Wednesday, November 5, 2014

013 - Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor

What They Wanted to Know
Pau et al. knew that Azotobacter vinelandii had three versions of nitrogenase, including one with no heterometal (Mo or V), only iron. All of these had similar requirements for energy and conditions. They're all similar in structure too, except that the alternatives both have an extra subunit.

So Pau and colleagues wanted to purify the iron-only dinitrogenase from A. vinelandii and analyze its structure and such.

What They Did
They used a strain of A. vinelandii with the genes for Mo and V nitrogenases deleted, so the only one it could produce was the iron-only one. Since this strain couldn't fix nitrogen in the presence of Mo (it represses the alternatives), they selected for a mutant that didn't have this limitation: RP306. They grew large amounts of this strain (in a 400-L fermenter) and purified the nitrogenase from it. Then they analyzed the enzymatic activity and chemical structure of protein and metallic cofactor.

What They Saw
The parent of strain RP306 couldn't grow by fixing nitrogen when molybdenum (Mo) was higher than 5nM in the medium, but RP306 actually grew better as Mo increased, up to 20nM.

Since the V nitrogenase has an extra subunit (δ) encoded by the vnfG gene, and the Fe nitrogenase has a homologous gene, anfG, Pau et al. thought that it might encode a δ subunit also. So they analyzed the subunits of the dinitrogenase with SDS-PAGE, and did indeed see a third small subunit as expected, whose amino acid sequence corresponds to the sequence of the anfG gene.

In terms of metal content, the dinitrogenase seemed to have about 24 atoms of iron and 18 of sulfur, which corresponded well to previous work. Not surprisingly, it had negligible V, but surprisingly it had 1 atom of Mo. So they analyzed it with electron paramagnetic resonance or EPR spectroscopy, which gives different curves depending on the chemical composition, and it seemed like the iron-only nitrogenase actually had a Mo-containing cofactor! Though it seemed like only one of the two cofactors in the dinitrogenase contained Mo. They were able to extract this cofactor, observed that it had a Mo-to-Fe ratio of 1:4.3, and could insert into a cofactor-less Mo nitrogenase from Klebsiella pneumoniae and make it active.

This Fe dinitrogenase with a Mo cofactor could reduce acetylene, but only to ethylene, not to ethane like regular V and Fe nitrogenases could produce. This activity, or any other, was only present when the enzyme was paired with the iron-only version of dinitrogenase reductase, not with the other versions.

With other substrates (N2) or no substrate (just argon), this Fe nitrogenase didn't perform as well as the Mo nitrogenase. With argon, it produced 350 nmol hydrogen per minute per mg of enzyme, compared to 2220 from the Mo version; with nitrogen, it produced about 100 times less ammonia than the Mo version, but twice as much hydrogen as ammonia. This is about 4 times as much as expected from the Mo version, which produces one hydrogen per nitrogen fixed. So about 57% or 4/7ths of its electron flux goes to hydrogen, compared to 25% of the Mo nitrogenase's. They also saw some ethane produced from acetylene somehow, especially when the ratio of dinitrogenase reductase to dinitrogenase was higher; at least half the electron flux went to ethane.

What This Means
Apparently the allegedly iron-only nitrogenase can incorporate Mo-containing cofactor, at least partially, and this affects its activity. I wouldn't expect this to happen much in nature, since in the presence of Mo the Fe nitrogenase wouldn't be produced, so it's not clear what this really means in terms of enzyme activity. It seems important to exclude Mo from the medium when studying the real activity of the Fe nitrogenase though.

From other results, it seems like the cofactors, despite their differences in metal content, can substitute for each other in the holoenzymes, though the resulting activity changes (not surprisingly). The cells rely on regulation of genes that produce the proteins and cofactors to keep things running the way they should be, rather than specificity of cofactor for protein. But it's probably usually not disastrous if there are a few mix-ups. The activity is best with the right match, but it still works somewhat with some mismatches.

Citation: Pau, R. N., Eldridge, M. E., Lowe, D. J., Mitchenall, L. A. & Eady, R. R. Molybdenum-independent nitrogenases of Azotobacter vinelandii: a functional species of alternative nitrogenase-3 isolated from a molybdenum-tolerant strain contains an iron-molybdenum cofactor. Biochem. J. 293, 101–107 (1993).

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