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:
- nif+ hup-: 100%
- nif+ hup+: 75%
- nif- hup-: 50%
- nif- hup+: 25%
- nif+ hup-: 62%
- nif+ hup+: 5%
- nif- hup-: 100%
- nif- hup+: 3%
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).