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).