The Concentration of Cellular Nitrogenase Proteins inAzotobacter vinelandiiWhole Cells as Determined by Activity Measurements and Electron Paramagnetic Resonance Spectroscopy

The concentration of MoFe protein (Av1) inAzotobacter vinelandiiwhole-cell crude extract was measured by electron paramagnetic resonance spectroscopy at g = 3.7 resonance. The Av1 concentration was also measured from the activity of crude extract to which increasing amounts of purified Av1 and Av2 were added. The Av2 concentration was determined by fitting activity measurements of crude extract and crude extract to which purified Av2 was added. The Av1 concentration was found to be 26–28 μMand that for Av2 was 42–45 μMin whole cells, with a Av2/Av1 ratio of 1.6.In vitroactivity measurements carried out as a function of Av1 concentration at Av2/Av1 ratios of 1 and 4 showed a dilution effect below 0.08 μM, a factor of 2 below that observed for nitrogenase reactivity forKlebsiella pneumoniae.No deviations from linearity were observed up to 26 μMfor the Av1–Av2 interaction. The flavoprotein (AvFlp) was shown to enhance nitrogenase reactivity at low Av2/Av1 ratios, a result attributed to decreasing theKmfor Av2–Av1 interaction. Direct reduction of bound Av2 is possibly the source of this kinetic enhancement. The kinetic results are considered in terms of the Thorneley and Lowe scheme.