610 - A Nitrogen Pressure of 50 Atmospheres does not Prevent Evolution of Hydrogen by Nitrogenase

Nitrogenase always seems to produce at least one hydrogen molecule per nitrogen fixed when fixing nitrogen. This study looked at whether very high pressures of pure nitrogen could prevent this wasteful side reaction.

What They Saw
They purified nitrogenase from Azotobacter vinelandii and put it in a chamber with 50-51 atmospheres of pure nitrogen gas. The chamber was designed so they could start and stop the reaction by tipping the chamber to add reagents that would start or stop it, without changing the atmosphere. Then they measured pressure in the chamber and amount of hydrogen.

Even at the high pressure of nitrogen in this experiment, the amount of hydrogen produced was about one-to-one with the amount of nitrogen fixed. So it must be pretty essential to the reaction.

Reference:

Simpson, F. B. & Burris, R. H. A Nitrogen Pressure of 50 Atmospheres does not Prevent Evolution of Hydrogen by Nitrogenase. Science 224, 1095–1097 (1984).

598 - Nitrogenase-catalyzed reactions

This paper looks at a bunch of possible substrates and reactions that nitrogenase catalyzes.

What They Saw
They extracted nitrogenase from Azotobacter vinelandii. ATP was necessary for any activity, of course; 3 mM was the optimal amount in their in vitro reactions, possibly because of the amount of magnesium ions that were present (5 mM). They saw 1.6 hydrogens per nitrogen fixed, on average, a bit higher than the normal figure of 1; about 33% of the energy flux went to hydrogen (normal figure is 25%).

With azide added, 1 mole azide is made into 1 mole ammonia and 1 mole dinitrogen. Lower levels allowed production of hydrogen instead, but that wasn't investigated much. The same was true with acetylene; both seem better substrates than nitrogen.

Cyanide is reduced to methane and ammonia; optimal concentrations were 2-4 mM. At other concentrations, there are other products formed: ethylene, ethane, methylamine (CH₃NH₂). They don't discuss hydrogen.

However, their calculations of ATP per electron pair transferred were skewed because they didn't measure all the products (i.e. hydrogen gas).

Reference:

Hwang, J. C. & Burris, R. H. Nitrogenase-catalyzed reactions. Biochim Biophys Acta 283, 339–350 (1972).

516 - Role of magnesium adenosine 5'-triphosphate in the hydrogen evolution reaction catalyzed by nitrogenase from Azotobacter vinelandii

Nitrogenase evolves hydrogen in a reaction that depends on ATP. The amount of ATP varies from 2 ATP per electron to over 20 (supposedly). The 4Fe-4S cluster in the dinitrogenase reductase donates one electron to the dinitrogenase, binding 2 ATP to do so. It seems like the ATP-powered flow of electrons can determine how many go toward hydrogen and how many toward nitrogen fixation. This study wanted to see if powering this flux was ATP's only role.

What They Saw
They purified enzyme from Azotobacter vinelandii and separated the two components, then mixed them with MgATP in in vitro assays. They observed that there was a burst of rapid ATP hydrolysis at first, which leveled off and remained fairly constant for the rest of the time. It seemed like the rate was the same as the rate of electron transfer between components. They confirmed the rate of about 2 ATP per electron; this makes sense with the dinitrogenase accepting two electrons at a time, one for each of its molybdenum atoms. The rate depended on the concentration of ATP, which makes sense and indicates that ATP is essential. This is also true when measuring the amount of hydrogen produced.

What This Means
They appeared to succeed in their goal of showing that ATP's only role in this reaction was powering the electron transfer, though some ATP can be used up to no purpose in some conditions.

Reference:

Hageman, R. V., Orme-Johnson, W. H. & Burris, R. H. Role of magnesium adenosine 5’-triphosphate in the hydrogen evolution reaction catalyzed by nitrogenase from Azotobacter vinelandii. Biochemistry 19, 2333–2342 (1980).

056 - Properties of Hydrogenase from Azotobacter vinelandii

Another study tries to purify the uptake hydrogenase from Azotobacter vinelandii.

What They Saw
They were unable to get pure, soluble enzyme; they couldn't separate it from the membrane, so it remained in insoluble particles.

They observed that hydrogenase activity of these particles didn't decrease after 18 days of exposure to air, unlike particles from other species. They did lose activity within a few weeks though.

They also observed that the enzyme reduces cytochrome C, possibly as part of its electron transport role. They found that carbon monoxide and cyanide inhibit the enzyme. Azide does not, at least not at pH 8.

What This Means
Some of the observations here may be due to the presence of other membrane-bound proteins, not just hydrogenase.

Reference:

Hyndman, L. A., Burris, R. H. & Wilson, P. W. Properties of Hydrogenase from Azotobacter vinelandii. J. Bacteriol. 65, 522–531 (1953).

049 - Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii

Azotobacter has a hydrogenase. Some organisms have hydrogenases that produce hydrogen, or sometimes produce and sometimes break down (reversible). Azotobacter's had not been observed to break down hydrogen (so was considered unidirectional). This study purified the membrane-bound uptake hydrogenase from A. vinelandii and tested its properties.

What They Saw
They measured activity of purified enzyme with an electrode measuring hydrogen oxidation in the presence of methylene blue dye (an electron acceptor).

They measured stability of the enzyme in the presence of oxygen, and found that crude extracts were very stable (and could go for weeks without losing activity), but the more pure the preparation, the less oxygen-tolerant it was: the most pure lost half its activity in 20 minutes at 20% oxygen. This inactivation was irreversible.

With a very good electron donor (methyl viologen), the hydrogenase could produce hydrogen. The highest rate they saw was 3.4 μmol hydrogen per minute per mg protein, which peaked at a fairly low pH (around 4). The rate was almost 0 closer to neutral. Also, the presence of hydrogen in the environment inhibits its production by the enzyme.

Even when electron acceptors were not present, the enzyme could combine one deuterium from D₂ with one hydrogen from water to make HD.

The enzyme seemed to be good at donating electrons to acceptors with positive mid-point potentials but not to negative ones, so it seems to have a higher potential than reversible hydrogenases.

Reference:

Kow, Y. W. & Burris, R. H. Purification and properties of membrane-bound hydrogenase from Azotobacter vinelandii. J Bacteriol 159, 564–569 (1984).