Wednesday, August 19, 2015

571 - Oxygen effects on the nickel- and iron-containing hydrogenase from Azotobacter vinelandii

This study looks at how oxygen affects the uptake hydrogenase of Azotobacter vinelandii.

What They Saw
They grew A. vinelandii OP (aka CA) and purified its membrane-bound hydrogenase. When purified anaerobically, it was fully active with an electron acceptor other than oxygen (methylene blue or benzylviologen). Added oxygen appeared to inhibit this reduction of methylene blue, and this was noncompetitive inhibition (adding extra methylene blue didn't relieve it).

When oxygen was removed by adding an oxygen-binding protein (leghemoglobin), the inhibition was reversed and activity recovered.

They claim the membrane-associated hydrogenase in these experiments was incapable of reducing the oxygen; it's not clear if being more capable would change the results, but it seems likely.

There was also slower, irreversible inactivation, shown by adding oxygen to an assay and adding enough dithionite to consume all of it to remove any effect of reversible inhibition. Over time, the enzyme lost activity, whether aerobically or anaerobically purified. Purified enzyme lost more activity more quickly than membrane-bound. It seemed like activity was only lost when the enzymes were exposed when active, but simply activating them didn't reproduce the effect. It was a confusing assay.

They did find that adding hydrogen could provide protection from inactivation, up to almost 100% protection, but neither hydrogen nor oxygen was consumed during this process. Super weird.

Finally, carbon monoxide didn't help protect the enzyme from oxygen at all, nor did affect protection by hydrogen.

What This Means
It's interesting, but probably not that important physiologically. A. vinelandii is capable of withstanding high levels of oxygen, and such high levels are just as likely to inhibit the nitrogenase which produces the hydrogen as the hydrogenase which consumes it. It might be interesting to study whether oxygen inhibits the oxidation of added hydrogen though.

Seefeldt, L. C. & Arp, D. J. Oxygen effects on the nickel- and iron-containing hydrogenase from Azotobacter vinelandii. Biochemistry 28, 1588–1596 (1989).

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