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 with g|| at 2.21(8) and g_^ at 2.04(5). The magnitude of the hyperfine splitting (A_|| = 6.36 mT) indicated the presence of type 1 copper only. The electronic spectrum had maxima at 280 nm, 474 nm and 595 nm (e = 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 (e = 2.5 mM^-1 cm^-1) and pronounced structures in the ultraviolet region. The EPR parameters were g_|| = 2.26(6) and g_^ = 2.05(6), with A_|| = 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 µmol 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.