Dependence of nitrogenase switch-off upon oxygen stress on the nitrogenase activity in Azotobacter vinelandii

Abstract

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

Nitrogenase activity and regeneration of the cellular ATP pool in Azotobacter vinelandii adapted to different oxygen concentrations

“…Specific rates of dinitrogen fixation were calculated on the basis of D and the total amounts of dinitrogen fixed by the cells under steady-state conditions (2). Since, under steady-state conditions, cells fixed 6.7 Ϯ 0.4 mol of dinitrogen per mg of protein irrespective of the dissolved oxygen concentration, the specific rate of dinitrogen fixation increased linearly with increasing D. A comparable result was shown before with cultures of A. vinelandii grown under conditions of either sucrose, citrate, or acetate limitation (13).…”

“…Additional protection of nitrogenase may be provided by the decrease of the cellular surface area per cell volume (24) as well as by the increase of the cellular content of superoxide dismutase (6) occurring when the dissolved oxygen concentration of chemostat cultures of A. vinelandii is increased. Moreover, it has been proposed that nitrogenase stays active in aerobic cultures as long as the low redox potential necessary for its function is warranted by a sufficiently high flux of electrons through the enzyme complex (13). This is in accord with the hypothesis of autoprotection of nitrogenase, assuming that oxygen enters the cells where it is reduced by nitrogenase (31).…”

“…This hypothesis postulates that reduction of nitrogenase and, thus, its function at ambient oxygen concentrations are warranted as long as the enzyme is supplied with sufficiently high amounts of ATP (13,16). In agreement with this hypothesis and irrespective of the oxygen concentration and the type of growth limitation, uniform relationships became apparent either when the size of the ATP pool was plotted versus the concentrations of dinitrogen fixed by nitrogenase or when the specific rates of regeneration of the ATP pool were plotted versus the specific rates of dinitrogen fixation.…”

“…For example, it was shown with A. vinelandii growing at high dissolved oxygen concentrations that the respiratory activity does not significantly increase with an increase in the dissolved oxygen concentration (25). Moreover, cultures of A. vinelandii exhibited comparable cellular contents and nitrogenase activities in spite of considerable differences in cellular oxygen consumption (7,13). Finally, it has been demonstrated that oxygen enters the cells of A. vinelandii without impairing the function of nitrogenase (21).…”

Cellular ATP levels and nitrogenase switchoff upon oxygen stress in chemostat cultures of Azotobacter vinelandii

“…Subsequently, the lack of proportionality between ambient oxygen concentrations, cellular respiratory rates, and the function of nitrogenase was confirmed by a number of independent observations (13). In this context it is interesting to note that the above-mentioned cytochrome o-deficient mutant of A. vinelandii exhibited the same oxygen tolerance as the wild type but only 45% of its respiratory activity (15).…”