Respiratory protection of nitrogenase in Azotobacter species: is a widely held hypothesis unequivocally supported by experimental evidence?

The hypothesis of respiratory protection, originally formulated on the basis of results obtained with Azotobacter species, postulates that consumption of O 2 at the surface of diazotrophic prokaryotes protects nitrogenase from inactivation by O 2 . Accordingly, it is assumed that, at increased ambient O 2 concentrations, nitrogenase activity depends on increased activities of a largely uncoupled respiratory electron transport system. The present review compiles evidence indicating that cellular O 2 consumption as well as both the activity and the formation of the respiratory system of Azotobacter vinelandii are controlled by the C/N ratio, that is to say the ratio at which the organism consumes the substrate (i.e. the source of carbon, reducing equivalents and ATP) per source of compound nitrogen. The maximal respiratory capacity which can be attained at increased C/N ratios, however, is controlled, within limits, by the ambient O 2 concentration. When growth becomes N‐limited at increased C/N ratios, cells synthesize nitrogenase and fix N 2 . Under these diazotrophic conditions, cellular O 2 consumption remains constant at a level controlled by the O 2 concentration. Control by O 2 has been studied on the basis of both whole cell respiration and defined segments of the respiratory electron transport chain. The results demonstrate that the effect of O 2 on the respiratory system is restricted to the lower range of O 2 concentrations up to about 70 μM. Nevertheless, azotobacters are able to grow diazotrophically at dissolved O 2 concentrations of up to about 230 μM indicating that respiratory protection is not warranted at increased ambient O 2 concentrations. This conclusion is supported and extended by a number of results largely excluding an obvious relationship between nitrogenase activity and the actual rate of cellular O 2 consumption. On the basis of theoretical calculations, it is assumed that the rate of O 2 diffusion into the cells is not significantly affected by respiration. All of these results lead to the conclusion that, in the protection of nitrogenase from O 2 damage, O 2 consumption at the cell surface is less effective than generally assumed. It is proposed that alternative factors like the supply of ATP and reducing equivalents are more important.