The central role of heme-copper oxidases in biological energy production has made them the targets of intense study in many laboratories across the globe. Since the crystal structures have now defined the coordination of the redox centers and the identity of proton pumping channels, attention has focused on determining mechanistic details. Unraveling these mechanisms has required application of specialized spectroscopic and biophysical techniques. Our contribution has been the application of X-ray absorption spectroscopy (XAS) to uniquely define the coordination of the copper centers in a number of derivatives of different members of the oxidase family. The lack of optical or magnetic signals from CuB makes XAS particularly relevant to testing proposed structures for resting and intermediate states of the binuclear center. In this renewal application, we will extend our studies to include novel members of the oxidase family and catalytic intermediates. First, we will investigate resting states of the CuB centers of quinol oxidases from Bacillus subtilis, Acidianus ambivalens, Thioalcalomicrobium aerophilum, and a CuA-deleted mutant of Thermus thermophilus. The reduced forms will be examined carefully for evidence of functionally important histidine dissociation and chloride ligation as recently observed for the b(03) enzyme. We will next determine the structures of key intermediates in the reaction pathway, again searching for evidence of histidine dissociation from CuB proposed to be an important element of proton pumping. These data will be backed up by studies on inorganic and engineered protein models for cytochrome oxidase. Finally, we will begin to explore the mechanism of interaction of cytochrome oxidase with recently discovered metallochaperones, which are believed to be essential for proper maturation of the intact oxidase in the membrane.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM054803-08S1
Application #
7197887
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Preusch, Peter C
Project Start
1997-05-01
Project End
2006-11-30
Budget Start
2004-05-01
Budget End
2006-11-30
Support Year
8
Fiscal Year
2006
Total Cost
$74,240
Indirect Cost
Name
Oregon Health and Science University
Department
Biochemistry
Type
Schools of Engineering
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
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
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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
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
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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