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

In order to elucidate the mechanism of inhibition, by nitrite,of the formation of oxyleghemoglobin (LbO₂ and the mechanismof generation of nitrosylleghemoglobin (LbNO), kinetic analysesof results of measurements of oxygen uptake and spectrophotometricassays of leghemoglobin were performed. The decrease, by nitrite, in the oxygen-binding capacity ofleghemoglobin was caused by the increase in levels of LbNO formedfrom ferrous leghemoglobin. In this case, the oxygen-bindingsite of leghemoglobin was competitively occupied by nitric oxideproduced from nitrite. The kinetic constants for the generation of LbNO from leghemoglobinand nitrite were 5.7 ? 10 for the association rate constant,4.4 ? 10^–5 for the dissociation rate constant, and 1.3? 10⁶ for the equilibrium constant. From calculations basedon the equilibrium constant, it appears that LbO₂ is presentin small amounts in nodules of plants supplied with nitrate.Furthermore, the dissociation rate constant for LbNO was muchsmaller than that for LbO₂ or carboxyleghemoglobin (LbCO). Thisdifference indicates that, once formed, LbNO is harder to dissociatethan LbO₂ or LbCO. Thus, the accumulation of LbNO in the nodule cytosol, as a resultof the supply of nitrate, would inhibit nitrogenase activitythrough a decrease in the diffusion of oxygen that results froma lack of LbO₂. (Received December 18, 1989; Accepted April 13, 1990)