Reversible and irreversible inactivation of cellular nitrogenase upon oxygen stress in Azotobacter vinelandii growing in oxygen controlled continuous culture

Abstract

Azotobacter vinelandii growing in oxygen controlled chemostat culture was subjected to sudden increases of ambient oxygen concentrations (oxygen stress) after adaptation to different oxygen concentrations adjustable with air (100% air saturation corresponds to 225±14 μM O₂). Inactivations of cellular nitrogenase during stress (switch off) as well as after release of stress (switch on) were evaluated in vivo as depending on stress duration and stress height (ΔpO₂). Switch off was at its final extent within 1 min of stress. The extent of switch off, however, increased with stress height and was complete at ΔpO₂ between 8–10% air saturation irrespective of different oxygen concentrations the organisms were adapted to before stress, indicating that switch off is adaptable. Inactivation of nitrogenase measurable after switch on represents irreversible loss of activity. Irreversible inactivation was at its characteristic level within less than 3 min of stess and at a ΔpO₂ of less than 1% air saturation. The level of irreversible inactivation increased linearly with the oxygen concentration the organisms were adapted to before stress. Thus adaptation of cells to increased oxygen concentrations did not prevent increased susceptibility of nitrogenase to irreversible inhibition during oxygen stress. The fast response of irreversible inactivation at low stress heights suggests that it takes place already during stress. Thus switch off comprised both a reversible and an irreversible phase. The data showed that reversible inactivation of nitrogenase was less susceptible to oxygen stress than irreversible inactivation. A basic pre-requisite of the hypothesis of respiratory protection of nitrogenase, i.e. the proposed relationship between respiratory activities and the protection of nitrogenase from irreversible inhibition by oxygen, was not supported by the results of this report.