307 - The Relationship Between Hydrogenase and Nitrogenase in Azotobacter chroococcum: Effect of Nitrogen Sources on Hydrogenase Activity

This study looked at the influence of different sources of nitrogen on the activity of nitrogenase and hydrogenase in Azotobacter chroococcum.

What They Saw
They grew cells in batch or continuous culture with sodium nitrate, ammonium acetate or chloride, or dinitrogen gas. Cultures were either carbon- or sulfate-limited. Dissolved oxygen was kept above zero. Nitrogenase activity was measured by acetylene reduction and hydrogenase by methylene blue (or by adding H-T with radioactive tritium and measuring radioactivity of resulting water when oxygen was the electron acceptor).

There was about twice as much hydrogenase activity when cells were fixing nitrogen in batch than when they had either ammonium or nitrate. They cite other results in A. chroococcum and A. vinelandii that showed higher activity with nitrate than ammonium though, but still less than when fixing nitrogen. This might be because cells have to adapt to use nitrate, and they'll be fixing nitrogen before that happens. But care is necessary because activity changes over the course of a batch culture, increasing throughout exponential phase even when standardized by protein concentration.

In continuous cultures, they started growing with ammonium, then switched to nitrogen-free, watched what happened with hydrogenase and nitrogenase, and then pulsed a limited amount of ammonium. As expected, nitrogenase activity started up and rose to a plateau, then immediately stopped when ammonium was added, and restarted when it was removed. Hydrogenase activity showed a similar pattern, though more delayed, and it never went all the way to zero.

In sulfate-limited culture, hydrogenase activity was lower even when fixing nitrogen, about the same as with ammonium in carbon limitation, and when going from fixing to non-fixing (with ammonium), the activity declined a bit but then went back up to about the same level. Results were similar going from non-fixing to fixing. So with sulfate limitation, nitrogen source doesn't matter much.

Also with nitrogen-fixing cells in sulfate limitation, when they increased the dilution rate, hydrogenase activity decreased but nitrogenase increased. The decrease wasn't linear, though; it leveled off.

They also tested whether adding hydrogen to the atmosphere of an ammonium-grown culture would influence hydrogenase activity, and it did! Hydrogenase activity doubled. This was not the case with sulfate limitation though, only carbon limitation. They didn't test nitrogen-fixing cells.

What This Means
The continuous culture experiments helped overcome the constantly changing activity in batch cultures.

That hydrogenase activity lags behind nitrogenase activity increase when ammonium runs out could be explained by the last observation: maybe nitrogenase starts producing hydrogen (as it does) and this stimulates hydrogenase activity.

They reasoned from the data that excess carbon might inhibit hydrogenase activity somehow (like catabolite repression). I'm not sure that makes sense, but it seems possible, and does fit with the data from batch cultures, sulfate limitations, and increasing dilution rates. Interesting.

Reference:

Partridge, C. D. P., Walker, C. C., Yates, M. G. & Postgate, J. R. The Relationship Between Hydrogenase and Nitrogenase in Azotobacter chroococcum: Effect of Nitrogen Sources on Hydrogenase Activity. J Gen Microbiol 119, 313–319 (1980).

113 - The hydrogen cycle in nitrogen-fixing Azotobacter chroococcum

Azotobacter chroococcum makes hydrogen when fixing nitrogen, but its uptake hydrogenase reoxidizes hydrogen. It wasn't clear what the purpose of this reoxidation is, or in what conditions it's useful, or how much hydrogen is produced in different conditions, so this study aimed to find out.

What They Saw
When they had bacteria in carbon-free broth, with hydrogen present in the atmosphere, the bacteria were able to reduce acetylene with nitrogenase using only energy from the hydrogen. No such activity was observed without hydrogen present. Even when mannitol was added up to 2 g/L, adding hydrogen still increased the acetylene reduction activity, though the proportion of activity attributable to hydrogen decreased as mannitol increased, though surprisingly it leveled off above zero even when mannitol wasn't the limiting nutrient anymore; it's possible that electron transfer from hydrogen works differently.

They also tried increasing oxygen levels with a little mannitol; when hydrogen was absent, oxygen became inhibitory about twice as fast as when hydrogen was present, so hydrogenase seems to help protect the nitrogenase. The effect went down to around zero as mannitol increased though.

They looked at hydrogen production when fixing nitrogen with various limitations (carbon, nitrogen, oxygen) in continuous culture; unlike in batch culture, cells seemed to evolve significant hydrogen. They compared hydrogen produced in air to that produced when replacing air with argon to get the proportion of nitrogenase activity going to hydrogen (presumably in air, the remainder goes to actually fixing nitrogen), and found that under oxygen or nitrogen limitation (whatever that means here), the proportion was 40-50% going to hydrogen. In carbon limitation, it was lower, around 13%, but they said that hydrogenase activity was higher in this case (for some reason) so it doesn't represent the true proportion (since not all hydrogen is observed).

What This Means
Since hydrogen could protect nitrogenase from oxygen, it seems like its electrons go to oxygen through the respiratory chain rather than to power nitrogenase activity.

It is somewhat puzzling that the hydrogenase would work so well when acetylene is present, since acetylene has been shown to inhibit the hydrogenase (112). They observed that in this study too. So it's possible that the hydrogenase might be even more useful when acetylene is not present. But 40% acetylene is required to completely inactivate hydrogenase, whereas they only used 8% in the activity assays.

40-50% electron flux going to hydrogen is higher than estimated by others, at least for the molybdenum nitrogenase, but it's unclear the effect of the limitations imposed.

Here's the model they propose:

Walker and Yates, 1978

Reference:

Walker, C. C. & Yates, M. G. The hydrogen cycle in nitrogen-fixing Azotobacter chroococcum. Biochimie 60, 225–231 (1978).

112 - The Effect of Nutrient Limitation on Hydrogen Production by Nitrogenase in Continuous Cultures of Azotobacter chroococcum

That nitrogenase produces hydrogen gas had been known for a while; this study wanted to see how different nutrient limitations affected this phenomenon.

They grew Azotobacter chroococcum on mannitol B medium in continuous culture, with limitations in carbon, sulfate, oxygen, or dinitrogen. Then they measured acetylene reduction, hydrogen production, and oxygen consumption in vivo and the former two on purified nitrogenase. Also update hydrogenase activity directly.

What They Saw

There was very little hydrogen evolved by oxygen-limited cultures in air, unless the uptake hydrogenase was inhibited by acetylene first. Replacing air with an argon/oxygen/CO₂ mix (without nitrogen) also increased hydrogen evolution to a similar level. With both treatments, the hydrogen seen was much higher.

When limited in sulfate, the hydrogen produced under air (with hydrogenase active) or under argon mix seemed much higher (at least proportional to the amount of protein in cells).

Under argon mixes with different proportions of oxygen, hydrogen evolution seemed highest around 10% oxygen. With nitrogen instead of argon, the peak was similar. Too much or too little oxygen was not good. And despite the high amount of hydrogen, around 10% (actually between 6-12%) was when the most nitrogen was being fixed too, such that the ratio of hydrogen produced to nitrogen fixed was as low as 1 (or 0.5, when sulfate-limited).

In carbon-limited cultures, though, the optimum oxygen value was 3%, though oxygen consumption increased as oxygen increased, at least up to 6%.

In vitro, the hydrogen-nitrogen ratio increased as the ratio of dinitrogenase to dinitrogenase reductase increased, though higher levels of ATP decreased this effect. Sulfate limitation didn't really affect this finding.

What This Means
This makes sense; some oxygen is required to generate ATP to power the nitrogenase, but too much oxygen requires extra carbon to detoxify it, so there's less energy for nitrogenase.

It seems like a lack of dinitrogenase reductase or ATP reduces the ability to fix nitrogen instead of just producing hydrogen. I wonder if dinitrogenase has any effect on its own in the absence of ATP or its other component.

Chemically it's unclear how or why nitrogenase produces hydrogen, but it seems to be an essential part of the nitrogen fixation process.

Reference:

Walker, C. C., Partridge, C. D. P. & Yates, M. G. The Effect of Nutrient Limitation on Hydrogen Production by Nitrogenase in Continuous Cultures of Azotobacter chroococcum. J. Gen. Microbiol. 124, 317–327 (1981).