045 - Tungsten incorporation into Azotobacter vinelandii nitrogenase

Tungsten is known to cause problems for molybdenum-containing enzymes. This report looks into its effect on Azotobacter vinelandii's Mo nitrogenase.

What They Saw
They grew A. vinelandii OP (aka CA) in Burk without Mo, with added ammonium phosphate. Because it's really hard to get rid of every little bit of Mo, they added lots of tungsten (W) to make sure that they could see it if it got incorporated into enzymes. Some of the W was radioactive.

W didn't inhibit growth when ammonia was present, which makes sense. But it did inhibit it, about the same, with N2, nitrate, or urea. The enzymes that use these N sources all need Mo. When Mo was about 0.1 μM, it took 20 μM W to inhibit growth 50%; when Mo was 10 μM, it took 4 mM W. When just a little ammonia was added, it took about 5000 times more W than Mo to stop growth.

When they purified nitrogenase from these W-grown cells, they actually did see acetylene reduction activity, though not nearly as much as with normal Mo nitrogenase. The W content of extracts was very high, though it seemed to be easily removable. Specifically purifying Fe-W protein and comparing to the Fe-Mo version, all activities seemed relatively low: acetylene reduction, hydrogen production, and ATP hydrolysis.

What This Means
A. vinelandii might treat W the same as it treats Mo: taking up as much as it can and storing what it doesn't incorporate. But it does seem to incorporate some into the Mo nitrogenase. This seems to result in a poorly functional enzyme, but is that enough to stop cells from growing entirely? Maybe W's effects on other enzymes cause some problems too.

Reference:

Benemann, J. R., Smith, G. M., Kostel, P. J. & McKenna, C. E. Tungsten incorporation into Azotobacter vinelandii nitrogenase. FEBS Lett 29, 219–221 (1973).

182 - The electron transport system in nitrogen fixation by Azotobacter. III. Requirements for NADPH-supported nitrogenase activity

Nitrogenase requires a steady flow of reductant to reduce nitrogen gas to ammonia. This study looks at how electrons move from catabolism to the nitrogenase in Azotobacter vinelandii. Previous studies seemed to isolate two components of the transport chain to nitrogenase: azotoflavin and azotobacter ferredoxin. But it was still unknown how these compounds themselves were reduced, since A. vinelandii can't reduce them directly from pyruvate as others can (such as Clostridium pasteurianum).

What They Saw
They extracted cell components and fractionated them, separating the carriers mentioned above from nitrogenase. Then they tested the potential of NADH and NADPH to reduce these carriers, hypothesizing that these reductants are used for many other redox reactions.

This reaction seems energetically unfavorable because the redox potential of NAD(P)H is higher than that of ferredoxins, but it has been shown to happen before, especially when the NAD(P)H/NAD(P)⁺ ratio is high. So it's plausible.

They found that NADPH seemed to be able to reduce azotoflavin, and could support nitrogenase activity, at least in vitro. NADH did not seem to have the same activity. Adding extra azotoflavin or ferredoxin increased activity too.

There was one more factor that they didn't identify or include in their assays, one that they replaced with spinach ferredoxin-NADP⁺ reductase. Presumably it is some reductase in A. vinelandii that they didn't purify right. They did figure out which fraction contained it though, and it restored most activity. It could be denatured by mild heating, apparently.

Of other substrates that might support nitrogenase activity, only those linked to NADP⁺ seemed to be useful in vitro: malate, glucose-6-phosphate, alpha-ketoglutarate, and isocitrate. And apparently NADP⁺-linked isocitrate dehydrogenase is pretty common in A. vinelandii cells.

So it seems like NADPH is the donor that starts the electron transport branch to nitrogenase.

Reference:

Benemann, J. R., Yoch, D. C., Valentine, R. C. & Arnon, D. I. The electron transport system in nitrogen fixation by Azotobacter. III. Requirements for NADPH-supported nitrogenase activity. Biochim. Biophys. Acta 226, 205–212 (1971).