KINETIC AND SPECTROSCOPIC ANALYSIS OF THE INACTIVATING EFFECTS OF NITRIC-OXIDE ON THE INDIVIDUAL COMPONENTS OF AZOTOBACTER-VINELANDII NITROGENASE

The effects of nitric oxide (NO) on the individual components of Azotobacter vinelandii nitrogenase have been examined by kinetic and spectroscopic methods. Incubation of the Fe protein (Av2) for 1 h with stoichiometries of 4- and 8-fold molar excesses of NO to Av2 dimer resulted in a complete loss of activity of Av2 in C2H2-reduction assays. The kinetics of inactivation indicated that the minimum stoichiometry of NO to Av2 required to fully inactivate Av2 lies between 1 and 2. The rate of inactivation of Av2 activity by NO was stimulated up to 2-fold by the presence of MgATP and MgADP but was unaffected by the presence of sodium dithionite. Unexpectedly, complete inactivation of Av2 by low ratios of NO to Av2 also resulted in a complete loss of its ability to bind MgATP and MgADP. UV-visible spectroscopy indicated that the effect of NO on Av2 involves oxidation of the [4Fe-4S] center. EPR spectroscopy revealed that the loss of activity during inactivation of Av2 by NO correlated with the loss of the S = 1/2 and S = 3/2 Signals. Appearance of the classical and intense iron-nitrosyl signal (g = 2.03) was only observed when Av2 was incubated with large molar excesses of NO and the appearance of this signal did not correlate with the loss of Av2 activity. The effects of NO on the MoFe protein (Avl) were more complex than for Av2. A time-dependent inactivation of Avl activity (C2H2 reduction) was observed which required considerably higher concentrations of NO than those required to inactivate Av2 (up to 10 kPa). In addition, the effects of NO on Avl were significantly affected by the presence of sodium dithionite. In fact, kinetic evidence suggests that an Avl-catalyzed, NO-dependent consumption of dithionite occurs before Avl is inactivated by NO. A correlation between UV-visible and EPR spectral features and the extent of NO inactivation has been established. The inactivation of either nitrogenase component by NO did not lead to aggregation or dissolution into their constitutive subunits. However, NO inactivation did cause changes in both proteins since neither NO-treated protein inhibited C2H2-reducing activity in assays containing equimolar concentrations of untreated protein. The effects of NO on both nitrogenase components are interpreted in terms of the known reactivity of NO with Fe-S centers.