Isolation and characterization of nitric oxide reductase from Paracoccus halodenitrificans.
Nitric oxide reductase was isolated from the membrane fraction of a denitrifying bacterium, Paracoccus halodenitrificans, in the presence of n-dodecyl beta-D-maltoside. A relatively simple and effective procedure to purify NO reductase using DEAE-Toyopearl and hydroxyapatite (ceramic) chromatographies has been developed. The enzyme consisted of two subunits with molecular masses of 20 and 42 kDa associated with the c-type heme and two b-type hemes, respectively. The optical and magnetic circular dichroism (MCD) spectra of the oxidized (as isolated) and reduced enzymes indicated that the heme c is in the low-spin state and the hemes b are in the high- and low-spin states. The EPR spectrum also showed the presence of the split high-spin component (g perpendicular = 6.6, 6.0) and two low spin components (gz,y,x = 2.96, 2.26, 1.46, gz = 3.59). Although the presence of an extra iron was suggested from atomic absorption spectroscopy, a non-heme iron could not be detected by colorimetric titrations using ferene and 2-(5-nitro-2-pyridylazo)- 5-(N-propyl-N-sulfopropylamino)phenolate (PAPS). One of the extra signals at g = 4.3 and 2.00 might come from a non-heme iron, while they may originate from an adventitious iron and a certain nonmetallic radical, respectively. When CO acted on the reduced enzyme, both of the low-spin hemes were not affected, and when NO acted on the reduced enzyme, the optical and MCD spectra were of a mixture of the oxidized and reduced enzymes. Consequently, the reduction of NO was supposed to take place at the high-spin heme b. The heme c and the low-spin heme b centers were considered to function as electron mediators during the intermolecular and intramolecular processes.
Sakurai
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Institute for Molecular Science, Okazaki National Research Institutes, Japan.
Sakurai
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D002942:Circular Dichroism; D003573:Cytochrome b Group / Q000737:chemistry; D003574:Cytochrome c Group / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D010230:Paracoccus / Q000201:enzymology*; D013053:Spectrophotometry
D003573:Cytochrome b Group; D003574:Cytochrome c Group; D006418:Heme; D010088:Oxidoreductases; C034747:nitric-oxide reductase