Later investigators criticized the previous study (182) as too simplistic, not explaining the total potential of the nitrogenase system. Azotobacter seems to make three flavodoxins, not just azotoflavin; flavodoxin II seems like the important one but it wasn't known how it got reduced; and ferredoxin might not be involved at all.
So this study grew A. vinelandii with and without ammonium, then observed the differences in its redox systems.
What They Saw
They grew cells in a chemostat with ammonium, and then removed samples and washed with nitrogen-free medium to remove the fixed nitrogen. They measured nitrogenase activity of these samples and labeled newly formed proteins with radioactive sulfur compounds.
They observed that nitrogenase activity correlated well with rate of respiration in different conditions, so they wondered if the two might be linked.
After they removed the fixed nitrogen from cells that had been growing with it, they observed nitrogenase activity within 20 minutes. Then the activity increased linearly over time for at least 40 minutes in this condition. On protein gels, they observed the nitrogenase proteins produced quickly, within 5 minutes, and flavodoxin II showed up some time later. There are some others of uncertain identity, and some interesting ones showing up only in the membrane protein fraction.
What This Means
The linear relationship between respiration and nitrogenase activity has a number of possible explanations. Extra respiration could mean higher membrane potential or ATP levels, so more energy for nitrogenase, or there could be more enzymes (or enzymes that are more active) to transport electrons to nitrogenase. The former seems unlikely, since Azotobacter uncouples respiration from energy generation at higher oxygen levels, so extra respiration doesn't necessarily mean more energy. And extra enzymes seems unlikely too, based on the protein results.
So it seems like increased nitrogenase activity might be due to increased transport of electrons to the enzyme, though it's not clear how that happens.
Reference:
So this study grew A. vinelandii with and without ammonium, then observed the differences in its redox systems.
What They Saw
They grew cells in a chemostat with ammonium, and then removed samples and washed with nitrogen-free medium to remove the fixed nitrogen. They measured nitrogenase activity of these samples and labeled newly formed proteins with radioactive sulfur compounds.
They observed that nitrogenase activity correlated well with rate of respiration in different conditions, so they wondered if the two might be linked.
After they removed the fixed nitrogen from cells that had been growing with it, they observed nitrogenase activity within 20 minutes. Then the activity increased linearly over time for at least 40 minutes in this condition. On protein gels, they observed the nitrogenase proteins produced quickly, within 5 minutes, and flavodoxin II showed up some time later. There are some others of uncertain identity, and some interesting ones showing up only in the membrane protein fraction.
What This Means
The linear relationship between respiration and nitrogenase activity has a number of possible explanations. Extra respiration could mean higher membrane potential or ATP levels, so more energy for nitrogenase, or there could be more enzymes (or enzymes that are more active) to transport electrons to nitrogenase. The former seems unlikely, since Azotobacter uncouples respiration from energy generation at higher oxygen levels, so extra respiration doesn't necessarily mean more energy. And extra enzymes seems unlikely too, based on the protein results.
So it seems like increased nitrogenase activity might be due to increased transport of electrons to the enzyme, though it's not clear how that happens.
Reference:
Klugkist, J., Haaker, H. & Veeger, C. Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii. European Journal of Biochemistry 155, 41–46 (1986).
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