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).

062 - Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability

This study looked at superoxide dismutase in Azotobacter vinelandii, an Fe-SOD encoded by sodB, and its importance.

What They Saw
Running proteins on a gel testing for SOD activity, they saw two bands: one was Fe-SOD and the other CuZnSOD (which sits in the periplasm). They tried knocking out sodB from A. vinelandii by introducing a kanamycin resistance cassette, and isolated a kan-resistant strain, but it appeared to have two copies of sodB (only one of which was knocked out). They tried increasing the concentration of kanamycin (presumably to force the strain to have multiple copies of the resistance gene), and got one that grew slowly at 100x more kanamycin than I use, but they couldn't get rid of the SOD. Seems like it's essential.

This also supports the idea that A. vinelandii can have multiple copies of its chromosome, since they saw multiple PCR products from the same locus, with and without the resistance marker. The genome only has one copy of sodB, so there must be multiple genome copies.

Reference:

Qurollo, B. A., Bishop, P. E. & Hassan, H. M. Characterization of the iron superoxide dismutase gene of Azotobacter vinelandii: sodB may be essential for viability. Can. J. Microbiol. 47, 63–71 (2001).

663 - Azotobacter vinelandii Vanadium Nitrogenase: Formaldehyde Is a Product of Catalyzed HCN Reduction, and Excess Ammonia Arises Directly from Catalyzed Azide Reduction

The V nitrogenase is similar to but distinct from the Mo nitrogenase in various ways. This study tests its activity and patterns with cyanide and azide as substrates; previously this had only been tested with the Mo nitrogenase.

What They Saw
As with the Mo version, cyanide inhibited hydrogen production and the overall electron flux through the V nitrogenase, though less than with the Mo nitrogenase; more cyanide had more effect, up to 75% inhibition of hydrogen and electrons at 50 mM cyanide. Methane formation from cyanide increased at first, and then decreased with more cyanide, while ammonia continued to increase. They didn't measure methylamine in most conditions though, so the electron flux numbers might be off. With low cyanide, there was significant methylamine relative to the methane, about 0.66 to 1, higher than the Mo nitrogenase; this ratio seems to increase to 1:1 as methane decreases.

Interestingly, the enzyme also produces formaldehyde from cyanide. It's not clear whether the Mo version does too and it's too hard to detect, or if it just doesn't. It can be tricky, since other components in the reaction react with it, and cyanide inhibits the Mo enzyme a lot more.

Azide inhibited hydrogen production from the V nitrogenase about 50%. Hydrazine was more of a product from azide than it is for the Mo nitrogenase, relative to dinitrogen and ammonia. Overall activity was less than the Mo version, which is typical, but also because azide seems to reduce the total electron flux in this version. The ammonia seems to come from azide directly, not from the dinitrogen produced, because adding hydrogen gas (which specifically inhibits nitrogen reduction) didn't change the values.

They tried seeing what adding carbon monoxide (CO) might do to affect these reactions. With azide, it rescued hydrogen production, though not the electron flux, and CO didn't entirely prevent some azide reduction. With cyanide, the effects were similar, except the electron flux inhibition was relieved too.

What This Means
It seems that these chemicals have similar effects on the V nitrogenase as they do on the Mo version, but not completely the same. I wonder, though, about the CO assays: CO can be a substrate for the V nitrogenase, reduced mostly to ethylene but also some propylene and methane; how did this affect their assays?

Reference:

Fisher, K., Dilworth, M. J. & Newton, W. E. Azotobacter vinelandii Vanadium Nitrogenase: Formaldehyde Is a Product of Catalyzed HCN Reduction, and Excess Ammonia Arises Directly from Catalyzed Azide Reduction. Biochemistry 45, 4190–4198 (2006).

615 - ATP-dependent reduction of azide and HCN by N2-fixing enzymes of Azotobacter vinelandii and Clostridium pasteurianum

This study again looks at nitrogenase in Azotobacter vinelandii and also Clostridium pasteurianum and how it affects/is affected by stuff like azide and cyanide.

What They Saw
They were looking at cell extracts, protein activity in vitro. Nitrogenase can reduce azide to ammonia (and other stuff); carbon monoxide can inhibit this process completely, and nitrous oxide (N₂O) partially. More ammonia is formed from azide than from dinitrogen even. However, each azide molecule is reduced to one N₂ and one NH₃, so two thirds of the nitrogen becomes gas instead of being fixed. Presumably if left long enough, all the N₂ would become ammonia though.

In terms of amount of hydrogen produced with azide present, it varied from between 28 and 35% of what was produced with no substrate present, similar to dinitrogen.

With cyanide, the enzyme reduces it to methane, ammonia, and methylamine (CH₃NH₂). CO inhibits this reaction completely, and azide and nitrous oxide partially. When nitrogen gas and cyanide are present, less base is formed than when only nitrogen is present, so there is competition.

In terms of hydrogen again, with cyanide there was 17% as much hydrogen as with no substrate, but it also seemed like cyanide reduced the electron flux to the enzyme overall, to about 30% of when fixing nitrogen. If CO was added with cyanide, it restored hydrogen production up to 58% of what it was with neither. This is pretty consistent with previous results.

They also tried methylamine reduction directly, but it was a very poor substrate. The same was true of cyanate (NCO^-), and they didn't detect any reduction of CO to methane. (Though they maybe should've looked for ethylene, since the Mo nitrogenase produces mostly that and none of methane from CO).

What This Means
So nitrogenase can reduce a bunch of things pretty well: nitrogen, nitrous oxide, azide, cyanide, and acetylene. It's an interesting enzyme.

This study has a good summary table of different substrates for nitrogenase, products made from them, and their effects on hydrogen production/electron flux.

Reference:

Hardy, R. W. F. & Knight Jr., E. ATP-dependent reduction of azide and HCN by N₂-fixing enzymes of Azotobacter vinelandii and Clostridium pasteurianum. Biochim Biophys Acta 139, 69–90 (1967).

630 - Diastereomer-dependent substrate reduction properties of a dinitrogenase containing 1-fluorohomocitrate in the iron-molybdenum cofactor

The normal nitrogenase central cofactor contains homocitrate near the central metal atom; it is required for formation of the cofactor. This study looks at compounds similar to homocitrate (diastereomers of fluorohomocitrate) incorporated into the cofactor, and how they affect the enzyme's activity.

What They Saw
They purified enzyme from Klebsiella pneumoniae and Azotobacter vinelandii I think; it's hard to tell because this paper (and others they cite) are really badly written with regard to methods. (They seriously put in a footnote saying "The experimental details of the synthesis of 1-fluorohomocitrate may be obtained from [author initials]." How is that acceptable, especially in a PNAS paper?). Somehow they got FeMo-cofactor with different analogs of homocitrate, and tested their activity with different substrates.

With threo-fluorohomocitrate, there was hardly any nitrogen fixation, similar to with citrate, but with erythro-fluorohomocitrate, there was about 3.5x less than with plain homocitrate, so it was somewhat active. Both were about half as good at acetylene reduction (measured by ethylene), and just as good at cyanide reduction (measured by methane).

In terms of hydrogen production, if there was a difference between plain homocitrate and the fluoros, it wasn't very big; they produced almost as much as the normal one. The same was true of other analogs (homoisocitrate, isocitrate, etc), though some were impaired (such as citrate, producing only about half as much).

The addition of inhibitors, carbon monoxide (CO) or carbonyl sulfide (COS), affected these different enzymes differently. The amount of hydrogen from homocitrate increased (maybe significantly) but decreased some or none for analogs, depending on the analog, up to 63%. The inhibitors reduced acetylene reduction from 35-100% in all cases, and cyanide reduction a little or a lot too.

Cyanide itself affected hydrogen production. (Dang, this paper is so badly written, it's giving me a headache trying to figure it out.) With all tested analogs (and homocitrate), cyanide inhibited hydrogen production 85-95%. Adding CO with cyanide prevented this inhibition with homocitrate and partially with fluorohomocitrate, but not much the other analogs. CO also inhibits cyanide reduction, at least with some analogs (especially the more active ones).

What This Means
This study can tell a lot about the biochemistry of different substrates binding to the enzyme and the enzyme acting on them. I wonder if the results are skewed somewhat because they only measured some of the products, not all possible ones (i.e. might some analog-based cofactors produce ethane from acetylene in addition to ethylene?). In terms of application, even if some of these analogs have desirable properties, it seems like it would be difficult to get them incorporated in vivo.

Reference:

Madden, M. S., Kindon, N. D., Ludden, P. W. & Shah, V. K. Diastereomer-dependent substrate reduction properties of a dinitrogenase containing 1-fluorohomocitrate in the iron-molybdenum cofactor. PNAS 87, 6517–6521 (1990).

610 - A Nitrogen Pressure of 50 Atmospheres does not Prevent Evolution of Hydrogen by Nitrogenase

Nitrogenase always seems to produce at least one hydrogen molecule per nitrogen fixed when fixing nitrogen. This study looked at whether very high pressures of pure nitrogen could prevent this wasteful side reaction.

What They Saw
They purified nitrogenase from Azotobacter vinelandii and put it in a chamber with 50-51 atmospheres of pure nitrogen gas. The chamber was designed so they could start and stop the reaction by tipping the chamber to add reagents that would start or stop it, without changing the atmosphere. Then they measured pressure in the chamber and amount of hydrogen.

Even at the high pressure of nitrogen in this experiment, the amount of hydrogen produced was about one-to-one with the amount of nitrogen fixed. So it must be pretty essential to the reaction.

Reference:

Simpson, F. B. & Burris, R. H. A Nitrogen Pressure of 50 Atmospheres does not Prevent Evolution of Hydrogen by Nitrogenase. Science 224, 1095–1097 (1984).

598 - Nitrogenase-catalyzed reactions

This paper looks at a bunch of possible substrates and reactions that nitrogenase catalyzes.

What They Saw
They extracted nitrogenase from Azotobacter vinelandii. ATP was necessary for any activity, of course; 3 mM was the optimal amount in their in vitro reactions, possibly because of the amount of magnesium ions that were present (5 mM). They saw 1.6 hydrogens per nitrogen fixed, on average, a bit higher than the normal figure of 1; about 33% of the energy flux went to hydrogen (normal figure is 25%).

With azide added, 1 mole azide is made into 1 mole ammonia and 1 mole dinitrogen. Lower levels allowed production of hydrogen instead, but that wasn't investigated much. The same was true with acetylene; both seem better substrates than nitrogen.

Cyanide is reduced to methane and ammonia; optimal concentrations were 2-4 mM. At other concentrations, there are other products formed: ethylene, ethane, methylamine (CH₃NH₂). They don't discuss hydrogen.

However, their calculations of ATP per electron pair transferred were skewed because they didn't measure all the products (i.e. hydrogen gas).

Reference:

Hwang, J. C. & Burris, R. H. Nitrogenase-catalyzed reactions. Biochim Biophys Acta 283, 339–350 (1972).

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).

563 - Construction and Characterization of Hybrid Component 1 from V-Nitrogenase Containing FeMo Cofactor

This study looked at a purified V nitrogenase from Azotobacter vinelandii with the FeMo cofactor instead of FeVco.

What They Saw
They detected only Mo, no V, in the preparation. The activities they saw were pretty weird in some ways: the electron flux going to nitrogen was almost as much as in the Mo nitrogenase (~70%, with the rest going to hydrogen), whereas with the normal FeVco, 50% went to each; in previous studies, V nitrogenase with FeMoco couldn't fix nitrogen at all. On the other hand, with acetylene, almost all electrons went to ethylene in the Mo nitrogenase, whereas only 30-35% did in the V nitrogenase with either cofactor, while 3% went to ethane with FeVco but 10% did with FeMoco, which is consistent with previous studies. So I'm confused.

When they added carbon monoxide, there was inhibition of all nitrogenase versions, as expected.

What This Means
I think something was weird in this study, but I don't know what. It seems very questionable. However, most of the later studies citing this one either accept it without question, or confirm its results, so maybe it's not as questionable as it seems.

Also, it's worth noting that the results only show the electron flux going to each substrate, not the total electron flux, so even if most electrons go to nitrogen in the FeMoco-substituted V nitrogenase, it's still possible that it's fixing a lot less nitrogen overall than either Mo or V versions proper.

Reference:

Moore, V. G., Tittsworth, R. C. & Hales, B. J. Construction and Characterization of Hybrid Component 1 from V-Nitrogenase Containing FeMo Cofactor. J. Am. Chem. Soc. 116, 12101–12102 (1994).

558 - Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii

This study is the first to fully purify the vanadium nitrogenase from Azotobacter vinelandii.

What They Saw
They used strain UW (aka CA) and a nifHDK knockout. It didn't seem like they scrubbed the medium for Mo, but they did test the purified product for metals and didn't see a detectable amount of Mo, only V and Fe (in a 1-to-13 ratio), so I guess it's good.

They did acetylene reduction assays on the V and Mo nitrogenases, and found that 90% of the electrons went to acetylene in the former, but only 12% in the latter. It seems like they only measured ethylene production though, so they might've underestimated the total activity by neglecting the ethane produced by the V nitrogenase.

Under argon, the Mo nitrogenase produced 1.6x more hydrogen than the V version. This was the same ratio as that of nitrogen fixed by each version. I'm not sure how much it's possible to compare these enzymes in vitro though. But it seems like the V nitrogenase is better at nitrogen and hydrogen than at acetylene.

As mentioned, ICP emission spectroscopy didn't detect any metals other than V or Fe: no Mo, Cr, Co, Ni, Cu, or W, so that's good.

What This Means
This shows how the Mo and V nitrogenases are similar in some ways and yet different in others. They work in similar ways and are sensitive to similar things, but the products of their reactions and such are different (though possible substrates seem largely the same). By comparing the two, we can learn more about the process of nitrogen fixation in general.

Reference:

Hales, B. J., Case, E. E., Morningstar, J. E., Dzeda, M. F. & Mauterer, L. A. Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii. Biochemistry 25, 7251–7255 (1986).

555 - Differential Effects on N2 Binding and Reduction, HD Formation, and Azide Reduction with α-195His- and α-191Gln-Substituted MoFe Proteins of Azotobacter vinelandii Nitrogenase

Similar to 554, this study looked at mutated versions of the Mo nitrogenase in Azotobacter vinelandii, but different reactions this time: interactions with nitrogen gas (hooray), with dihydrogen and dideuterium, and with azide (N₃^-).

What They Saw
Adding nitrogen gas revealed that Asn 195 couldn't fix nitrogen; Gln 195 had a slight ability. However, replacing argon with pure nitrogen reduced hydrogen production by Asn 195 about 30%, though increasing the pressure with additional nitrogen didn't affect things further except maybe to increase the ATP required for each electron transfer, almost double what it is at 100% argon. Nitrogen didn't seem to affect hydrogen from Lys 191 at all.

Adding nitrogen also inhibited acetylene reduction in Asn 195, 28%; and again, did not inhibit Lys 191. For the former, nitrogen seems to inhibit the reaction competitively but reversibly; removing the nitrogen restored the rate almost to what it had been.

They tried adding hydrogen to acetylene reduction assays with Asn 195, enough to raise the pressure to two atmospheres. This didn't affect anything with argon; ethylene and ethane were both produced about the same amount. But when nitrogen was present, hydrogen restored most or all the activity that nitrogen would've inhibited.
They also saw that having 50% deuterium with the rest nitrogen doesn't really result in inhibition of hydrogen production by Asn 195.

When they tried adding sodium azide, this didn't really affect hydrogen production, but the activity reducing it to ammonia or hydrazine (N₂H₄) was much less for all the mutants than for the wild-type, at least 8x less. Adding carbon monoxide to Asn 195 assays abolished any azide reduction activity, but adding hydrogen had no effect. The azide might've reduced electron flux through Asn 195 a little (20%) but CO prevented this reduction too.

What This Means
Even some of the mutants that can't fix nitrogen seem to interact with it to some extent, as evidenced by its inhibiting other reactions. The other findings are pretty interesting too. It all relates to how the mutations affect the activity: in affinity, in substrate fit in the active site, and in electron flux through the whole complex.

Reference:

Fisher, K., Dilworth, M. J. & Newton, W. E. Differential Effects on N₂ Binding and Reduction, HD Formation, and Azide Reduction with α-195^His- and α-191^Gln-Substituted MoFe Proteins of Azotobacter vinelandii Nitrogenase. Biochemistry 39, 15570–15577 (2000).

554 - Azotobacter vinelandii Nitrogenases Containing Altered MoFe Proteins with Substitutions in the FeMo-Cofactor Environment: Effects on the Catalyzed Reduction of Acetylene and Ethylene

This is another study looking at mutating the Mo nitrogenase protein in Azotobacter vinelandii to see how it changes the enzyme's activity. Here's a picture they gave of the active center, with the FeMo cofactor in the middle and the protein surrounding it, with certain amino acids they were targeting:

What They Saw
They targeted conserved amino acids, common to homologs in many organisms, such as Gln 191 and His 195, with three mutations: Lys 191 (seen before in 536), Asn 195, and Gln 195. These proteins were extracted and purified.

Under argon, Gln 195 produced about as much hydrogen as the wild-type, while the others only about half as much (though apparently they only contained half as much FeMo cofactor). Under 10% acetylene, the wild-type put most electrons toward producing ethylene, while the others only devoted 55% at most, the others going to hydrogen (Gln 195) or hydrogen and ethane (others).

The mutations also affected how the protons were added to acetylene, whether in cis or in trans, as determined by using C₂D₂ instead of C₂H₂ and looking at where there was hydrogen or deuterium. The mutants had higher proportions of trans-C₂D₂H₂ compared to the wild-type, except Gln 195 which had lower. This seems related to their ability to reduce acetylene all the way to ethane or not.

Then they tested whether ethylene instead of acetylene, with or without CO or acetylene too, could be a substrate or inhibitor of activity. Having 50% ethylene with the rest argon led to ethane production from all versions, including the wild-type, except not Lys 191. It seemed to inhibit overall flux a little, though for most versions it didn't inhibit as much when CO was present (the exception was Lys 191, which had about 3x less flux with CO present compared to just argon, though in both cases it all went to hydrogen). Interestingly, adding 50% hydrogen with 50% ethylene increased the amount of ethane for all versions. Strangely, though it didn't reduce ethylene to ethane, adding 10% acetylene to the mix with Lys 191 showed some ethane production; so it doesn't reduce ethylene, only acetylene. Acetylene also increased the rate of ethane production with Asn 195.

What This Means
This helps understand the precise reaction that takes place in nitrogenase. It seems like the affinity of the enzyme for the substrate affects how far that substrate is reduced before being replaced by a fresh molecule. Still, it's hard to make real comparisons from in vitro assays, but I don't know that there's a good alternative.

Reference:

Fisher, K., Dilworth, M. J., Kim, C.-H. & Newton, W. E. Azotobacter vinelandii Nitrogenases Containing Altered MoFe Proteins with Substitutions in the FeMo-Cofactor Environment: Effects on the Catalyzed Reduction of Acetylene and Ethylene. Biochemistry 39, 2970–2979 (2000).

544 - Nitrogenase from vanadium-grown Azotobacter: Isolation, characteristics, and mechanistic implications

It was known that Mo seemed important for nitrogen fixation in Azotobacter vinelandii. This study looked at substituting V for Mo for nitrogen fixation. It wasn't discovered until about a decade later that there were two separate sets of genes for two versions of nitrogenase with different metals, so here they thought it was a substitution of metals in the same protein.

What They Saw
They grew A. vinelandii OP (aka CA) with Mo or V and extracted and purified its nitrogenase. They said it didn't grow without either Mo or V, which seems weird because it should be able to grow with just iron.

The V nitrogenase activity (measured as hydrogen production) was lower than that of the Mo nitrogenase, about 22% of it, though it's hard to compare in vitro assays. It also seemed less stable and more prone to heat inactivation.

They did detect traces of Mo in the V purification, so it's not clear exactly what's happening. There was about 20x more V than Mo. Both purifications could reduce acrylonitrile, propionitrile, and acetonitrile in addition to the more familiar substrates, and hydrogen was produced at the same time. Hydrogen inhibited nitrogen reduction and carbon monoxide inhibited everything except hydrogen production.

In terms of efficiency, they observed that the Mo nitrogenase allocated 70% of its electrons to nitrogen and only 30% to hydrogen (similar to the typical 75%/25% numbers), while electrons in the V nitrogenase only went to nitrogen 25% of the time, which works out to 6 electrons making 2 ammonia, and another 18 making 9 hydrogen. Other substrates gave different numbers, but the V nitrogenase always had higher flux to hydrogen.

It seemed like CO inhibition of the V version was competitive but it wasn't clear that the same was true of the Mo version. And acrylonitrile reduction was different between them: V nitrogenase produced about twice as much propane as opposed to propylene compared to the Mo version.

What This Means
It seems, from the results, pretty likely that they were studying the V nitrogenase (Vnf) in this study, despite slight contamination with Mo. The CO inhibition pattern, and other activity patterns, support this conclusion.

I didn't know that acrylonitrile and such could be substrates for nitrogenase, but perhaps the cyanide residue is reduced to make it a hydrocarbon, either propylene or propane. Not sure this seems more useful than other substrates.

Overall, it's interesting how much this study revealed that wasn't really known until later studies confirmed it.

Reference:

Burns, R. C., Fuchsman, W. H. & Hardy, R. W. F. Nitrogenase from vanadium-grown Azotobacter: Isolation, characteristics, and mechanistic implications. Biochem Biophys Res Commun 42, 353–358 (1971).

542 - Purification and Characterization of the vnf-encoded Apodinitrogenase from Azotobacter vinelandii

This study looks at the vanadium nitrogenase apoprotein and how it works with its own cofactors or those of the other versions.

What They Saw
Azotobacter vinelandii strains possessing or lacking genes for one or more of the nitrogenases were grown and their protein was extracted. They also extracted central cofactors for each type separately.

Without nifB, a strain can't produce any of the cofactors, so it's easier to get apoproteins. They took vanadium aponitrogenase (Vnf version) and activated it with FeVco. Not surprisingly, they only saw activity in strains containing vnf when vanadium was present and Mo or ammonium was not. Extracts of active strains lost activity when treated with heat or exposed to air for 45 minutes, though the holoenzyme seemed more stable to heat.

When they purified the enzyme as much as possible, the delta subunit (vnfG-encoded) seemed only loosely attached to the others, unlike in A. chroococcum where it purifies together with the others; though it's not clear that conditions were the same. It was necessary for active enzyme though, and seems to be involved in inserting the cofactor into the enzyme, but also something else.

They tried replacing FeVco with FeMoco in the V nitrogenase (or vice versa in the Mo version). With the V version and FeVco, carbon monoxide inhibited about 70% of the acetylene reduction activity but no hydrogen production activity. The Mo nitrogenase with FeVco had a fraction of the V version's acetylene activity but no nitrogen fixation; sadly they didn't test hydrogen production.

With the V nitrogenase and FeMoco, it had a bit (1/6th) of the acetylene reduction activity, which seemed insensitive to CO. Ethane production was proportionally higher (1:4 instead of 1:12). Hydrogen production was reduced about 40%. Nitrogen fixation was pretty much abolished.

With the correct cofactors, the V nitrogenase had about 31% the acetylene reduction activity, 34% of the hydrogen production activity, and 22% of the nitrogen fixation activity of the Mo nitrogenase, but it's not clear if these in vitro assays allow for accurate comparisons.

It didn't seem like FeFeco allowed any activity in the V aponitrogenase.

What This Means
It seems like nitrogen fixation requires a very specific environment, and messing with it in various ways (mutations, different cofactors) messes it up while allowing the enzymes to still do less strict activities.

Reference:

Chatterjee, R., Allen, R. M., Ludden, P. W. & Shah, V. K. Purification and Characterization of the vnf-encoded Apodinitrogenase from Azotobacter vinelandii. J. Biol. Chem. 271, 6819–6826 (1996).

536 - Nitrogenase-catalyzed Ethane Production and CO-sensitive Hydrogen Evolution from MoFe Proteins Having Amino Acid Substitutions in an α-Subunit FeMo Cofactor-binding Domain

To figure out which parts of the nitrogenase protein are important, this study made very specific mutations to amino acids in the protein in Azotobacter vinelandii to see how they affected the catalysis.

What They Saw
They grew cells, wild-type and mutants, with molybdenum, then extracted and tested their nitrogenase. There was a nifEN knockout strain, a nifDK knockout, and others with specific changes in nifD, sometimes combined with nifN knockout.

None of these had nitrogen-fixing activity. All had just as much dinitrogenase reductase activity as the wild-type; some had more. But regarding acetylene reduction, all nifN knockouts had about zero, but the single-mutation strains all had some, though none nearly as much as the wild-type. They each had more ethane production than the wild-type though, so although total reduction and ethylene production were lower, ethane production was higher.

The temperature stability of mutants wasn't all the same either; some were more sensitive to heat. Lowering the temperature below 30ºC also led to a lower proportion of electron flux going to ethane instead of ethylene (in the mutants that produced ethane). No ethane was seen at any temperature in the wild-type. Though these measurements may not have been reliable, so the trend might not be real.

Then they tried adding carbon monoxide (CO) to inhibit the enzymes. The pattern was the same for each strain (they say), but the amount of total inhibition was different; some were less sensitive than the wild-type, some more, some equal to wild-type.

The FeMo cofactor didn't seem to be different in the mutants; extracting it and using it to restore activity to an apoprotein gave the same results from each strain.

After these results on crude extracts, they purified wild-type nitrogenase and the mutants' most stable enzyme (that replaced the glutamine in NifD position 191 with a lysine). Under acetylene, the wild-type enzyme had about the same electron flux with or without 0.2% CO, but more went to ethylene (vs hydrogen) when CO was absent. 3% CO completely inhibited nitrogen fixation (under nitrogen, obviously), but didn't inhibit hydrogen production: about as much was produced with nitrogen and CO as with argon and CO (or argon without CO). Incidentally, this study gave a NH₃ to H₂ ratio of 1.4 to 1 in 100% nitrogen, which is somewhat lower than the normal 2 to 1.

With the mutant, there was at least 4x less electron flux overall. With CO absent, most of it went to hydrogen when acetylene was present, but what did go to acetylene produced some ethane and more ethylene (as usual). With argon or nitrogen, it all went to hydrogen. When CO was present, the electron flux seemed even more reduced, but the patterns of product were similar.

What This Means
The 191 glutamine residue seems involved in the catalysis, positioned near the FeMo cofactor active center as it is. I am curious about several things, considering nitrogenase's already interesting catalytic abilities: what would similar studies of the other nitrogenases show? What kind of activity might result from other modifications? And, does this kind of modification allow for the reduction of new substrates, such as carbon monoxide itself? These would be interesting studies, if they haven't been done already. Apparently the vanadium nitrogenase is less sensitive to CO than the Mo version here, and it has already been shown to reduce CO to hydrocarbons, at least in vitro.

The difference in acetylene reduction could be due to different affinities: in the wild-type, a new acetylene replaces an old as soon as it is reduced to ethylene, whereas in a mutant, the ethylene remains long enough to be reduced further to ethane. It seems like this difference is due to a difference in the enzyme itself, rather than the cofactor; so the enzyme itself affects the catalysis (though I guess that's not surprising).

Reference:

Scott, D. J., Dean, D. R. & Newton, W. E. Nitrogenase-catalyzed Ethane Production and CO-sensitive Hydrogen Evolution from MoFe Proteins Having Amino Acid Substitutions in an α-Subunit FeMo Cofactor-binding Domain. J. Biol. Chem. 267, 20002–20010 (1992).

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).

524 - In vivo and in vitro nickel-dependent processing of the [NiFe] hydrogenase in Azotobacter vinelandii

This study looked at Azotobacter vinelandii's hydrogenase again, its post-translational processing, and whether nickel influenced this process.

What They Saw
The normal Azotobacter medium (Burk's) has enough contaminating nickel that adding it is unnecessary. But when they added a chelator (nitrilotriacetate) to bind it up, the hydrogenase activity decreased by 80% without affecting growth. This inhibition was lessened by adding nickel.

Nickel availability seemed to affect which form of the alpha subunit was present: the larger, unprocessed form, or the smaller, mature form. With nickel available, only the smaller form was seen; when it was bound up, only the larger. But when excess nickel was added, following the proteins over time showed that gradually the population shifted from larger to smaller as the nickel was used. These two forms are found in different places: the smaller is bound to the membrane (as it should be), and the larger is soluble.

Inhibiting protein synthesis, such as with chloramphenicol, and then adding nickel led to a similar increase in activity as a control without an inhibitor, up to 70 minutes; so for this period, increasing activity wasn't due to protein synthesis. But after this point, the inhibited cultures stopped increasing while the uninhibited continued. The processing of the large form into the small continued regardless of inhibition. So it seems that nickel is important partially for processing and partially for stimulating protein synthesis.

In vitro, ATP or GTP was important for processing. Membranes and oxygen (or lack thereof) were not important. No divalent cation could substitute for nickel: zinc inhibited processing completely, and cobalt or calcium some too. The only protease inhibitor that prevented processing was 1,10-phenanthroline, which inhibits metal-activated proteases.

What This Means
It seems that nickel and processing are both essential for hydrogenase activity, and apparently they are interrelated. It's possible that the processing is regulated by the presence of nickel; without the metal, there isn't much point. Or maybe processing without nickel available will lead to nonfunctional product that can't be fixed. Alternatively, the protease that does the processing could require nickel. It's hard to distinguish these possibilities though. Anyway, it seems like when nickel is absent, the hydrogenase subunits are present but in a premature form, waiting for nickel. How poetic.

Reference:

Menon, A. L. & Robson, R. L. In vivo and in vitro nickel-dependent processing of the [NiFe] hydrogenase in Azotobacter vinelandii. J. Bacteriol. 176, 291–295 (1994).

523 - Carboxyl-terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii

Based on amino acid prediction from gene sequence, the HoxG alpha subunit of the uptake hydrogenase should be about 66.6 kDa, but in the wild-type it appears smaller. In some mutants with accessory genes knocked out, the size matches this number. So this study tried to figure out if post-translational processing was involved in producing active enzyme. N-terminal modification was already ruled out, as that sequence matches the prediction.

What They Saw
They grew Azotobacter vinelandii CA and purified its hydrogenase, then studied its subunits with mass spectrometry.

They observed that the actual size of the larger subunit was 64.9 kDa, smaller than the 66.6 predicted size. The N-terminal was still the same as predicted, so they concluded that about 15 amino acids had been removed from the C-terminal of the subunit. This appears to be necessary for it to function.

Reference:

Gollin, D. J., Mortenson, L. E. & Robson, R. L. Carboxyl-terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii. FEBS Letters 309, 371–375 (1992).