The respiratory nitric oxide reductase (NorBC) from Paracoccus denitrificans.
The two subunit cytochrome bc complex (NorBC) isolated from membranes of the model denitrifying soil bacterium Paracoccus denitrificans is the best characterized example of the bacterial respiratory nitric oxide reductases. These are members of the superfamily of heme-copper oxidases and are characterized by the elemental composition of their active site, which contains nonheme iron rather than copper, at which the reductive coupling of two molecules of nitric oxide to form nitrous oxide is catalyzed. This chapter describes methods for the purification and characterization of both native nitric oxide reductase from P. denitrificans and a recombinant form of the enzyme expressed in Escherichia coli, which enables site-directed mutagenesis of the catalytic subunit NorB. Examples are given of electronic absorption and electron paramagnetic resonance spectra that characterize the enzyme in a number of redox states, along with a method for the routine assay of the complex using its natural electron donor pseudoazurin.
Field
Sarah J
SJ
Center for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich, United Kingdom.
Thorndycroft
Faye H
FH
Matorin
Andrey D
AD
Richardson
David J
DJ
Watmough
Nicholas J
NJ
D001400:Azurin / Q000378:metabolism; D015374:Biosensing Techniques; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D050901:Nitrate Reductase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000378:metabolism; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification; D013057:Spectrum Analysis / Q000379:methods
D020030:Nitric Oxide Donors; D011994:Recombinant Proteins; C047163:pseudoazurin; D001400:Azurin; D009569:Nitric Oxide; D050901:Nitrate Reductase