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).

557 - Detection of the in vivo incorporation of a metal cluster into a protein - The FeMo cofactor is inserted into the FeFe protein of the alternative nitrogenase of Rhodobacter capsulatus

This is another study looking at central cofactors from some nitrogenase versions inserted into other apoproteins than usual, but this time in Rhodobacter capsulatus.

What They Saw
They looked at purified enzymes from wild-type R. capsulatus and a nifHDK deletion mutant. The latter should only produce the iron-only nitrogenase, if anything. They had to treat the medium to remove as much Mo as possible so as to be able to control the concentration; this reduced the Mo present from around 1 ppb to less than 0.05 ppb (the detection limit).

They found that adding 10μM Mo to cultures growing with no Mo (and thus producing the Fe nitrogenase) greatly increased the amount of ethane produced from acetylene reduction (up to 40% of the ethylene produced). Without Mo, ethane remained constant at about 2% of ethylene. The amount of ethane increased over 72 hours too, up to 68% of ethylene; however, total activity decreased greatly over that time, down to only about 5% of what it had been before adding Mo. The ethane production rate increased for 24 hours and then decreased more slowly than the ethylene rate. This may be due to repression of the Fe nitrogenase, but the authors claim it is not, because the rate decreases more quickly in late-log cultures with chloramphenicol + Mo than with just chloramphenicol (or neither, which was about the same as with chloramphenicol alone). This shows that no new nitrogenase protein is being made even when chloramphenicol is absent, but adding Mo speeds the loss of it.
They also found that the more Mo they added, the higher the proportion of ethane produced (and also the lower the total acetylene reduction activity).

They tested the sensitivity of the system to oxygen, both with and without Mo: in both cases, more oxygen meant less acetylene reduction, though the system with Mo seemed a bit more sensitive (dropping to almost 0% with 1% oxygen while that without Mo only fell to about 20%), but also they noticed that increased oxygen increased the proportion of ethane produced after Mo was added. So somehow oxygen enhanced the Mo effect.

Rhenium, tungsten, and vanadium did not cause anything similar to the Mo effect. The Mo effect was also absent in mutants unable to produce FeMo cofactor (nifE knockouts), so it seems like the cofactor is part of the system. nifQ knockouts seemed to show the effect only at high concentrations of Mo (0.1-1mM); this gene's product is involved in cofactor synthesis at a different step. Mo uptake wasn't an issue; all strains had the same intracellular concentrations.

Using EPR spectroscopy on purified enzyme, they claim to show that the spectrum for Fe nitrogenase with added Mo is similar to that of the Mo nitrogenase from the wild-type, so it seems like the FeMo cofactor is incorporated into the Fe nitrogenase. I would've liked to see their result for the Fe nitrogenase without added Mo as a control, but I'll have to take their word for it. Though they did do metal analysis that found ratios of Fe to Mo similar to that of the Mo nitrogenase.

The fact that chloramphenicol didn't prevent the Mo effect seemed to show that the proteins required to make FeMoco were present before Mo was added. This was confirmed with lacZ fusions to related genes to observe expression more directly.

What This Means
Is FeMoco actually replacing FeFeco in completed enzymes? Seems more likely that FeMoco is inserting into incomplete apoprotein, but it's hard to distinguish between these possibilities. Considering that the process seems to continue over several days, maybe the FeMoco is actually displacing FeFeco from completed proteins over time. This is supported by the observation of oxygen enhancement of the Mo effect; oxygen seems to make the enzyme more labile.

Reference:

Gollan, U., Schneider, K., Müller, A., Schüddekopf, K. & Klipp, W. Detection of the in vivo incorporation of a metal cluster into a protein - The FeMo cofactor is inserted into the FeFe protein of the alternative nitrogenase of Rhodobacter capsulatus. Eur. J. Biochem. 215, 25–35 (1993).

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).

541 - Diversity of Nitrogenase Systems in Diazotrophs

This review looks at different kinds of nitrogen-fixing enzymes, real and theoretical. Azotobacter vinelandii itself has three genetically distinct versions, with different central metals in their central cofactors: molybdenum, vanadium, and iron.

The molybdenum version is most common in nature, and used preferentially in organisms that possess it. It has also been studied the most. No known organism possesses either of the other two versions while lacking this one. Protein sequences of this enzyme in different organisms are remarkably similar to each other. It uses 2 ATP to transfer one electron from the dinitrogenase reductase protein to the dinitrogenase complex, and 6 such transfers reduce one dinitrogen to two ammonia (along with 2 electrons going to hydrogen).

The other versions were discovered when Mo or its enzyme were unavailable yet nitrogen fixation continued. They have an extra subunit of unknown function (perhaps cofactor insertion), but otherwise seem pretty similar in structure and function.

Other than those three, Streptomyces thermoautotrophicus has a novel system: it has Mo in the dinitrogenase and the dinitrogenase reductase equivalent is a manganese-superoxide oxidoreductase with no iron or sulfur. It doesn't do acetylene reduction, but far from being oxygen-sensitive, it depends on oxygen for its activity. The overall stoichiometry is similar to the Azotobacter Mo nitrogenase though, including the hydrogen production, but the minimum ATP requirement is only 4, instead of 16.

Then the authors go into some discussion of other nitrogenases, with the same apoenzymes as those in A. vinelandii but with different central cofactors that may or may not be found in nature. For example, a tungsten-iron cofactor, which has been studied before: it doesn't permit nitrogen fixation, but some proton reduction is possible. Then there were chromium- or manganese-iron cofactor proteins: when extracts were treated with o-phenanthroline and purified as apoproteins, activity could be partially restored with solutions including salts of Mn, V, Mo, Cr, or Re. W failed to give this effect, but the others permitted some acetylene and proton reduction activity. I don't have access to the citations for these claims though.

These in vitro proteins, even if they contain weird metals, also seem to contain Mo in equal proportions to the others. A. vinelandii UW3 lacks nif genes required for the primary nitrogenase, but can use alternatives, and some suggest that the V nitrogenase may use Re or Mn or Cr instead; or maybe the Mo nitrogenase proteins are expressed but use these metals. It doesn't seem very clear, but it's interesting.

Reference:

Zhao, Y., Bian, S.-M., Zhou, H.-N. & Huang, J.-F. Diversity of Nitrogenase Systems in Diazotrophs. J Integr Plant Biol 48, 745–755 (2006).

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).

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).

457 - Hydrogenase and Nitrogen Fixation by Azotobacter

This study looked at hydrogenase in different Azotobacter species (A. vinelandii, A. chroococcum, A. agile whatever that is).

What They Saw
They looked at different kinds and amounts of fixed nitrogen and their effect and different gases in the atmosphere. Many experiments used ammonium phosphate or other forms of ammonium, and they thought maybe the drop in pH seen as ammonium was consumed led to decreased hydrogenase activity, but actually even when they used forms that didn't allow a pH drop, they still saw the same decrease, suggesting that it's the fixed nitrogen itself that leads to decreased activity. Which makes sense.

They found, consistent across species, that ammonium led to the biggest activity decrease, about 60-80%; nitrate as little as 20%; and glutamate hardly at all. I think these cultures were not adapted to these compounds though.

So they tried adapted cultures too. They found that the more fixed nitrogen they added, the less hydrogenase activity they saw. Adaptation didn't matter with ammonium, but cultures adapted to nitrate had more of a decrease in activity. Apparently they didn't test glutamate.

Then they compared cells with various nitrogen sources grown in air or in a hydrogen-oxygen mixture. They didn't test cells without a nitrogen source in this gas mixture though, maybe because they couldn't grow. Anyway, the hydrogenase was always more active in air with no fixed nitrogen than with any kind of fixed nitrogen (as seen before), and with H₂-O₂ the activity seemed even lower, even than with the same fixed nitrogen source in air. Activity was almost zero in nitrate-adapted cells given nitrate. This seems odd; previous studies seemed to show that hydrogen stimulated hydrogenase activity.

What This Means
I'd say other studies showing stimulation by hydrogen were more convincing, but at least this one was consistent showing an adaptation effect and down-regulation in the presence of fixed nitrogen.

Reference:

Lee, S. B. & Wilson, P. W. Hydrogenase and Nitrogen Fixation by Azotobacter. J. Biol. Chem. 151, 377–385 (1943).

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).

448 - Hydrogen-deuterium exchange reactions catalysed by nitrogenase

Previous reports suggested that nitrogenase could convert dideuterium (D₂) to hydrogen-deuterium (HD) by swapping one hydrogen from water with one deuterium atom. This only happened in the presence of nitrogen. This was tested in various organisms with D₂ or D₂O as a source of deuterium.

Azotobacter species didn't make HD unless ATP and electrons were present. They actually made less hydrogen and HD with nitrogen than with argon, contradicting earlier findings. 10% CO did inhibit HD formation but not hydrogen formation. Acetylene and methyl isocyanide inhibited HD completely. Cyanide partially or fully inhibited.

There was more exchange with D₂O than with D₂ for some reason, but this was inhibited more by nitrogen or cyanide.

This seems to have implications for nitrogenase's functions relating to hydrogen.

Reference:

Kelly, M. Hydrogen-deuterium exchange reactions catalysed by nitrogenase. Biochem J 109, 322–324 (1968).

365 - Mechanism of biological nitrogen fixation VII. Molecular H2 and the pN2 function of Azotobacter

They looked at the amount of nitrogen fixed with different concentrations in the atmosphere, in Azotobacter vinelandii and chroococcum cells. They were looking for K_N2: the concentration of nitrogen at which the amount of nitrogen fixed is half the maximum.

With inert gases (argon, helium) or with a partial vacuum, the concentration of nitrogen had to get down below 0.15 atm before the fixation decreased much. It gets to 50% around 0.01 atm. But when there was hydrogen, the rate decreased more quickly; the more, the faster. So hydrogen seems to inhibit the nitrogenase, but only at very high concentrations (over 20% of the atmosphere). This inhibition is competitive and reversible.

Reference:

Wyss, O., Lind, C. J., Wilson, J. B. & Wilson, P. W. Mechanism of biological nitrogen fixation VII. Molecular H₂ and the pN₂ function of Azotobacter. Biochem J 35, 845–854 (1941).

362 - Mutants of Azotobacter chroococcum Defective in Hydrogenase Activity

This study isolated some hydrogenase-negative mutants of Azotobacter chroococcum by chemical mutagenesis and looked at how they behaved.

What They Saw
Almost all of the 16 mutants had almost no hydrogenase activity, as expected. Some had a little, <2% of wild-type. Some more had a little hydrogen-producing activity in the right conditions, usually less than 7% of the wild-type, but one had 40% of wild-type. That one also seemed to have a relatively active soluble hydrogenase (possibly the uptake hydrogenase in soluble form). All of them seemed able to take up nickel.

The one weirdest mutant, MCD-124, showed max activity at a different pH (5.5 instead of 8) and was weird in other ways.

Also, the authors were surprised by the frequency with which they could get hydrogenase mutants. They wondered whether the relevant genes were just more susceptible, or if the growth medium was more favorable to mutants somehow, or if there were just that many necessary genes. But judging from the genome sequence, this isn't quite a sufficient explanation.

Overall, it's hard to know exactly what's going on in this study.

Reference:

Yates, M. G. & Robson, R. L. Mutants of Azotobacter chroococcum Defective in Hydrogenase Activity. J Gen Microbiol 131, 1459–1466 (1985).

358 - Molecular H2 and the pN2 function of Azotobacter

There was a question at this time of whether hydrogen in the air could inhibit nitrogen fixation in Azotobacter vinelandii, and in what conditions this might happen. There were various complicating factors though. This study attempted to do a more controlled investigation using purified enzyme.

What They Saw

They observed that the higher the concentration of nitrogen in the atmosphere, the more nitrogen was fixed in a given time (thus the higher the specific activity was). From this they could calculate the K_N2, by plotting the inverse of the specific activity over the inverse of the concentration and taking the slope of the line. This is an indication of the enzyme's affinity for nitrogen, I think. This is higher than seen from intact cells (0.01).

Then they got to the hydrogen inhibition experiments. As they increased the amount of hydrogen, the nitrogen-fixing activity did seem to decrease, indicating competitive inhibition.

One issue that they didn't control for was the production of hydrogen by nitrogenase itself; this could've influenced the numbers. It could also influence the K_N2 numbers, come to think of it. It's also unclear whether the extracts had any hydrogenase activity, which could influence things in the opposite direction.

Reference:

Strandberg, G. W. & Wilson, P. W. Molecular H₂ and the pN₂ function of Azotobacter. Proc Natl Acad Sci U S A 58, 1404–1409 (1967).

340 - Kinetic studies of the nitrogenase-catalyzed hydrogen evolution and nitrogen reduction reactions

This study looked at the kinetics of hydrogen production from purified nitrogenase; also, the nitrogen fixation reaction.

What They Saw
This nitrogenase was purified from Azotobacter vinelandii; they don't give details on culture conditions, so presumably it's the molybdenum version.

First, they observed that more ATP meant more hydrogen over time. The free phosphate-to-hydrogen ratio was similar at all levels though, so that makes sense. The same pattern was seen for nitrogen fixation, except the proportion of electron flux going to ammonia increased as ATP increased; more hydrogen was produced at lower ATP, relative to ammonia.

They found that whether under nitrogen or argon, the electron flux was the same; this was true over multiple ATP concentrations.

Reference:

Silverstein, R. & Bulen, W. A. Kinetic studies of the nitrogenase-catalyzed hydrogen evolution and nitrogen reduction reactions. Biochemistry 9, 3809–3815 (1970).

335 - ATP-Dependent hydrogen evolution by cell-free preparations of Azotobacter vinelandii

This study looked at hydrogen production in Azotobacter vinelandii strain O, to see what induced it in cell extracts.

What They Saw
Hydrogen production depended on ATP. Argon or hydrogen in the atmosphere didn't matter. The higher the protein concentration, the more hydrogen was produced. At 0ºC in air, the enzyme was pretty stable; over 90% activity was left after 3 days.

Hydrogen oxidation activity was found in separate fractions from the production activity, so they concluded it was a different enzyme.

Comparing extracts from cells grown with urea or no fixed nitrogen, they saw no hydrogen production activity in urea samples, but the hydrogen oxidation activity with urea was lower than that without.

What This Means
We know now that the hydrogen production comes from nitrogenase and the hydrogen oxidation from hydrogenase. It's surprising how air-stable the nitrogenase seemed to be outside the context of the cell, but I guess it was still surrounded by cellular elements in the crude extract. Also I'm not sure how oxygen inactivation affects nitrogenase hydrogen production.

Reference:

Burns, R. C. & Bulen, W. A. ATP-Dependent hydrogen evolution by cell-free preparations of Azotobacter vinelandii. Biochim Biophys Acta 105, 437–445 (1965).

308 - Hydrogen-mediated enhancement of hydrogenase expression in Azotobacter vinelandii

This study looked at whether added hydrogen could stimulate hydrogenase activity in Azotobacter vinelandii.

What They Saw
They grew cells with or without ammonium, then added argon or hydrogen to their headspace, and measured whole-cell or purified hydrogenase activity. Oxygen or methylene blue were electron acceptors.

With ammonium, there was a little activity, but adding hydrogen gas increased it about 2.5 to 5 times. As a control, injecting the same amount of argon didn't change anything. In nitrogen-fixing cells, adding hydrogen didn't affect activity.

As with others, activity increased over time in the culture, even corrected by biomass; the hypothesis was that excess carbon inhibits it somehow.

If they added an mRNA or protein synthesis inhibitor (rifampin or chloramphenicol) before adding the hydrogen, activity didn't increase with either case, so it seemed like the regulation was transcriptional.

Also, since the effect was the same with methylene blue (which doesn't require electron transport chain components to act as electron acceptor), it seemed that the regulation was at the hydrogenase directly rather than a related component.

Comparing a couple of Mo nitrogenase-deficient strains (CA11 and CA30) to their parent, they saw that hydrogen didn't affect hydrogenase activity in CA much (in nitrogen-fixing conditions), but it did increase the activity a lot in the mutants. The hydrogenase protein abundance increased too. But in conditions with ammonium, CA and CA11 behaved pretty similar.

Reference:

Prosser, J., Graham, L. & Maier, R. J. Hydrogen-mediated enhancement of hydrogenase expression in Azotobacter vinelandii. J. Bacteriol. 170, 1990–1993 (1988).

307 - The Relationship Between Hydrogenase and Nitrogenase in Azotobacter chroococcum: Effect of Nitrogen Sources on Hydrogenase Activity

This study looked at the influence of different sources of nitrogen on the activity of nitrogenase and hydrogenase in Azotobacter chroococcum.

What They Saw
They grew cells in batch or continuous culture with sodium nitrate, ammonium acetate or chloride, or dinitrogen gas. Cultures were either carbon- or sulfate-limited. Dissolved oxygen was kept above zero. Nitrogenase activity was measured by acetylene reduction and hydrogenase by methylene blue (or by adding H-T with radioactive tritium and measuring radioactivity of resulting water when oxygen was the electron acceptor).

There was about twice as much hydrogenase activity when cells were fixing nitrogen in batch than when they had either ammonium or nitrate. They cite other results in A. chroococcum and A. vinelandii that showed higher activity with nitrate than ammonium though, but still less than when fixing nitrogen. This might be because cells have to adapt to use nitrate, and they'll be fixing nitrogen before that happens. But care is necessary because activity changes over the course of a batch culture, increasing throughout exponential phase even when standardized by protein concentration.

In continuous cultures, they started growing with ammonium, then switched to nitrogen-free, watched what happened with hydrogenase and nitrogenase, and then pulsed a limited amount of ammonium. As expected, nitrogenase activity started up and rose to a plateau, then immediately stopped when ammonium was added, and restarted when it was removed. Hydrogenase activity showed a similar pattern, though more delayed, and it never went all the way to zero.

In sulfate-limited culture, hydrogenase activity was lower even when fixing nitrogen, about the same as with ammonium in carbon limitation, and when going from fixing to non-fixing (with ammonium), the activity declined a bit but then went back up to about the same level. Results were similar going from non-fixing to fixing. So with sulfate limitation, nitrogen source doesn't matter much.

Also with nitrogen-fixing cells in sulfate limitation, when they increased the dilution rate, hydrogenase activity decreased but nitrogenase increased. The decrease wasn't linear, though; it leveled off.

They also tested whether adding hydrogen to the atmosphere of an ammonium-grown culture would influence hydrogenase activity, and it did! Hydrogenase activity doubled. This was not the case with sulfate limitation though, only carbon limitation. They didn't test nitrogen-fixing cells.

What This Means
The continuous culture experiments helped overcome the constantly changing activity in batch cultures.

That hydrogenase activity lags behind nitrogenase activity increase when ammonium runs out could be explained by the last observation: maybe nitrogenase starts producing hydrogen (as it does) and this stimulates hydrogenase activity.

They reasoned from the data that excess carbon might inhibit hydrogenase activity somehow (like catabolite repression). I'm not sure that makes sense, but it seems possible, and does fit with the data from batch cultures, sulfate limitations, and increasing dilution rates. Interesting.

Reference:

Partridge, C. D. P., Walker, C. C., Yates, M. G. & Postgate, J. R. The Relationship Between Hydrogenase and Nitrogenase in Azotobacter chroococcum: Effect of Nitrogen Sources on Hydrogenase Activity. J Gen Microbiol 119, 313–319 (1980).

297 - The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H2-uptake hydrogenase of Azotobacter chroococcum

And finally, seeming to complete our journey back in time through the discovery of hydrogenase genetics in Azotobacter chroococcum, this study looks at the structural genes, hupSL.

What They Saw
The sequences were similar to A. vinelandii's hoxKG structural genes. They tried knocking each out, then measuring hydrogen oxidation (with methylene blue) and hydrogen production (with methyl viologen). As expected, hydrogen oxidation in mutants was no higher than negative controls. Surprisingly, they did see hydrogen production in the some of the different mutants with a strong electron donor, but it was less than in the wild-type. Only the mutant with an insertion very close to the start of the hupS gene had no hydrogen production.

What This Means
It seems like a fragment of HupS is sufficient to produce hydrogen with a strong electron donor, but not as much as with both HupS and HupL completely intact.

Reference:

Ford, C. M. et al. The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H₂-uptake hydrogenase of Azotobacter chroococcum. Mol Microbiol 4, 999–1008 (1990).

296 - The Azotobacter chroococcum hydrogenase gene cluster: sequences and genetic analysis of four accessory genes, hup A, hupB, hupY and hupC

So at this point, hupSL (hydrogenase structural genes) and hupDE (accessory genes at the end of the operon) had already been identified. This study found a few more upstream of hupDE.

What They Saw
They sequenced the DNA upstream of hupDE and found four open reading frames, which they called hupABYC. The AB and C were similar to E. coli genes, but the Y wasn't, so they called it Y (for Ynknown, I guess). These were all homologous to A. vinelandii genes though, and in the same order.

Then they tried knocking out each of these. Each knockout was unable to oxidize hydrogen, even in the presence of methylene blue as an electron acceptor.

They also made a fusion of HupL (the structural subunit) and beta-galactosidase, then knocked out hupY or hupB to see if this changed the expression of hupL. Beta-galactosidase activity rose a little bit, like 25% in each, but it didn't seem either was an important regulator.

Reference:

Tibelius, K. H., Du, L., Tito, D. & Stejskal, F. The Azotobacter chroococcum hydrogenase gene cluster: sequences and genetic analysis of four accessory genes, hup A, hupB, hupY and hupC. Gene 127, 53–61 (1993).

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).

190 - Characterization of a Tungsten-Substituted Nitrogenase Isolated from Rhodobacter capsulatus

The three normal nitrogenases have molybdenum and iron, vanadium and iron, or just iron in their central cofactors. This study looked at whether it's possible to have a version that only has tungsten in it though, and if it has any activity, in Rhodobacter capsulatus.

What They Saw
This strain only has Mo and Fe nitrogenases, and they knocked out the latter. So when they removed all Mo from the medium and added W, the only thing it would produce would be Mo nitrogenase with W in it, theoretically.

Adding tungsten almost completely stopped acetylene reduction activity in the wild-type, unless 10x more Mo was added. So tungsten doesn't allow that kind of activity.

Mo also induces production of the Mo nitrogenase (which makes sense), but W also seems to have that effect, though not to the same extent.

Then they purified nitrogenase that had been produced when only W was present (in significant amounts) in the mutant. Purification was the same with this as with Mo in the wild-type, though of course the yield was lower. But the protein had 1 atom of W and none of Mo pretty much, as hoped. Still, it seems like there's only one FeW cofactor, not two, but at least there aren't any FeMos.

This protein still wasn't very active in acetylene reduction, and it didn't seem to be able to fix nitrogen at all. What it could do, though, was produce hydrogen when under an argon atmosphere, so electron flux wasn't totally abolished. And this activity wasn't inhibited by acetylene, unlike in the Mo version. Still, the amount of hydrogen it produced was only 1/4th the amount the normal enzyme could produce in the same conditions.

Finally they wanted to see if rhenium (Re) could take the place of Mo as a FeReco and be functional, since Re is not far from Mo and W (one more proton than W), so they tried growing cells with perrhenate (KReO₄), but it didn't seem to help at all. The cells didn't even appear to be able to assimilate it, so it wasn't possible to test whether the nitrogenase could use it.

What This Means
It's difficult to do this kind of study, because Mo and other metals are almost impossible to eliminate completely from the medium. But they seem to have succeeded as much as possible, and still the nitrogenase only had one FeW cofactor rather than two. But surprisingly it showed some proton reduction activity, though not an exceptional amount. It seems like W in the protein is much more difficult to reduce, an essential step in the catalysis. Maybe the amount of reduction that's possible is only enough for some proton reduction activity.

One interesting speculation is based on an observation that Methanococcus thermolithotrophicus can fix nitrogen in the presence of tungstate at 60ºC, a pretty high temperature, so they wonder whether the nitrogenase with FeWco in R. capsulatus might also have more activity at higher temperature, but they didn't actually test this. Maybe another study.

Reference:

Siemann, S., Schneider, K., Oley, M. & Müller, A. Characterization of a Tungsten-Substituted Nitrogenase Isolated from Rhodobacter capsulatus. Biochemistry 42, 3846–3857 (2003).

162 - Hydrogen evolution: A major factor affecting the efficiency of nitrogen fixation in nodulated symbionts

In the nitrogen fixation process, hydrogen gas is produced. This consumes extra ATP, about 4 per molecule of hydrogen (for reference, a molecule of glucose can be fully oxidized to produce about 30 ATP). This paper looks at this process in legume nodules and its effects on yield.

What They Saw
They got nodules from different kinds of plants, infected with either wild bacteria or commercial strains, and measured hydrogen production in air or argon, and acetylene reduction.

In an in vitro nitrogenase system, activity depended on the presence of ATP, reducing equivalents, and the enzyme. The ratio of hydrogen in argon to hydrogen in air here was about 3.

There was a pretty wide variety of values they saw, but most seemed to cluster around a measure of 50% of the hydrogen produced in argon was produced in air; so the presence of nitrogen reduced the hydrogen production by half. It's not clear how uptake hydrogenases might have affected these numbers though. They got similar results with whole plants. This means that each nitrogen gets 6 electrons to become 2 ammonia, and another 6 electrons go to hydrogen; that seems like a lot.

They also noticed that adding enough acetylene stopped the hydrogen production, so it seemed like hydrogen is not a necessary byproduct of that reaction.

What This Means
Correlation between amounts of acetylene reduced and nitrogen fixed are not necessarily accurate, because of the hydrogen produced as a byproduct. But the hydrogen production seems to be a big waste of efficiency; reducing this could help increase yield, maybe by choosing good nodulating bacteria. They wonder whether the hydrogen could be captured from plants somehow and used for energy.

Reference:

Schubert, K. R. & Evans, H. J. Hydrogen evolution: A major factor affecting the efficiency of nitrogen fixation in nodulated symbionts. Proc Natl Acad Sci 73, 1207–1211 (1976).

159 - Effect of chelating agents on hydrogenase in Azotobacter chroococcum: Evidence that nickel is required for hydrogenase synthesis

This study used chelating (metal-binding) compounds to study the cofactor of hydrogenase in Azotobacter chroococcum.

What They Saw
All the chelators they added (NTA, EDTA, etc.) decreased the hydrogenase activity in batch cultures, though to different extents. NTA was much stronger than EDTA. The effect was not inhibition of already-formed enzyme (since adding chelators to resting cells or extracts didn't affect activity), so it must be from preventing formation of additional enzyme.

They tried adding trace metal salts along with the chelators to see if pure excess of whatever was missing could restore activity. Copper, zinc, and manganese didn't really do anything. Cobalt helped if it was added in fairly large amounts, but the most helpful was nickel. Adding extra iron boosted this effect even more.

Monitoring nickel uptake by adding radioactive nickel, they saw that cyanide completely wiped out uptake (possibly by binding the nickel), but juglone and 2,4-dinitrophenol enhanced it (despite inhibiting respiration). Sodium azide didn't really affect either. The chelators they tested earlier generally seemed to inhibit nickel uptake too, generally in the same proportions as they had inhibited hydrogenase activity.

What This Means
Chelators seem to inhibit hydrogenase, but rather than acting on the enzyme directly, it seems to be by inhibiting its synthesis, and even this mechanism seems to be by inhibiting nickel uptake in most cases, rather than something more direct. Nickel is important for synthesizing the enzyme; it's a part of its essential cofactor. It seems like cobalt might be able to substitute for nickel somewhat though. I wonder if palladium would work too, since it has similar orbital arrangements. But apparently cobalt doesn't help in the absence of chelators or contaminating trace metals, so maybe it only helped here because it distracted the chelators away from nickel (so to speak).

Reference:

Partridge, C. D. & Yates, M. G. Effect of chelating agents on hydrogenase in Azotobacter chroococcum: Evidence that nickel is required for hydrogenase synthesis. Biochem. J. 204, 339–344 (1982).

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).

067 - Analysis of a gene region required for dihydrogen oxidation in Azotobacter vinelandii

This study looks more closely into the genes discovered in previous studies (518,050,051,052) to be required for hydrogenase in Azotobacter vinelandii.

What They Saw
They knocked out different genes (or the whole operon) in the hyp operons of A. vinelandii CA by inserting resistance + lacZ cassettes, then tested these strains for hydrogen oxidation and expression of the genes (via beta-galactosidase activity). The medium they used had a lot more trace elements than typical Burk medium, including nickel.

They found that when lacZ was inserted in the same direction as the gene, they saw expression in all cases, whether fixing nitrogen or not, but if it was inserted in the opposite direction, they didn't. So apparently the genes are expressed to some extent even when not fixing nitrogen.

When they measured hydrogen oxidation, there was about 6x more when fixing nitrogen though, in the wild-type, and none in the mutants. The growth rates of the mutants were similar to the wild-type though, or so they claim without reporting any details.

Finally they tried growing the strains with extra added nickel, because the hydrogenase is a nickel-containing enzyme. This didn't have much effect on the wild-type, but the hypB mutant actually showed some hydrogenase activity in nitrogen-fixing conditions with the extra nickel, and activity was higher with more nickel added.

What This Means
It seems like extra added nickel can substitute for the lack of HypB, so maybe the enzyme has a role in nickel cofactor processing somehow. This makes sense considering the multiple histidines it contains. It's interesting that nickel didn't help activity in non-fixing conditions; maybe there are multiple hydrogenases, active in different conditions, and hypB is required for all but nickel doesn't help some of them.

Reference:

Chen, J. C., Mortenson, L. E. & Seefeldt, L. C. Analysis of a gene region required for dihydrogen oxidation in Azotobacter vinelandii. Curr. Microbiol. 30, 351–355 (1995).

056 - Properties of Hydrogenase from Azotobacter vinelandii

Another study tries to purify the uptake hydrogenase from Azotobacter vinelandii.

What They Saw
They were unable to get pure, soluble enzyme; they couldn't separate it from the membrane, so it remained in insoluble particles.

They observed that hydrogenase activity of these particles didn't decrease after 18 days of exposure to air, unlike particles from other species. They did lose activity within a few weeks though.

They also observed that the enzyme reduces cytochrome C, possibly as part of its electron transport role. They found that carbon monoxide and cyanide inhibit the enzyme. Azide does not, at least not at pH 8.

What This Means
Some of the observations here may be due to the presence of other membrane-bound proteins, not just hydrogenase.

Reference:

Hyndman, L. A., Burris, R. H. & Wilson, P. W. Properties of Hydrogenase from Azotobacter vinelandii. J. Bacteriol. 65, 522–531 (1953).

054 - Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium

This study investigates how the alternative (vanadium) nitrogenase interacts with tungsten.

What They Saw
They grew Azotobacter vinelandii UW (aka CA) or a tungsten-tolerant mutant with tungsten and Mo or V, then extracted the nitrogenase.

This mutant was derived by growing UW with W and ammonium over several passages to try to remove all the Mo the cells might be storing, and then plating the cells on plates without ammonium, so they would have to fix nitrogen in the presence of W and absence of Mo to survive. Stuff that grew was tungsten-tolerant: strain LM2.

Then to get nitrogenase, they grew LM2 with tungsten and UW with tungsten and ammonium (not sure why it would produce nitrogenase in that condition, though I guess it might when it ran out of fixed nitrogen).

Both UW and LM2 could grow on medium with Mo, though LM2 was sometimes slower. With W, LM2 grew faster or slower depending on amount of W, while UW did not grow.

They used something called rocket immunoelectrophoresis to measure how much MoFe nitrogenase components each strain produced with W. Each produced about the same amount of dinitrogenase reductase in all conditions, but UW with 1mM W produced about 56% the amount of dinitrogenase as it produced with Mo, and LM2 with 10mM W produced about 7% of the amount that UW produced with Mo. I wonder how accurate this technique is. But they also note that the specific acetylene reduction activity of crude extracts was about 6-8% for each strain grown in W compared to UW in Mo. Does this include alternative nitrogenase activity? They say no. But overall, it seems that both strains produce less Mo nitrogenase with W, LM2 less than UW, but LM2's is relatively more active.

I think what happened next was that they couldn't isolate nitrogenase well from LM2, so they studied it from UW. They had two kinds: typical Mo nitrogenase, and Mo/W nitrogenase that had one FeW-cofactor and one FeMo-cofactor. This latter showed less activity in every way: hydrogen under argon, nitrogen, or acetylene atmospheres, nitrogen reduction, and acetylene reduction. This fits with at least one previous study (045). There is still activity though, even with nitrogen reduction, so it's not clear why the cells can't grow in W; maybe its interaction with other important proteins?

What This Means
It's possible that instead of each molecule of Mo/W protein containing one W and one Mo, half the protein could have all Mo and the other half all W; this would give the same results, but seems a less likely explanation. Of course, both possibilities are pretty weird and confusing.

According to some chemistry stuff they did, it seems like the enzyme can't reduce FeW-cofactors, which could reduce the possible electron flux by half, I think. There are a lot of mysteries here.

Reference:

Hales, B. J. & Case, E. E. Nitrogen fixation by Azotobacter vinelandii in tungsten-containing medium. J. Biol. Chem. 262, 16205–16211 (1987).