This research aims to elucidate reaction intermediates of NO-reductase activity in diiron proteins, with the ultimate goal of understanding how the metal clusters catalyze this reaction. Our studies will focus on three enzymes: 1) denitrifying NO reductases cNOR from Paracoccus denitrificans and qCuANOR from Bacillus azotoformans, 2) the [heme-copper] ba3 terminal oxidase from Thermus thermophilus, and 3) detoxifying NO reductase flavoprotein A (FprA) from Moorella thermoacetica. A better understanding of microbial NO reductases is highly desirable considering that these enzymatic reactions provide a resistance to the mammalian immune response. Although crystal structures exist for some of these enzymes, the structure and reactivity of their NO-complexes are not known. Diiron proteins participate in both detoxifying and denitrifying NO reductase reactions and are thought to react with NO to form [FeNO]2 intermediates. Alternative mechanistic models are considered and tested in this proposal. Resonance Raman and FTIR spectroscopies have the unique capability to identify nitrosyl intermediates and to define their NO-binding geometries with regard to the two metal ions. Novel rapid freeze-quench (RFQ) instrumentation that can trap intermediates within a sub-ms timescale provides new opportunities to characterize reaction intermediates that were previously inaccessible to spectroscopic methods. FTIR spectroscopy, in conjunction with low temperature photolysis of N3~ and CO, acting as NO surrogates, offers insight into binding geometries, potential hydrogen bond interactions and proton transfers relevant to these NO reductase mechanisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM074785-05
Application #
7782670
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2006-04-01
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2012-03-31
Support Year
5
Fiscal Year
2010
Total Cost
$217,138
Indirect Cost
Name
Oregon Health and Science University
Department
Engineering (All Types)
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
Bhagi-Damodaran, Ambika; Reed, Julian H; Zhu, Qianhong et al. (2018) Heme redox potentials hold the key to reactivity differences between nitric oxide reductase and heme-copper oxidase. Proc Natl Acad Sci U S A 115:6195-6200
Sabuncu, Sinan; Reed, Julian H; Lu, Yi et al. (2018) Nitric oxide reductase activity in heme-nonheme binuclear engineered myoglobins through a one-electron reduction cycle. J Am Chem Soc :
Reed, Julian H; Shi, Yelu; Zhu, Qianhong et al. (2017) Manganese and Cobalt in the Nonheme-Metal-Binding Site of a Biosynthetic Model of Heme-Copper Oxidase Superfamily Confer Oxidase Activity through Redox-Inactive Mechanism. J Am Chem Soc 139:12209-12218
Bhagi-Damodaran, Ambika; Michael, Matthew A; Zhu, Qianhong et al. (2017) Why copper is preferred over iron for oxygen activation and reduction in haem-copper oxidases. Nat Chem 9:257-263
Sharma, Savita K; Schaefer, Andrew W; Lim, Hyeongtaek et al. (2017) A Six-Coordinate Peroxynitrite Low-Spin Iron(III) Porphyrinate Complex-The Product of the Reaction of Nitrogen Monoxide (·NO(g)) with a Ferric-Superoxide Species. J Am Chem Soc 139:17421-17430
Nilsson, Zach N; Mandella, Brian L; Sen, Kakali et al. (2017) Distinguishing Nitro vs Nitrito Coordination in Cytochrome c' Using Vibrational Spectroscopy and Density Functional Theory. Inorg Chem 56:13205-13213
Confer, Alex M; McQuilken, Alison C; Matsumura, Hirotoshi et al. (2017) A Nonheme, High-Spin {FeNO}8 Complex that Spontaneously Generates N2O. J Am Chem Soc 139:10621-10624
McQuilken, Alison C; Matsumura, Hirotoshi; Dürr, Maximilian et al. (2016) Photoinitiated Reactivity of a Thiolate-Ligated, Spin-Crossover Nonheme {FeNO}(7) Complex with Dioxygen. J Am Chem Soc 138:3107-17
Basudhar, Debashree; Madrona, Yarrow; Yukl, Erik T et al. (2016) Distal Hydrogen-bonding Interactions in Ligand Sensing and Signaling by Mycobacterium tuberculosis DosS. J Biol Chem 291:16100-11
Matsumura, Hirotoshi; Chakraborty, Saumen; Reed, Julian et al. (2016) Effect of Outer-Sphere Side Chain Substitutions on the Fate of the trans Iron-Nitrosyl Dimer in Heme/Nonheme Engineered Myoglobins (Fe(B)Mbs): Insights into the Mechanism of Denitrifying NO Reductases. Biochemistry 55:2091-9

Showing the most recent 10 out of 38 publications