Inhibition of Nitrogen Fixation in Soybean Plants Supplied with Nitrate III. Kinetics of the Formation of Nitrosylleghemoglobin and of the Inhibition of Formation of Oxyleghemoglobin

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In order to elucidate the mechanism of inhibition, by nitrite, of the formation of oxy-leghemoglobin (LbO_2) and the mechanism of generation of nitrosylleghemoglobin (LbNO), kinetic analyses of results of measurements of oxygen uptake and spectrophotometric assays of leghemoglobin were performed. The decrease, by nitrite, in the oxygen-binding capacity of leghemoglobin was caused by the increase in levels of LbNO formed from ferrous leghemoglobin. In this case, the oxygen-binding site of leghemoglobin was competitively occupied by nitric oxide produced from nitrite. The kinetic constants for the generation of LbNO from leghemoglobin and nitrite were 5.7 × 10 for the association rate constant, 4.4 × 10^<-5> for the dissociation rate constant, and 1.3 × 10^6 for the equilibrium constant. From calculations based on the equilibrium constant, it appears that LbO_2 is present in small amounts in nodules of plants supplied with nitrate. Furthermore, the dissociation rate constant for LbNO was much smaller than that for LbO_2 Or carboxyleghemoglobin (LbCO). This difference indicates that, once formed, LbNO is harder to dissociate than LbO_2 Or LbCO. Thus, the accumulation of LbNO in the nodule cytosol, as a result of the supply of nitrate, would inhibit nitrogenase activity through a decrease in the diffusion of oxygen that results from a lack of LbO_2.
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