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.

Reference:

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

297 - The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H2-uptake hydrogenase of Azotobacter chroococcum

And finally, seeming to complete our journey back in time through the discovery of hydrogenase genetics in Azotobacter chroococcum, this study looks at the structural genes, hupSL.

What They Saw
The sequences were similar to A. vinelandii's hoxKG structural genes. They tried knocking each out, then measuring hydrogen oxidation (with methylene blue) and hydrogen production (with methyl viologen). As expected, hydrogen oxidation in mutants was no higher than negative controls. Surprisingly, they did see hydrogen production in the some of the different mutants with a strong electron donor, but it was less than in the wild-type. Only the mutant with an insertion very close to the start of the hupS gene had no hydrogen production.

What This Means
It seems like a fragment of HupS is sufficient to produce hydrogen with a strong electron donor, but not as much as with both HupS and HupL completely intact.

Reference:

Ford, C. M. et al. The identification, characterization, sequencing and mutagenesis of the genes (hupSL) encoding the small and large subunits of the H₂-uptake hydrogenase of Azotobacter chroococcum. Mol Microbiol 4, 999–1008 (1990).

067 - Analysis of a gene region required for dihydrogen oxidation in Azotobacter vinelandii

This study looks more closely into the genes discovered in previous studies (518,050,051,052) to be required for hydrogenase in Azotobacter vinelandii.

What They Saw
They knocked out different genes (or the whole operon) in the hyp operons of A. vinelandii CA by inserting resistance + lacZ cassettes, then tested these strains for hydrogen oxidation and expression of the genes (via beta-galactosidase activity). The medium they used had a lot more trace elements than typical Burk medium, including nickel.

They found that when lacZ was inserted in the same direction as the gene, they saw expression in all cases, whether fixing nitrogen or not, but if it was inserted in the opposite direction, they didn't. So apparently the genes are expressed to some extent even when not fixing nitrogen.

When they measured hydrogen oxidation, there was about 6x more when fixing nitrogen though, in the wild-type, and none in the mutants. The growth rates of the mutants were similar to the wild-type though, or so they claim without reporting any details.

Finally they tried growing the strains with extra added nickel, because the hydrogenase is a nickel-containing enzyme. This didn't have much effect on the wild-type, but the hypB mutant actually showed some hydrogenase activity in nitrogen-fixing conditions with the extra nickel, and activity was higher with more nickel added.

What This Means
It seems like extra added nickel can substitute for the lack of HypB, so maybe the enzyme has a role in nickel cofactor processing somehow. This makes sense considering the multiple histidines it contains. It's interesting that nickel didn't help activity in non-fixing conditions; maybe there are multiple hydrogenases, active in different conditions, and hypB is required for all but nickel doesn't help some of them.

Reference:

Chen, J. C., Mortenson, L. E. & Seefeldt, L. C. Analysis of a gene region required for dihydrogen oxidation in Azotobacter vinelandii. Curr. Microbiol. 30, 351–355 (1995).

053 - Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing αβ dimer

This study purified and characterized the uptake hydrogenase from Azotobacter vinelandii.

What They Saw
As a single unit, not denatured, the hydrogenase ran as a single band on a gel. On a denaturing gel it was two bands though, 31 and 67 kDa, indicating that it had two subunits, a larger and a smaller (HoxG and HoxK). The whole thing is about 98.6 kDa. This contrasts with previous reports that it was a single subunit of 60 kDa (049), but it's not really clear why there's this discrepancy.

They also measured metal contents, and found 6.6 mol iron and 0.68 mol nickel per mol hydrogenase, so they rounded up to 1 mol Ni and 10 mol Fe, 1:10 ratio.

They also contradicted the previous result that the hydrogenase couldn't donate to acceptors with negative potential. But they confirmed the possibility that hydrogenase produces hydrogen when paired with a very reduced donor, except this peaked at a higher pH (6 to 8.5).

Reference:

Seefeldt, L. C. & Arp, D. J. Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing αβ dimer. Biochimie 68, 25–34 (1986).