Properties of a Soluble Domain of Subunit C of a Bacterial Nitric Oxide Reductase

Bacterial nitric oxide reductases are integral membrane proteins that catalyze the reduction of two molecules of nitric oxide to nitrous oxide and water. They are diverged members of the superfamily of heme/copper oxidases. The enzyme from Paracoccus denitrificans (NorBC) contains two subunits; NorB...

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Bibliographic Details
Published in:Biochemistry (Easton) 2002-09, Vol.41 (35), p.10858-10865
Main Authors: Oubrie, Arthur, Gemeinhardt, Sabine, Field, Sarah, Marritt, Sophie, Thomson, Andrew J, Saraste, Matti, Richardson, David J
Format: Article
Language:English
Subjects:
Bacterial Proteins - biosynthesis
Bacterial Proteins - chemistry
Bacterial Proteins - genetics
Bacterial Proteins - isolation & purification
Circular Dichroism
Cloning, Molecular
Cytochrome b Group - chemistry
Cytochrome c Group - biosynthesis
Cytochrome c Group - chemistry
Cytochrome c Group - genetics
Cytochrome c Group - isolation & purification
Electron Spin Resonance Spectroscopy
Magnetics
Nuclear Magnetic Resonance, Biomolecular
Oxidoreductases - biosynthesis
Oxidoreductases - chemistry
Oxidoreductases - genetics
Paracoccus denitrificans - enzymology
Paracoccus denitrificans - genetics
Peptide Fragments - biosynthesis
Peptide Fragments - chemistry
Peptide Fragments - genetics
Peptide Fragments - isolation & purification
Potentiometry
Protein Structure, Tertiary - genetics
Protein Subunits
Solubility
Spectrophotometry, Ultraviolet
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Summary:Bacterial nitric oxide reductases are integral membrane proteins that catalyze the reduction of two molecules of nitric oxide to nitrous oxide and water. They are diverged members of the superfamily of heme/copper oxidases. The enzyme from Paracoccus denitrificans (NorBC) contains two subunits; NorB comprises the membrane-integrated active site, which harbors a heme iron/non-heme iron dinuclear center. NorC is a membrane-anchored c-type cytochrome and presumably the site of electron uptake. A DNA construct encoding the water-soluble domain of NorC (NorCsol) was coexpressed with the cytochrome c maturation genes in Escherichia coli. Using redox potentiometry, electronic absorption, circular dichroism (CD), magnetic CD (MCD), nuclear magnetic resonance, and electron paramagnetic resonance (EPR) spectroscopy the following observations were made:  (i) NorCsol was folded into a α-helical structure. (ii) The low-spin heme iron was coordinated by histidine and methionine in both redox states. (iii) The midpoint redox potential of the NorCsol heme was 183 mV, much lower than the corresponding value of 275 mV in the NorBC complex. This points to an increased solvent exposure of the NorCsol heme compared to in the native NorBC complex and shows that the electronic properties of NorC are modulated by NorB in the complex. (iv) The EPR and MCD spectra of NorCsol were considered alongside the spectra of NorBC, which has helped to resolve the contribution that different redox centers make in the holo-enzyme complex.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi026140y