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).

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).

020 - Comparative characterization of H2 production by the conventional Mo nitrogenase and the alternative "iron-only" nitrogenase of Rhodobacter capsulatus hup- mutants

As I've mentioned before, hydrogen gas is a byproduct of nitrogen fixation (which makes sense; nitrogenase adds protons and electrons to N₂ to form NH₃, and in the process some protons and electrons stick to each other, forming H₂. And the different kinds of nitrogenase have different efficiencies—that is, different proportions of protons and electrons that come off as hydrogen instead of actually useful stuff. The molybdenum-containing nitrogenase is most efficient, with one H₂ per N₂ fixed, and the others have higher ratios.

So this study intended to compare different nitrogenases to find out how much hydrogen they produced. It was done with Rhodobacter capsulatus, not Azotobacter, but the enzymes are similar. R. capsulatus is a type of phototrophic bacterium that possesses the primary Mo nitrogenase and also the iron-only alternative, as well as an uptake hydrogenase.

In order to get around the confounding effects of an uptake hydrogenase, which would significantly reduce the amount of measurable hydrogen given off by all nitrogenases, the scientists used a hydrogenase-negative strain they had generated using a transposon (jumping gene). Besides this strain and its parent, they had a strain with the Mo nitrogenase and Mo transport genes deleted (so it could use only the iron nitrogenase), and a hydrogenase-negative mutant of this strain.

The strains were each grown in broth and then exposed to an atmosphere of argon or nitrogen, sometimes mixed with acetylene or oxygen. The purpose of argon is that, when the nitrogenase enzyme lacks any other substrate (nitrogen, acetylene, etc), it will still work but just devote all its protons and electrons to producing hydrogen gas, producing a lot more than in any other condition. Adding acetylene measured the enzyme activity converting acetylene to ethylene (and a little ethane too, in the case of the iron nitrogenase), and adding oxygen measured its effect on the enzymes. Then after some time for the reaction to occur, concentrations of ethylene, ethane, and hydrogen in the headspace were measured.

In an argon atmosphere with nothing else, the hydrogenase-negative Mo nitrogenase strain produced the most hydrogen. In the parent strain that was hydrogenase-positive, the hydrogenase consumed about 1/4 the hydrogen produced. The hydrogenase-negative strain using the iron nitrogenase produced about 1/2 the hydrogen of the top producer, and when present, hydrogenase consumed about 1/2 its hydrogen, resulting in 1/4 the amount of the top producer. So like this:

Under argon:

• nif+ hup-: 100%
• nif+ hup+: 75%
• nif- hup-: 50%
• nif- hup+: 25%

This may seem odd because the iron nitrogenase is supposed to produce more hydrogen (relative to other substrates), but that is not the only difference between the enzymes; the Mo nitrogenase's rate of production (productivity) is also higher, such that it produces more of any product in a given time. Since these reactions were measured after 1 hour, the Mo nitrogenase was able to produce more hydrogen in that time than the alternative, though they might have produced the same amount (or the iron version more) if allowed to consume all their substrate.

The story of hydrogen production is somewhat different in a nitrogen gas atmosphere:

• nif+ hup-: 62%
• nif+ hup+: 5%
• nif- hup-: 100%
• nif- hup+: 3%

Not surprisingly, when nitrogen is present for the enzymes to fix, the alternative nitrogenase produces a lot more hydrogen than the Mo nitrogenase. However, even in the hydrogenase-negative iron nitrogenase strain's case, the hydrogen produced is a bit more than 1/4 of that produced by the top producer under argon. When hydrogenase is present, it is able to consume most of the hydrogen. Similar results are obtained when acetylene is added to an argon atmosphere.

When acetylene was added, the hydrocarbon results were as expected also. Presence or absence of hydrogenase didn't make much difference regarding ethylene and ethane produced. Mo nitrogenase produced much more ethylene than iron nitrogenase, and hardly any ethane; while the iron nitrogenase produced about 17 times more ethane than the Mo nitrogenase, and 11 times less ethylene. Total products for Mo nitrogenase were also about 11 times more than for iron nitrogenase. More efficient, I say.

In terms of protons and electrons, these results suggest that 80% of Mo nitrogenase's electrons go toward nitrogen fixation (that is, 1 hydrogen for every nitrogen fixed, as I said), but only 45% of the alternative's electrons (so, it makes 3-4 hydrogens for each nitrogen).

Oxygen had different effects on the different enzymes also. In the hydrogenase-negative strains, higher levels of oxygen inhibited each, but the alternative nitrogenase's activity dropped to below 40% with very small increases in oxygen levels, whereas the decrease in Mo nitrogenase activity was almost linear with increasing O₂, retaining as much activity as the alternative at more than five times the level of oxygen. So the iron nitrogenase seems to be about 4 times more sensitive to oxygen. Understandably alternative.

So, at least in this species, the iron nitrogenase is 3-4 times less efficient in terms of electrons going to hydrogen, about 11 times slower, and 4 times more sensitive to oxygen than the Mo nitrogenase.

Citation: Krahn, E., Schneider, K. & Müller, A. Comparative characterization of H₂ production by the conventional Mo nitrogenase and the alternative ‘iron-only’ nitrogenase of Rhodobacter capsulatus hup^- mutants. Appl. Microbiol. Biotechnol. 46, 285–290 (1996).