217 - Short-Term Effect of Ammonium Chloride on Nitrogen Fixation by Azotobacter vinelandii and by Bacteroids of Rhizobium leguminosarum

The question here is how fixed nitrogen regulates nitrogenase in Azotobacter vinelandii. Adding ammonium above a certain concentration immediately shuts off nitrogen fixation, but the organism doesn't have the DRAT/DRAG enzyme system for ADP-ribosylation of the nitrogenase for posttranscriptional regulation that other organisms have.

This shut-off in A. vinelandii is not due solely to repression of nitrogenase synthesis, since that would not show an effect so quickly. To test alternative mechanisms, this paper took cells grown without fixed nitrogen, suspended them in buffer with a carbon source (sucrose or succinate) and oxygen, and measured acetylene reduction before and after adding ammonium. They also measured nucleotide phosphate levels and respiration.

As expected, adding ammonium greatly lowered nitrogen fixation. The ratio of ATP to ADP seemed mostly to increase though, so lack of ATP didn't seem to cause the inhibition. So what's left? Maybe lack of reducing equivalents used to reduce nitrogen.

Then they did a very confusing and poorly explained experiment (Fig 2) showing uptake of ammonium, which doesn't seem very surprising or informative. However, apparently adding a compound that dissipates membrane potential (valinomycin) caused the opposite effect (loss of ammonium), and another (nigericin) does the opposite (enhancing uptake), so that's somewhat interesting.

They measured proton motive force by a gradient of lipophilic cations, such as tetraphenylphosphonium, across the membrane. They also used a weak acid, 5,5-dimethyloxazolidine-2,4-dione to measure the pH gradient. The sum of these measurements was the proton motive force, in mV.

What they saw was that increasing amounts of ammonium chloride decreased the total proton motive force, but not the pH gradient part or the internal pH of the cells. So the electrical gradient was decreased. This could be because taking up a lot of ammonium, a cation, affects the charge of the membrane. I wonder if they could've tested this further using a different cation though.

Anyway, that's pretty interesting. Nitrogen fixation requires a fairly delicate redox balance, but this can be beneficial for the cells if they use imbalance to regulate their metabolism.

Reference:

Laane, C., Krone, W., Konings, W., Haaker, H. & Veeger, C. Short-Term Effect of Ammonium Chloride on Nitrogen Fixation by Azotobacter vinelandii and by Bacteroids of Rhizobium leguminosarum. Eur J Biochem 103, 39–46 (1980).

183 - Studies on the mechanism of electron transport to nitrogenase in Azotobacter vinelandii

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:

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).

161 - On the Efficiency of Oxidative Phosphorylation in Membrane Vesicles of Azotobacter vinelandii and of Rhizobium leguminosarum Bacteroids

The authors wanted to study oxidative phosphorylation (generating ATP through respiration) in Azotobacter vinelandii and another related to oxygen, nitrogen fixation, and hydrogen oxidation by hydrogenase.

What They Saw
They grew A. vinelandii strain OP (aka CA) in chemostats, at D = 0.1 h^-1, limited in oxygen or nitrogen, then broke up the cells and isolated membrane vesicles anaerobically.

The P/O ratio is how much ATP is produced by moving 2 electrons through the electron transport chain to reduce one oxygen atom. The pattern of P/O over a range of oxygen levels is similar for different electron donors (NADH, malate, hydrogen, and NADH + hydrogen): it goes up to a peak, then falls as dissolved oxygen levels rise above the limit of detection. ATP production mostly levels off near that peak too (except with hydrogen, where it declines). The peak occurs at higher oxygen levels with NADH and malate than with hydrogen though. The height of the peak is 0.7 (ATPs per O reduced) for NADH and hydrogen, but only 0.5 with malate.

They tried adding acetylene up to 20%, but didn't see any indication that hydrogenase was inhibited. Previous studies showed that 40% acetylene was required to show inhibition, so it's not surprising. They also found that the hydrogen branch seemed to be very efficiently coupled to phosphorylation, and doesn't seem to involve flavoproteins.

What This Means
The lower values from hydrogen at high oxygen is probably due to inactivation of the hydrogenase, which is sensitive to oxygen.

The fall in P/O ratio is likely due to excess oxygen going through a different cytochrome branch, with cytochrome b to cytochrome d, which isn't involved in ATP production, so respiration and ATP are decoupled.

It also appears that the electrons from hydrogen oxidation don't travel through the same branch of the electron transport chain as electrons from carbon sources; hydrogenase has its own branch.

Reference:

Laane, C., Haaker, H. & Veeger, C. On the Efficiency of Oxidative Phosphorylation in Membrane Vesicles of Azotobacter vinelandii and of Rhizobium leguminosarum Bacteroids. European Journal of Biochemistry 97, 369–377 (1979).