KINETICS OF THE REACTIONS OF TRIOXODINITRATE AND NITRITE IONS WITH CYTOCHROME-D IN ESCHERICHIA-COLI

The rate of reaction of trioxodinitrate with reduced cytochrome oxidase d in membrane particles from Escherichia coli at pH 7 and 25-degrees-C depends linearly upon [HN2O3-] over the concentration range studied (up to 0.05 mM) and is also first-order in cytochrome d. The known rate of decomposition of trioxodinitrate to give NO- and NO2- is about 4.5-times faster than the rate of reaction of reduced cytochrome d with trioxodinitrate, implying that cytochrome d reacts directly with NO-, with a trapping ratio of between 0.20 and 0.25, rather than with trioxodinitrate. The implications of the facile formation of the NO-nitrosyl complex of cytochrome d for the mechanism of denitrification are discussed with particular reference to the mechanism of N-N bond formation. The reaction of reduced cytochrome d with nitrite (a decomposition product of trioxodinitrate) under these conditions is much slower than that with trioxodinitrate. The kinetics show a biphasic dependence of initial rate upon nitrite concentration. The rate data at low [NO2-] are consistent with saturation of a high affinity site for nitrite, having V(max) = 4.29.10(-9) M s-1 and K(m) = 0.034 mM. The existence of two binding sites for nitrite is consistent with the suggestion that the cytochrome bd complex contains two cytochrome d haems.