mechanism of effects of Hr on the function of cbb3 oxidase in Pa. The proposed studies of cbb3 oxidases will provide a foundation for understanding the molecular mechanisms that enable bacteria to thrive in low-O2 environments and identifying strategies that may disrupt bacterial growth in these settings. With our focus on redox cooperativity, conformational gating, proton-coupled processes, and regulatory protein networks, our studies will address important topics in biological redox mechanisms relevant to function of many other metalloproteins that play a role in human health.

Public Health Relevance

The project focuses on the characterization of proteins that function under conditions of low oxygen, which are common in dense bacterial communities, such as biofilms, and during host infections. The developed insights will shed light on the molecular processes that enable bacteria to thrive under these conditions and provide a necessary mechanistic foundation for identifying strategies to interrupt these pathways in the design of new therapies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Anderson, Vernon
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Dartmouth College
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United States
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Deng, Yunling; Zhong, Fangfang; Alden, Stephanie L et al. (2018) The K79G Mutation Reshapes the Heme Crevice and Alters Redox Properties of Cytochrome c. Biochemistry 57:5827-5840
Zhong, Fangfang; Pletneva, Ekaterina V (2018) Ligation and Reactivity of Methionine-Oxidized Cytochrome c. Inorg Chem 57:5754-5766
Amacher, Jeanine F; Zhong, Fangfang; Lisi, George P et al. (2018) Correction to ""A Compact Structure of Cytochrome c Trapped in a Lysine-Ligated State: Loop Refolding and Functional Implications of a Conformational Switch"". J Am Chem Soc 140:4466
Gu, Jie; Shin, Dong-Woo; Pletneva, Ekaterina V (2017) Remote Perturbations in Tertiary Contacts Trigger Ligation of Lysine to the Heme Iron in Cytochrome c. Biochemistry 56:2950-2966
Amacher, Jeanine F; Zhong, Fangfang; Lisi, George P et al. (2015) A Compact Structure of Cytochrome c Trapped in a Lysine-Ligated State: Loop Refolding and Functional Implications of a Conformational Switch. J Am Chem Soc 137:8435-49
Leung, Chris W T; Hong, Yuning; Hanske, Jonas et al. (2014) Superior fluorescent probe for detection of cardiolipin. Anal Chem 86:1263-8
Muenzner, Julia; Pletneva, Ekaterina V (2014) Structural transformations of cytochrome c upon interaction with cardiolipin. Chem Phys Lipids 179:57-63
Snider, Erik J; Muenzner, Julia; Toffey, Jason R et al. (2013) Multifaceted effects of ATP on cardiolipin-bound cytochrome c. Biochemistry 52:993-5
Muenzner, Julia; Toffey, Jason R; Hong, Yuning et al. (2013) Becoming a peroxidase: cardiolipin-induced unfolding of cytochrome c. J Phys Chem B 117:12878-86
Vincelli, Amber J; Pottinger, Danielle S; Zhong, Fangfang et al. (2013) Recombinant expression, biophysical characterization, and cardiolipin-induced changes of two Caenorhabditis elegans cytochrome c proteins. Biochemistry 52:653-66

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