Wednesday, August 5, 2015

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.

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).

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