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).

571 - Oxygen effects on the nickel- and iron-containing hydrogenase from Azotobacter vinelandii

This study looks at how oxygen affects the uptake hydrogenase of Azotobacter vinelandii.

What They Saw
They grew A. vinelandii OP (aka CA) and purified its membrane-bound hydrogenase. When purified anaerobically, it was fully active with an electron acceptor other than oxygen (methylene blue or benzylviologen). Added oxygen appeared to inhibit this reduction of methylene blue, and this was noncompetitive inhibition (adding extra methylene blue didn't relieve it).

When oxygen was removed by adding an oxygen-binding protein (leghemoglobin), the inhibition was reversed and activity recovered.

They claim the membrane-associated hydrogenase in these experiments was incapable of reducing the oxygen; it's not clear if being more capable would change the results, but it seems likely.

There was also slower, irreversible inactivation, shown by adding oxygen to an assay and adding enough dithionite to consume all of it to remove any effect of reversible inhibition. Over time, the enzyme lost activity, whether aerobically or anaerobically purified. Purified enzyme lost more activity more quickly than membrane-bound. It seemed like activity was only lost when the enzymes were exposed when active, but simply activating them didn't reproduce the effect. It was a confusing assay.

They did find that adding hydrogen could provide protection from inactivation, up to almost 100% protection, but neither hydrogen nor oxygen was consumed during this process. Super weird.

Finally, carbon monoxide didn't help protect the enzyme from oxygen at all, nor did affect protection by hydrogen.

What This Means
It's interesting, but probably not that important physiologically. A. vinelandii is capable of withstanding high levels of oxygen, and such high levels are just as likely to inhibit the nitrogenase which produces the hydrogen as the hydrogenase which consumes it. It might be interesting to study whether oxygen inhibits the oxidation of added hydrogen though.

Reference:

Seefeldt, L. C. & Arp, D. J. Oxygen effects on the nickel- and iron-containing hydrogenase from Azotobacter vinelandii. Biochemistry 28, 1588–1596 (1989).

570 - Kinetic analysis of the interaction of nitric oxide with the membrane-associated, nickel and iron-sulfur-containing hydrogenase from Azotobacter vinelandii

This study looked at the effect of nitric oxide (NO) on Azotobacter vinelandii's uptake hydrogenase.

What They Saw
They isolated hydrogenases from cells but didn't separate them from the membrane, because that made them too sensitive to oxygen. When activated in a reducing environment and then exposed to NO, hydrogenase activity was inhibited, but this inhibition could be almost completely reversed by adding iron EDTA, which reacts with NO. The more NO, the more inhibition (relative to the no NO control).

When NO was added and the hydrogenase wasn't active, the inactivation was irreversible; the longer the exposure, the more the inhibition, but it took larger amounts of NO to get the same amount of inhibition as the reversible kind. Hydrogen or carbon monoxide didn't protect against this inactivation; hydrogen even enhanced the effect.

Reference:

Hyman, M. R. & Arp, D. J. Kinetic analysis of the interaction of nitric oxide with the membrane-associated, nickel and iron-sulfur-containing hydrogenase from Azotobacter vinelandii. Biochim Biophys Acta 1076, 165–172 (1991).

569 - Hydrogen-oxidizing electron transport components in nitrogen-fixing Azotobacter vinelandii

This study looks at oxidation of hydrogen by Azotobacter vinelandii's uptake hydrogenase, and which proteins are involved in the electron transport chain.

What They Saw
They grew A. vinelandii CA fixing nitrogen, and isolated the membrane fraction from the cells. They looked at oxygen uptake, and saw that unless there were oxidizable substrates, there was no consumption of oxygen, which makes sense. Hydrogen fulfilled the requirement though, and there were two hydrogen molecules taken up for each molecule of oxygen, which makes sense: two hydrogen atoms for each atom of oxygen, to make H₂O.

Using spectrophotometry, they observed peaks that occurred when components in the membrane were reduced with hydrogen, malate, or dithionite. Hydrogen affected cytochrome d (showing a peak at 627nm), b (shoulder at 559nm) and c (peak at 550), but not a (595). The other reductants affected b a lot more, and a somewhat.

With carbon monoxide added, hydrogen only reduced cytochrome d. The others showed a new peak, cytochrome o, at 417nm, but hydrogen didn't. The same seemed true with low levels of cyanide; so hydrogenase's terminal oxidase seems to be cytochrome d type. Not really sure how they prevented these inhibitors from inhibiting the hydrogenase itself, like they seem to in other studies.

Reference:

Wong, T. Y. & Maier, R. J. Hydrogen-oxidizing electron transport components in nitrogen-fixing Azotobacter vinelandii. J. Bacteriol. 159, 348–352 (1984).

525 - Hydrogen-mediated mannose uptake in Azotobacter vinelandii

This study looked at Azotobacter vinelandii's ability to use hydrogen gas to power its uptake of the sugar mannose.

What They Saw
They grew A. vinelandii CA in Burk broth but with mannose instead of glucose or sucrose, and either hydrogen or argon in the atmosphere (along with nitrogen and oxygen). They used ¹⁴C mannose to observe its uptake via the radioactivity of the isotope.

The increase in radioactivity from mannose activity was a lot higher in cells given hydrogen than those without, up to 5-fold.

They tried inhibiting respiration to see if that was related to this effect, and found that usually by inhibiting respiration, they could inhibit the increased mannose uptake, so it seems to be respiration-dependent rather than some sort of regulatory effect.

So this seems to be another of hydrogen's possible roles in the energy metabolism of Azotobacter.

Reference:

Maier, R. J. & Prosser, J. Hydrogen-mediated mannose uptake in Azotobacter vinelandii. J. Bacteriol. 170, 1986–1989 (1988).

516 - Role of magnesium adenosine 5'-triphosphate in the hydrogen evolution reaction catalyzed by nitrogenase from Azotobacter vinelandii

Nitrogenase evolves hydrogen in a reaction that depends on ATP. The amount of ATP varies from 2 ATP per electron to over 20 (supposedly). The 4Fe-4S cluster in the dinitrogenase reductase donates one electron to the dinitrogenase, binding 2 ATP to do so. It seems like the ATP-powered flow of electrons can determine how many go toward hydrogen and how many toward nitrogen fixation. This study wanted to see if powering this flux was ATP's only role.

What They Saw
They purified enzyme from Azotobacter vinelandii and separated the two components, then mixed them with MgATP in in vitro assays. They observed that there was a burst of rapid ATP hydrolysis at first, which leveled off and remained fairly constant for the rest of the time. It seemed like the rate was the same as the rate of electron transfer between components. They confirmed the rate of about 2 ATP per electron; this makes sense with the dinitrogenase accepting two electrons at a time, one for each of its molybdenum atoms. The rate depended on the concentration of ATP, which makes sense and indicates that ATP is essential. This is also true when measuring the amount of hydrogen produced.

What This Means
They appeared to succeed in their goal of showing that ATP's only role in this reaction was powering the electron transfer, though some ATP can be used up to no purpose in some conditions.

Reference:

Hageman, R. V., Orme-Johnson, W. H. & Burris, R. H. Role of magnesium adenosine 5’-triphosphate in the hydrogen evolution reaction catalyzed by nitrogenase from Azotobacter vinelandii. Biochemistry 19, 2333–2342 (1980).

515 - Hydrogen Uptake and Methylene Blue Reduction Activities of Hydrogenase in Azotobacter agile

This study used tritium (³H, a radioactive isotope of hydrogen) uptake to look at hydrogenase and nitrogenase activity in Azotobacter agile (aka A. agilis I think).

What They Saw
The tritium was ditritium gas, similar to dihydrogen. They purified and fractionated protein from the bacteria and tested the fractions for tritium uptake, methylene blue reduction, and acetylene reduction. They found that acetylene reduction and the other two were found in separate fractions (makes sense; one's nitrogenase and the others hydrogenase). Tritium uptake could be stimulated with ATP somehow.

Tritium uptake and methylene blue reduction mostly went together in terms of fractionation, but there was a little of the latter in some fractions where the former wasn't observed. So there could be something else reducing methylene blue.

They found that carbon monoxide (CO) inhibited tritium uptake, though it was less inhibitory when ATP was present.

What This Means
This supports the idea that nitrogenase produces hydrogen while hydrogenase oxidizes it and reduces electron acceptors such as methylene blue. It's weird that ATP should stimulate that though, and also weird that some fractions had reduction activity but not hydrogen oxidation.

Reference:

Suzuki, T., Maruyama, Y. & Nakamura, M. Hydrogen Uptake and Methylene Blue Reduction Activities of Hydrogenase in Azotobacter agile. Agricultural and Biological Chemistry 43, 2067–2073 (1979).

454 - Activity of the H2-oxidizing hydrogenase in different N2-fixing bacteria

Despite some studies suggesting that hydrogen stimulates hydrogenase, other data suggested it does not. So the people who generated this data did this study on various species, including Azotobacter vinelandii CA, and claimed that low oxygen stimulated hydrogenase activity.

What They Saw
They grew A. vinelandii with ammonium chloride and measured hydrogenase activity with different electron acceptors (oxygen, methylene blue, etc). As A. vinelandii grew, it used up the dissolved oxygen, and hydrogenase activity went up but then back down after the oxygen was gone (when oxygen or iron cyanide were the electron acceptors, it went to zero; otherwise it didn't go all the way to zero). This was all in the presence of ammonium.

What This Means
Based on other studies, I wouldn't expect much activity from hydrogenase in general when growing with fixed nitrogen. I'm not sure how to interpret these results, especially with electron acceptors other than oxygen, but I guess it would make sense if hydrogenase were somewhat downregulated in low-oxygen conditions, even if other acceptors were present.

Reference:

Pinkwart, M., Bahl, H., Reimer, M., Wölfle, D. & Berndt, H. Activity of the H₂-oxidizing hydrogenase in different N₂-fixing bacteria. FEMS Microbiology Letters 6, 177–181 (1979).

453 - Direct mass-spectrometric determination of the relationship between respiration, hydrogenase and nitrogenase activities in Azotobacter chroococcum

This study looked at hydrogen and its relationship to different enzymatic processes in Azotobacter chroococcum.

What They Saw
The organism was grown in continuous culture with limited oxygen, 5% glucose. Samples were removed and sparged with different mixtures of argon, oxygen, and deuterium. Gases are measured by mass spectrometer.

Their figure is pretty confusing, poorly designed, and poorly described, but as far as I can tell, when they added either 80% argon with 20% oxygen or 70% argon, 20% oxygen, and 10% deuterium, the oxygen goes down to near zero within about 5 minutes either way, at which point hydrogen production starts increasing and deuterium uptake slows down or stops. So it seems like oxygen is required for hydrogenase to work.

They tried again with the addition of some carbon monoxide and acetylene to inhibit hydrogenase. This didn't really change the oxygen consumption, but deuterium consumption was a lot lower. Hydrogen evolution was the same.

What This Means
The deuterium consumption is by hydrogenase, of course, and it seems like oxygen is a necessary electron acceptor for it to function (in the absence of something else). But it seems like the increase in hydrogen evolution is not because hydrogenase stopped working, but rather because nitrogenase started, as oxygen stopped interfering. This is something we've seen before.

Oxygen is important for many things: it provides energy by accepting electrons, powering the nitrogenase and allowing more hydrogen production. It accepts electrons from hydrogenase, enabling hydrogen oxidation. It inhibits nitrogenase, reducing hydrogen production. Pretty confusing.

Reference:

Lespinat, P.-A., Gerster, R. & Berlier, Y. Direct mass-spectrometric determination of the relationship between respiration, hydrogenase and nitrogenase activities in Azotobacter chroococcum. Biochimie 60, 339–341 (1978).

295 - Sequences, organization and analysis of the hupZMNOQRTV genes from the Azotobacter chroococcum hydrogenase gene cluster

This study focuses on the hydrogenase genes in Azotobacter chroococcum. They had already found hupSL encoding the structural genes (equivalent to A. vinelandii's hoxKG I guess), and then the accessory genes hupABYCDE further downstream (homologous to A. vinelandii's hypABFCDE). Now, in between, are the hupZMNOQRTV genes, completing the 16-gene operon.

What They Saw
This set of genes seems to be homologous to A. vinelandii's hoxZMLOQRTV string, making the whole operon very similar in both organisms.

As in previous studies, HupZ (and its analog, HoxZ) seems to be an electron carrier in the membrane, possibly a cytochrome. When they knocked it out, they observed similar results to 070: hydrogenase could oxidize hydrogen with methylene blue as an electron acceptor, but not with oxygen, so HupZ seems to be part of the transport chain to oxygen.

Based on comparison with homologs in other organisms, HupM may help the hydrogenase attach to the membrane, or with processing the subunits. It's unclear. The other genes may be involved in processing or metal stuff. HupR may be another electron-carrying protein. Further study is required.

Reference:

Du, L., Tibelius, K. H., Souza, E. M., Garg, R. P. & Yates, M. G. Sequences, organization and analysis of the hupZMNOQRTV genes from the Azotobacter chroococcum hydrogenase gene cluster. Journal of Molecular Biology 243, 549–557 (1994).

071 - Two open reading frames (ORFs) identified near the hydrogenase structural genes in Azotobacter vinelandii, the first ORF may encode for a polypeptide similar to rubredoxins

This study looked at the genetics of the two open reading frames (ORFs) near the hydrogenase structural genes (hoxZM).

What They Saw
They sequenced the ORFs and compared to known genes. hoxZ seemed to have most homology with genes encoding proteins called rubredoxin from other species. These are typically small proteins that play roles in electron transport, which makes sense. And that's all.

Reference:

Chen, J. & Mortenson, L. Two open reading frames (ORFs) identified near the hydrogenase structural genes in Azotobacter vinelandii, the first ORF may encode for a polypeptide similar to rubredoxins. Biochim Biophys Acta 1131, 122–124 (1992).

070 - The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase

Here they wanted to look into the hoxZ gene more closely. Previous studies suggested that the product might be involved in electron transport for the hydrogenase.

What They Saw
They grew Azotobacter vinelandii DJ (an easy-to-transform strain) and knocked out hoxZ and hoxKG by transformation and screening for hydrogen production.

Comparing the hoxZ mutant to DJ (positive control) and the hoxKG mutant (negative control), they observed an intermediate rate of hydrogen oxidation, so there seemed to still be some activity. DJ consumed nearly all the hydrogen, and hoxKG consumed very little (the graph showed a decrease but it was apparently because gas leaked out of the vial, so it's a good thing they had good controls!).

Then they tried measuring short-term hydrogen oxidation with different electron acceptors: oxygen or methylene blue. DJ quickly oxidized all the hydrogen while reducing oxygen or methylene blue, as expected. Both mutants didn't show activity with either acceptor at first, despite the difference in the previous assay. But then they added sodium dithionite (a powerful reducer of oxygen) and more methylene blue, and the hoxZ mutant showed up to 80% of the activity of DJ. As far as I can tell, the hoxKG didn't show the same effect when they gave it the same treatment, but they don't say that explicitly. But it seems like the hydrogenase needs to be activated somehow, as by dithionite.

These results were confirmed by observing methylene blue color change too; DJ quickly started oxidizing hydrogen, but the hoxZ mutant did too after a longer lag period.

When they isolated membrane-bound hydrogenase from cells (still embedded in membranes), even DJ needed activation with dithionite. hoxKG mutants had no activity in any case, of course. But hoxZ mutant had more activity in the soluble supernatant portion than DJ did, at least when membranes were isolated aerobically; it seemed like lack of hoxZ led to more soluble enzyme. But it had low activity in general so this conclusion was uncertain. Though membrane-bound activity in general was higher when isolated anaerobically, and they didn't measure soluble activity in that case for some reason. So HoxZ may help stabilize hydrogenase in the presence of oxygen.

The increased presence of detached hydrogenase in the mutant was not confirmed by Western blot, so it seems like an artifact.

What This Means
HoxZ seems to have a role in shuttling electrons between hydrogenase and oxygen, though there may be other components involved in this path. It's possible that when HoxZ is missing, another acceptor can take the electrons, but isn't as good at it.

It also may be involved in activating the enzyme (which requires removing oxygen and providing reduction); somehow hydrogen is not enough for this. And it may help stabilize the hydrogenase to keep oxygen from inactivating it, maybe also using its role as electron transporter.

Reference:

Sayavedra-Soto, L. A. & Arp, D. J. The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase. J. Bacteriol. 174, 5295–5301 (1992).

052 - Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii

This study looks at the hydrogenase-related genes in Azotobacter vinelandii in between the structural genes and the hyp genes.

What They Saw
They already knew about the structural genes, hoxKG. So they got some overlapping fragments containing those and sequences downstream of them, and sequenced them. They found 5 new open reading frames (ORFs), ORF3-7. They named these hoxZMLOQ. There was also part of an eighth at the end. All of them were homologous to hydrogenase genes in other organisms.

Then they knocked out each gene by inserting kanamycin or kan+lacZ cassettes, though probably some of these insertions affected other genes (polar effects) so it wasn't possible to study each gene individually. Each insertion abolished hydrogen oxidation. All 5 new ORFs seem to be involved in subunit processing, as the unprocessed form was present in higher proportion (or alone) in their knockouts.

The knockouts didn't seem to be impaired in growth, even when fixing nitrogen, though they might show more effect in certain conditions (like carbon limitation).

HoxZ seems to be a membrane protein, possibly a cytochrome, so part of the electron transport chain. HoxL seems like it might be involved in metal-binding somehow.

What This Means
Knowing more of the sequence of the operon helps to study it. The whole thing, hox and hyp, is pretty big, kinda surprising for an enzyme that doesn't seem to help the organism in some conditions. Probably in nature, the likelihood of encountering carbon-limited circumstances is much higher, so having an uptake hydrogenase is important.

Reference:

Menon, A. L., Mortenson, L. E. & Robson, R. L. Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii. J Bacteriol 174, 4549–4557 (1992).

049 - Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii

Azotobacter has a hydrogenase. Some organisms have hydrogenases that produce hydrogen, or sometimes produce and sometimes break down (reversible). Azotobacter's had not been observed to break down hydrogen (so was considered unidirectional). This study purified the membrane-bound uptake hydrogenase from A. vinelandii and tested its properties.

What They Saw
They measured activity of purified enzyme with an electrode measuring hydrogen oxidation in the presence of methylene blue dye (an electron acceptor).

They measured stability of the enzyme in the presence of oxygen, and found that crude extracts were very stable (and could go for weeks without losing activity), but the more pure the preparation, the less oxygen-tolerant it was: the most pure lost half its activity in 20 minutes at 20% oxygen. This inactivation was irreversible.

With a very good electron donor (methyl viologen), the hydrogenase could produce hydrogen. The highest rate they saw was 3.4 μmol hydrogen per minute per mg protein, which peaked at a fairly low pH (around 4). The rate was almost 0 closer to neutral. Also, the presence of hydrogen in the environment inhibits its production by the enzyme.

Even when electron acceptors were not present, the enzyme could combine one deuterium from D₂ with one hydrogen from water to make HD.

The enzyme seemed to be good at donating electrons to acceptors with positive mid-point potentials but not to negative ones, so it seems to have a higher potential than reversible hydrogenases.

Reference:

Kow, Y. W. & Burris, R. H. Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii. J Bacteriol 159, 564–569 (1984).

215 - Respiratory protection of nitrogenase in Azotobacter vinelandii

This study is pretty similar to 214, on how oxygen levels influence the respiratory chain of Azotobacter vinelandii.

What They Saw
They grew cells in batch at low aeration, and then increased the aeration to expose cells to excess oxygen.

The results were similar to 214: when aeration increased, cells stopped growing until respiration had ramped up and leveled off. Despite the large increase in respiration, cellular ATP levels dropped 40%. P/O ratios decreased as respiration increased to accommodate the increased oxygen, at least for NADH dehydrogenase.

They tried adding chloramphenicol again, since last time it didn't affect the growth lag, but this time observed that it lessened the increase in respiration about half, and prevented cytochrome a₂ and other dehydrogenase increases.

What This Means
The M/N ratio, meaning maintenance M (moles ATP consumed per weight of cells over time) over phosphorylating efficiency N (P/O ratio x 2), explains doubling of respiratory activity upon increase in oxygen only if M increases or N decreases. It doesn't seem like M does increase, so N seems to be decreasing, because respiration uncouples from phosphorylation.

A. vinelandii seems to have different branches in its respiratory chain, which allows it to tolerate different and suddenly changing levels of oxygen.

Reference:

Jones, C. W., Brice, J. M., Wright, V. & Ackrell, B. A. C. Respiratory protection of nitrogenase in Azotobacter vinelandii. FEBS Letters 29, 77–81 (1973).

214 - The Respiratory System of Azotobacter vinelandii 2. Oxygen Effects

This study looks at how oxygen levels influence the respiratory chain in Azotobacter vinelandii.

What They Saw
They grew cells with high or low aeration (based on volume of culture in the same size flask), then isolated respiratory membranes and measured P/O ratios.

The cells grew and quickly used up all the dissolved oxygen. With high aeration, they grew much faster and leveled off once the oxygen was gone, and with low aeration the growth was slower (not even really logarithmic) but continued long after the oxygen was gone. Respiratory activities were 2-5x higher when cells were growing logarithmically with excess oxygen than when oxygen was limited.

If they suddenly increased the aeration when oxygen had run out, respiratory activity increased back up to high levels (1000 μl/h/mg dry weight). Cells didn't start growing again until it had leveled off. This lag was the same when chloramphenicol (which inhibits protein synthesis) was present, suggesting that it wasn't due to the synthesis of new enzymes for respiration.

When oxygen was being consumed, cytochrome and oxidase levels were pretty constant (except cyt o oxidase, which increased), but when oxygen ran out, levels of c₄, c₅, and b₁ increased quickly and a₂ more slowly. Cytochrome o oxidase and a₂ oxidase also increased a lot, a₁ less so. The increase of o was fastest and greatest.

P/O ratios were similar to those seen before at maximum, but they didn't reach this maximum until oxygen was mostly used up late in the logarithmic growth phase, at least for NADH dehydrogenase.

With low aeration, cytochrome levels were pretty constant (since oxygen ran out almost immediately), increasing just a little, except for cyt a₂ oxidase which went up pretty constantly. P/O ratios were pretty level too, and fairly high, similar to when oxygen ran out in high aeration.

What This Means
This kind of pattern fits in well with those seen in other obligate aerobes. Cytochromes probably increase during oxygen limitation to try to compensate for the limitation. But the low P/O ratios at high oxygen makes sense in light of respiratory protection; respiration is uncoupled from energy generation.

Reference:

Ackrell, B. A. C. & Jones, C. W. The Respiratory System of Azotobacter vinelandii 2. Oxygen Effects. Eur. J. Biochem. 20, 29–35 (1971).

352 - The Respiratory System of Azotobacter vinelandii: 1. Properties of Phosphorylating Respiratory Membranes

Similar to 161, this study looks at elements of Azotobacter vinelandii's respiratory chain and P/O ratios thereof.

What They Saw
They isolated respiratory membranes (basically pieces of cell with the respiratory chain intact but not much else, I think) and looked at oxygen reduction with different electron donors.

They saw P/O levels up to 1.1 with NADH and about 0.7 with malate, higher than in 161.

Reference:

Ackrell, B. A. C. & Jones, C. W. The Respiratory System of Azotobacter vinelandii: 1. Properties of Phosphorylating Respiratory Membranes. Eur. J. Biochem. 20, 22–28 (1971).

198 - Oxidation of nitrogenase iron protein by dioxygen without inactivation could contribute to high respiration rates of Azotobacter species and facilitate nitrogen fixation in other aerobic environments

This study looks at oxygen interactions with dinitrogenase reductase (DNR) in Azotobacter chroococcum (and Klebsiella pneumoniae) to see if it's possible that the enzyme can be oxidized without being totally inactivated.

When the DNR was present in high enough levels relative to the amount of oxygen (4-fold molar excess), it was protected from inactivation by oxygen: it seemed to reduce superoxides to peroxide (and then water if catalase doesn't get to it first) before the reactive oxygen species could harm it. Superoxide seems to be the harmful form of oxygen for DNR. They suggest this helps with high respiration rates (i.e. consumption of oxygen). They calculated that if 10% of the cell's protein were DNR, it could account for pretty much all of the oxygen consumption. This is unlikely, but it could still be a significant part of respiratory protection. They call this "autoprotection."

Reference:

Thorneley, R. N. & Ashby, G. A. Oxidation of nitrogenase iron protein by dioxygen without inactivation could contribute to high respiration rates of Azotobacter species and facilitate nitrogen fixation in other aerobic environments. Biochem J 261, 181–187 (1989).

197 - Superoxide dismutase and catalase in Azotobacter vinelandii grown in continuous culture at different dissolved oxygen concentrations

Considering how oxygen-sensitive nitrogenase is, it might be expected that enzymes specifically involved in oxygen detoxification (such as catalase and superoxide dismutase (SOD)) might be involved in protecting such oxygen-sensitive enzymes, especially since ramping up respiration in response to increased oxygen might also ramp up the production of reactive oxygen species. This study investigates the activity of SOD and catalase in Azotobacter vinelandii at different oxygen levels.

What They Saw
They grew A. vinelandii OP (aka CA) in chemostats with different levels of oxygen, with 3 or 15 g/L sucrose. They extracted enzymes from samples and assayed them for SOD or catalase activity, and also by electrophoresis.

As oxygen saturation increased from 1% to 90%, SOD activity increased linearly (when standardized to total protein); the increase was slightly faster at lower oxygen when standardized by number of cells (probably because cell size increases as oxygen increases, 098). The sucrose concentration didn't affect things. When cells were given ammonia, SOD activity was about 2x lower.

Based on electrophoresis, they concluded that the SOD is iron-containing, rather than manganese. They tried adding manganese but still didn't see any Mn-SOD.

It didn't seem like catalase activity increased with increasing oxygen, standardized by protein. The increase standardized by cells was much more apparent.

What This Means
It seems like SOD at least might contribute to A. vinelandii's protection of its nitrogenase enzyme from oxygen.

Reference:

Dingler, C. & Oelze, J. Superoxide dismutase and catalase in Azotobacter vinelandii grown in continuous culture at different dissolved oxygen concentrations. Arch. Microbiol. 147, 291–294 (1987).

195 - Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations

Obviously oxygen levels have a big effect on nitrogen-fixing Azotobacter. This study looked into specific effects on levels of different proteins related to nitrogen fixation, at different oxygen levels.

What They Saw
They grew A. vinelandii OP (aka CA) in chemostats with 3 g/L sucrose at different oxygen levels (or dilution rates). They measured nitrogenase activity and purified nitrogenase components as well as flavodoxin and FeSII protein.

Unlike in previous studies (111, 165, 183), increasing the oxygen levels didn't seem to reduce nitrogenase activity (in steady state), at least not in the range they tried (except maybe a little at low levels). At all different levels, nitrogenase activity (i.e. acetylene reduction) correlated only with dilution rate.

With Western blots, they found that levels of different nitrogenase proteins (and others) didn't really vary much across different oxygen levels; they were always about 10% of total protein. And if the activity doesn't vary, this means the proportion of active enzyme is constant too. The enzyme activity does match previous numbers though (106).

Trying to grow cells in ammonia, the two components of nitrogenase disappeared, flavodoxin decreased, but FeSII remained constant.

They tried measuring nitrogenase levels at different dilution rates and oxygen levels. At the lowest D, nitrogenase increased as oxygen increased, but it remained pretty constant at higher rates (as shown before). In contrast to the earlier data though, levels didn't seem to increase consistently with increasing D; it could be that the same quantity of enzyme is less active at lower D, probably related to the flow of electrons to the enzymes.

Then they tried inhibiting protein synthesis with chloramphenicol, at either low or high oxygen. The culture started to wash out, of course. Levels of the four proteins didn't change much with oxygen or with time passed after addition of the antibiotic, but nitrogenase activity decreased greatly over time (while respiratory activity didn't change much). This wasn't due to damage to the nitrogenase components; nitrogenase extracts had just as much activity as cells grown without chloramphenicol. Somehow the activity is inhibited.

What This Means
As suggested before, it seems like the absolute presence/concentration of oxygen doesn't determine its toxicity so much as the ratio of oxygen to availability of energy and reducing equivalents. So if there's enough energy and electrons available to nitrogenase, it can keep going up to high levels of oxygen.

Reference:

Dingler, C., Kuhla, J., Wassink, H. & Oelze, J. Levels and activities of nitrogenase proteins in Azotobacter vinelandii grown at different dissolved oxygen concentrations. J Bacteriol 170, 2148–2152 (1988).