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