340 - Kinetic studies of the nitrogenase-catalyzed hydrogen evolution and nitrogen reduction reactions

This study looked at the kinetics of hydrogen production from purified nitrogenase; also, the nitrogen fixation reaction.

What They Saw
This nitrogenase was purified from Azotobacter vinelandii; they don't give details on culture conditions, so presumably it's the molybdenum version.

First, they observed that more ATP meant more hydrogen over time. The free phosphate-to-hydrogen ratio was similar at all levels though, so that makes sense. The same pattern was seen for nitrogen fixation, except the proportion of electron flux going to ammonia increased as ATP increased; more hydrogen was produced at lower ATP, relative to ammonia.

They found that whether under nitrogen or argon, the electron flux was the same; this was true over multiple ATP concentrations.

Reference:

Silverstein, R. & Bulen, W. A. Kinetic studies of the nitrogenase-catalyzed hydrogen evolution and nitrogen reduction reactions. Biochemistry 9, 3809–3815 (1970).

335 - ATP-Dependent hydrogen evolution by cell-free preparations of Azotobacter vinelandii

This study looked at hydrogen production in Azotobacter vinelandii strain O, to see what induced it in cell extracts.

What They Saw
Hydrogen production depended on ATP. Argon or hydrogen in the atmosphere didn't matter. The higher the protein concentration, the more hydrogen was produced. At 0ºC in air, the enzyme was pretty stable; over 90% activity was left after 3 days.

Hydrogen oxidation activity was found in separate fractions from the production activity, so they concluded it was a different enzyme.

Comparing extracts from cells grown with urea or no fixed nitrogen, they saw no hydrogen production activity in urea samples, but the hydrogen oxidation activity with urea was lower than that without.

What This Means
We know now that the hydrogen production comes from nitrogenase and the hydrogen oxidation from hydrogenase. It's surprising how air-stable the nitrogenase seemed to be outside the context of the cell, but I guess it was still surrounded by cellular elements in the crude extract. Also I'm not sure how oxygen inactivation affects nitrogenase hydrogen production.

Reference:

Burns, R. C. & Bulen, W. A. ATP-Dependent hydrogen evolution by cell-free preparations of Azotobacter vinelandii. Biochim Biophys Acta 105, 437–445 (1965).