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Azotobacter vinelandii was grown diazotrophically at different dissolved oxygen concentrations (in the range of 3 to 216 FM) in sucrose-linited continuous culture. The specific nitrogenase activity, measured on the basis of acetylene reduction in situ, was dependent solely on the growth rate and was largely independent of oxygen and sucrose concentration. FeMo (Avl) and Fe (Av2) nitrogenase proteins were quantified after Western blotting (immunoblotting). When the cultures were grown at a constant dilution rate (D, representing the growth rate, ,u) of 0.15. h1, the cellular levels of both proteins were constant regardless of different dissolved oxygen concentrations. The same was true when the organisms were grown at D values above 0.15 h-1. At a lower growth rate (D = 0.09 h-1), however, and at lower oxygen concentrations cellular levels of both nitrogenase proteins were decreased. This means that catalytic activities of nitrogenase proteins were highest at low oxygen concentrations, but at higher oxygen concentrations they increased with growth rate. Under all conditions tested, however, the Av1:Av2 molar ratio was 1:(1.45 ± 0.12). Cellular levels of flavodoxin and FeS protein II were largely copstant as well. In order to estimate turnover of nitrogenase proteins in the absence of protein synthesis, chloramphenicol was added to cultures adapted to 3 and 216 ,uM oxygen, respectively.After 2 h of incubation, no significant decrease in the cellular levels of Avl and Av2 could be observed. Thbis suggests that oxygen has no significant effect on the breakdown of nitrogenase proteins.

Biological dinitrogen fixation is largely restricted to conditions of low aeration and complete anaerobiosis, because the enzyme catalyzing the reduction of atmospheric dinitrogen to ammonia, nitrogenase, is extremely oxygen labile (18,22). Nevertheless, members of the Azotobacteraceae are capable of fixing dinitrogen while growing at high ambient oxygen concentrations. As originally proposed by Postgate (22), protection of nitrogenase against oxygen damage may involve two mechanisms in Azotobacter chroococcum and related species. The hypothesis of respiratory protection postulates that the unusually high activities of cellular oxygen consumption, characteristic of azotobacters, prevent the diffusion of oxygen into the cells and thus to nitrogenase. Whenever this steady state becomes disturbed by an increase in the dissolved oxygen concentration, conformational protection, i.e., switch-off of nitrogenase activity, protects nitrogenase proteins (3,28).Studies of the molecular basis of the switch-off effect indicated that a low-molecular-weight (two atoms of iron and two atoms of sulfur) protein associates with the iron-molybdenum and iron proteins of nitrogenase to form a threecomponent complex (25). Formation of the complex depends on the oxidation of the electron donor to nitrogenase, as well as on the reversible oxidation of nitrogenase (25,27,29).Following a switch-off, nitrogenase activity resumes either when dissolved oxygen decreases to about its original concentration or after adaptation of the cultures to the elevated oxygen concentration. The latter is accompanied by an enhanced formation of constituents of the respiratory chain, increasing the cellular respiratory activity (13). However, detailed investigations with chemostat cultures of Azotobacter vinelandii which were adapted to different oxygen concentrations revealed that, particularly at high ambient oxygen concentrations, the activity of oxygen consumption * Corresponding author. increased with increasing oxygen concentrations, but not linearly (1, 21). Thus, additional or even alternative mechanisms are required to provide maximal protection of nitrogenase against an oxygen-induced switch-off and, moreover, against irreversible damage. Protection against the latter may involve superoxide dismutase, the activity of which increased when the dissolved oxygen concentration was raised (7). The present investigation, focusing on the stability of nitrogenase activity under oxygen stress, demonstrates that the switch-off behavior depends entirely on the rate of supply of the energy and carbon source rather than on the rate of total oxygen consumption by the cells. MATERIALS AND METHODS

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