Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens.
Pseudomonas aureofaciens truncates the respiratory reduction of nitrate (denitrification) at the level of N2O. The nitrite reductase from this organism was purified to apparent electrophoretic homogeneity and found to be a blue copper protein. The enzyme contained 2 atoms of copper/85 kDa, both detectable by electron paramagnetic resonance (EPR) spectroscopy. The protein was dimeric, with subunits of identical size (40 +/- 3 kDa). Its pI was 6.05. The EPR spectrum showed an axial signal g at 2.21(8) and g at 2.04(5). The magnitude of the hyperfine splitting (A parallel = 6.36 mT) indicated the presence of type 1 copper only. The electronic spectrum had maxima at 280 nm, 474 nm and 595 nm (epsilon = 7.0 mM-1 cm-1), and a broad shoulder around 780 nm. A copper protein of low molecular mass (15 kDa), with properties similar to azurin, was also isolated from P. aureofaciens. The electronic spectrum of this protein showed a maximum at 624 nm in the visible range (epsilon = 2.5 mM-1 cm-1) and pronounced structures in the ultraviolet region. The EPR parameters were g parallel = 2.26(6) and g perpendicular = 2.05(6), with A parallel = 5.8 mT. The reduced azurin transferred electrons efficiently to nitrite reductase; the product of nitrite reduction was nitric oxide. The specific nitrite-reducing activity with ascorbate-reduced phenazine methosulfate as electron donor was 1 mumol substrate min-1 mg protein-1. The reaction product again was nitric oxide. Nitrous oxide was the reaction product from hydroxylamine and nitrite and from dithionite-reduced methyl viologen and nitrite. No 'oxidase' activity could be demonstrated for the enzyme. Our data disprove the presumed exclusiveness of cytochrome cd1 as nitrite reductase within the genus Pseudomonas.
Zumft
W G
WG
Lehrstuhl für Mikrobiologie, Universität Karlsruhe, Federal Republic of Germany.
Gotzmann
D J
DJ
Kroneck
P M
PM
D001400:Azurin / Q000032:analysis; D001426:Bacterial Proteins; D002849:Chromatography, Gas; D004579:Electron Transport; D009682:Magnetic Resonance Spectroscopy; D008667:Metalloproteins / Q000032:analysis* / Q000502:physiology; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis*; D009572:Nitrite Reductases / Q000032:analysis*; D009573:Nitrites / Q000378:metabolism; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000032:analysis* / Q000201:enzymology
D001426:Bacterial Proteins; D008667:Metalloproteins; D009573:Nitrites; C017079:mauC protein, Methylobacterium extorquens; D001400:Azurin; D009247:NADH, NADPH Oxidoreductases; D009572:Nitrite Reductases