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).

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).

021 - Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii

People had known at this point that tungsten was poisonous to nitrogenase in Azotobacter vinelandii. It seemed to replace molybdenum in the primary nitrogenase, rendering it non-functional for nitrogen fixation. But then Paul Bishop and colleagues discovered some strains that could grow in the presence of tungsten, using alternative nitrogenases (001). So Riddle and colleagues wanted to characterize such strains and their weird nitrogenases.

What They Did

The scientists first isolated some tungsten-tolerant mutants of their own. This is pretty simple; just grow cells (in this case, A. vinelandii ATCC 12837) in the presence of tungsten, and some tolerant mutants are likely to grow. They chose one and called it WD2 (for Tungsten-Derived 2). For some reason they grew their bacteria at 35ºC, which seems odd because usually Azotobacter is grown at 30ºC, but whatever.

Then they confirmed WD2's ability to grow and fix nitrogen in the presence of tungsten, compared to the parent, did acetylene reduction assays to measure nitrogenase activity, and ran 2-D gels to compare proteomics of the cells in different conditions. Finally they used electron spin resonance (ESR) and other tests to see the chemical characteristics of the cells' enzymes.

What They Observed

They compared the growth of the wild-type and WD2 in broth with or without tungsten. WD2's growth in tungsten was in between the growth of the wild-type in tungsten-free broth and the wild-type in broth with tungsten. Wild-type with tungsten didn't grow hardly at all, as expected. So tungsten inhibits WD2's growth a little bit, compared to the wild-type. To be fair, the amount of tungsten they used was 1000 times more than the minimum needed to inhibit nitrogen fixation in the wild-type.

Then they grew WD2 with various combinations of Mo and W. The growth curves were all pretty similar, not clearly significantly different, but the one with Mo and no W grew best and the one with W and no Mo was worst. Others were in between as expected (more Mo = better, more W = worse), so it seems like a good trend.

They measured acetylene reduction (as a proxy for nitrogen in nitrogenase activity assays) with the wild-type and WD2 (with tungsten), with various concentrations of acetylene in the atmosphere. In each case, activity (as measured by ethylene produced) was highest early in the exponential growth phase. It was not clear (to my eyes) that different amounts of acetylene or the different strains had significant effects on activity. It seemed like perhaps the higher concentrations permitted more activity later in the growth phase. WD2 in W broth showed only 17% the activity of the wild-type in regular broth.

2-D gels showed different profiles for WD2 in regular Burk broth vs. broth with tungsten, though it's always tough to interpret gel images on their own (as two of the same rarely look alike). It did seem like WD2 and the wild-type with ammonium showed similar profiles, and WD2 with tungsten or even with Mo was different, indicating different proteins for tungsten-tolerant nitrogen fixation. ESR spectra of the different proteins were different too.

With heat-extracted proteins from Mo-grown wild-type or W-grown WD2 cells, the wild-type had about 1 nmol Mo per mg protein, whereas WD2 had negligible, but had 18 nmol W. Acetylene reduction activity was much different too: wild-type had more than 10x the activity of WD2, at least in terms of ethylene produced. Spectra were very different too, especially in the band associated with the Mo-iron cofactor.

What This Means
Overall, WD2's results seem to agree with those of others (001), that mutants of wild-type A. vinelandii can fix nitrogen and grow in the presence of tungsten. And the 2-D gels apparently look similar between these two groups of researchers.

Some of the results (growth curves with different Mo:W ratios) seem to indicate that WD2 was using Mo even when tungsten was present. Others (2-D gels) seemed to indicate otherwise.

Based on the acetylene reduction assay, cells might have lower affinity for acetylene later in their growth phase.

Not much new, but it corroborates some data.

Reference:

1. Riddle, G. D., Simonson, J. G., Hales, B. J. & Braymer, H. D. Nitrogen fixation system of tungsten-resistant mutants of Azotobacter vinelandii. J. Bacteriol. 152, 72–80 (1982).