Hemme-copper oxidases are the terminal oxidases of the electron transport chains of mammals and a wide variety of eukaryotic and prokaryotic organisms. They play a pivotal role in cellular bioenergetics and human health by converting the redox energy of oxygen reduction into a protonmotive force, which is ultimately used to drive the endogonic synthesis of ATP. As the structure and catalytic mechanism have become better understood, the pathways leading to the assembly and metallation of the critical redox centers are emerging as the next frontier. This proposal aims to extend our investigations on heme-copper oxidases into this area and will focus on the proteins involved in the assembly and metallation of the CuA and CuB centers. Our experimental program is divided into 4 areas. (i) We will characterize the copper centers of the oxidized and reduced forms of the family of Sco proteins involved in copper transfer to the CuA center. (ii) We will probe the mechanism of metal transfer between Sco and its partner protein, the CuA-containing CCO subunit II, using selenomethionine substitution to provide a site-specific XAS probe of metal transfer reactivity. (iii) We will probe the function of the Sco homologue of B. subtilis via in vivo assay of Cu(I) and Cu(II) site-specific mutations. (iv) We will apply expressed protein ligation as a tool for the incorporation of selenocysteine at the active centers of Sco, CuA and Cox 11 as an additional XAS spectroscopic probe of function. These studies will provide much-needed biophysical data on the proteins involved in cytochrome oxidase assembly, and will complement the extensive genetic evidence linking defects in CCO assembly with respiratory disease.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function A Study Section (MSFA)
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Anderson, Vernon
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Oregon Health and Science University
Schools of Medicine
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Yu, Yang; Petrik, Igor D; Chacón, Kelly N et al. (2017) Effect of circular permutation on the structure and function of type 1 blue copper center in azurin. Protein Sci 26:218-226
Fetherolf, Morgan M; Boyd, Stefanie D; Taylor, Alexander B et al. (2017) Copper-zinc superoxide dismutase is activated through a sulfenic acid intermediate at a copper ion entry site. J Biol Chem 292:12025-12040
Hosseinzadeh, Parisa; Marshall, Nicholas M; Chacón, Kelly N et al. (2016) Design of a single protein that spans the entire 2-V range of physiological redox potentials. Proc Natl Acad Sci U S A 113:262-7
Farashishiko, Annah; Chacón, Kelly N; Blackburn, Ninian J et al. (2016) Nano assembly and encapsulation; a versatile platform for slowing the rotation of polyanionic Gd(3+) -based MRI contrast agents. Contrast Media Mol Imaging 11:154-9
Tian, Shiliang; Liu, Jing; Cowley, Ryan E et al. (2016) Reversible S-nitrosylation in an engineered azurin. Nat Chem 8:670-7
Martin-Diaconescu, Vlad; Chacón, Kelly N; Delgado-Jaime, Mario Ulises et al. (2016) K? Valence to Core X-ray Emission Studies of Cu(I) Binding Proteins with Mixed Methionine - Histidine Coordination. Relevance to the Reactivity of the M- and H-sites of Peptidylglycine Monooxygenase. Inorg Chem 55:3431-9
Chakraborty, Saumen; Polen, Michael J; Chacón, Kelly N et al. (2015) Binuclear Cu(A) Formation in Biosynthetic Models of Cu(A) in Azurin Proceeds via a Novel Cu(Cys)2His Mononuclear Copper Intermediate. Biochemistry 54:6071-81
Clark, Kevin M; Yu, Yang; van der Donk, Wilfred A et al. (2014) Modulating the Copper-Sulfur Interaction in Type 1 Blue Copper Azurin by Replacing Cys112 with Nonproteinogenic Homocysteine. Inorg Chem Front 1:153-158
Chacón, Kelly N; Mealman, Tiffany D; McEvoy, Megan M et al. (2014) Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins. Proc Natl Acad Sci U S A 111:15373-8
Abriata, Luciano A; Alvarez-Paggi, Damian; Ledesma, Gabriela N et al. (2012) Alternative ground states enable pathway switching in biological electron transfer. Proc Natl Acad Sci U S A 109:17348-53

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