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).

570 - Kinetic analysis of the interaction of nitric oxide with the membrane-associated, nickel and iron-sulfur-containing hydrogenase from Azotobacter vinelandii

This study looked at the effect of nitric oxide (NO) on Azotobacter vinelandii's uptake hydrogenase.

What They Saw
They isolated hydrogenases from cells but didn't separate them from the membrane, because that made them too sensitive to oxygen. When activated in a reducing environment and then exposed to NO, hydrogenase activity was inhibited, but this inhibition could be almost completely reversed by adding iron EDTA, which reacts with NO. The more NO, the more inhibition (relative to the no NO control).

When NO was added and the hydrogenase wasn't active, the inactivation was irreversible; the longer the exposure, the more the inhibition, but it took larger amounts of NO to get the same amount of inhibition as the reversible kind. Hydrogen or carbon monoxide didn't protect against this inactivation; hydrogen even enhanced the effect.

Reference:

Hyman, M. R. & Arp, D. J. Kinetic analysis of the interaction of nitric oxide with the membrane-associated, nickel and iron-sulfur-containing hydrogenase from Azotobacter vinelandii. Biochim Biophys Acta 1076, 165–172 (1991).

569 - Hydrogen-oxidizing electron transport components in nitrogen-fixing Azotobacter vinelandii

This study looks at oxidation of hydrogen by Azotobacter vinelandii's uptake hydrogenase, and which proteins are involved in the electron transport chain.

What They Saw
They grew A. vinelandii CA fixing nitrogen, and isolated the membrane fraction from the cells. They looked at oxygen uptake, and saw that unless there were oxidizable substrates, there was no consumption of oxygen, which makes sense. Hydrogen fulfilled the requirement though, and there were two hydrogen molecules taken up for each molecule of oxygen, which makes sense: two hydrogen atoms for each atom of oxygen, to make H₂O.

Using spectrophotometry, they observed peaks that occurred when components in the membrane were reduced with hydrogen, malate, or dithionite. Hydrogen affected cytochrome d (showing a peak at 627nm), b (shoulder at 559nm) and c (peak at 550), but not a (595). The other reductants affected b a lot more, and a somewhat.

With carbon monoxide added, hydrogen only reduced cytochrome d. The others showed a new peak, cytochrome o, at 417nm, but hydrogen didn't. The same seemed true with low levels of cyanide; so hydrogenase's terminal oxidase seems to be cytochrome d type. Not really sure how they prevented these inhibitors from inhibiting the hydrogenase itself, like they seem to in other studies.

Reference:

Wong, T. Y. & Maier, R. J. Hydrogen-oxidizing electron transport components in nitrogen-fixing Azotobacter vinelandii. J. Bacteriol. 159, 348–352 (1984).

525 - Hydrogen-mediated mannose uptake in Azotobacter vinelandii

This study looked at Azotobacter vinelandii's ability to use hydrogen gas to power its uptake of the sugar mannose.

What They Saw
They grew A. vinelandii CA in Burk broth but with mannose instead of glucose or sucrose, and either hydrogen or argon in the atmosphere (along with nitrogen and oxygen). They used ¹⁴C mannose to observe its uptake via the radioactivity of the isotope.

The increase in radioactivity from mannose activity was a lot higher in cells given hydrogen than those without, up to 5-fold.

They tried inhibiting respiration to see if that was related to this effect, and found that usually by inhibiting respiration, they could inhibit the increased mannose uptake, so it seems to be respiration-dependent rather than some sort of regulatory effect.

So this seems to be another of hydrogen's possible roles in the energy metabolism of Azotobacter.

Reference:

Maier, R. J. & Prosser, J. Hydrogen-mediated mannose uptake in Azotobacter vinelandii. J. Bacteriol. 170, 1986–1989 (1988).

524 - In vivo and in vitro nickel-dependent processing of the [NiFe] hydrogenase in Azotobacter vinelandii

This study looked at Azotobacter vinelandii's hydrogenase again, its post-translational processing, and whether nickel influenced this process.

What They Saw
The normal Azotobacter medium (Burk's) has enough contaminating nickel that adding it is unnecessary. But when they added a chelator (nitrilotriacetate) to bind it up, the hydrogenase activity decreased by 80% without affecting growth. This inhibition was lessened by adding nickel.

Nickel availability seemed to affect which form of the alpha subunit was present: the larger, unprocessed form, or the smaller, mature form. With nickel available, only the smaller form was seen; when it was bound up, only the larger. But when excess nickel was added, following the proteins over time showed that gradually the population shifted from larger to smaller as the nickel was used. These two forms are found in different places: the smaller is bound to the membrane (as it should be), and the larger is soluble.

Inhibiting protein synthesis, such as with chloramphenicol, and then adding nickel led to a similar increase in activity as a control without an inhibitor, up to 70 minutes; so for this period, increasing activity wasn't due to protein synthesis. But after this point, the inhibited cultures stopped increasing while the uninhibited continued. The processing of the large form into the small continued regardless of inhibition. So it seems that nickel is important partially for processing and partially for stimulating protein synthesis.

In vitro, ATP or GTP was important for processing. Membranes and oxygen (or lack thereof) were not important. No divalent cation could substitute for nickel: zinc inhibited processing completely, and cobalt or calcium some too. The only protease inhibitor that prevented processing was 1,10-phenanthroline, which inhibits metal-activated proteases.

What This Means
It seems that nickel and processing are both essential for hydrogenase activity, and apparently they are interrelated. It's possible that the processing is regulated by the presence of nickel; without the metal, there isn't much point. Or maybe processing without nickel available will lead to nonfunctional product that can't be fixed. Alternatively, the protease that does the processing could require nickel. It's hard to distinguish these possibilities though. Anyway, it seems like when nickel is absent, the hydrogenase subunits are present but in a premature form, waiting for nickel. How poetic.

Reference:

Menon, A. L. & Robson, R. L. In vivo and in vitro nickel-dependent processing of the [NiFe] hydrogenase in Azotobacter vinelandii. J. Bacteriol. 176, 291–295 (1994).

523 - Carboxyl-terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii

Based on amino acid prediction from gene sequence, the HoxG alpha subunit of the uptake hydrogenase should be about 66.6 kDa, but in the wild-type it appears smaller. In some mutants with accessory genes knocked out, the size matches this number. So this study tried to figure out if post-translational processing was involved in producing active enzyme. N-terminal modification was already ruled out, as that sequence matches the prediction.

What They Saw
They grew Azotobacter vinelandii CA and purified its hydrogenase, then studied its subunits with mass spectrometry.

They observed that the actual size of the larger subunit was 64.9 kDa, smaller than the 66.6 predicted size. The N-terminal was still the same as predicted, so they concluded that about 15 amino acids had been removed from the C-terminal of the subunit. This appears to be necessary for it to function.

Reference:

Gollin, D. J., Mortenson, L. E. & Robson, R. L. Carboxyl-terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii. FEBS Letters 309, 371–375 (1992).

515 - Hydrogen Uptake and Methylene Blue Reduction Activities of Hydrogenase in Azotobacter agile

This study used tritium (³H, a radioactive isotope of hydrogen) uptake to look at hydrogenase and nitrogenase activity in Azotobacter agile (aka A. agilis I think).

What They Saw
The tritium was ditritium gas, similar to dihydrogen. They purified and fractionated protein from the bacteria and tested the fractions for tritium uptake, methylene blue reduction, and acetylene reduction. They found that acetylene reduction and the other two were found in separate fractions (makes sense; one's nitrogenase and the others hydrogenase). Tritium uptake could be stimulated with ATP somehow.

Tritium uptake and methylene blue reduction mostly went together in terms of fractionation, but there was a little of the latter in some fractions where the former wasn't observed. So there could be something else reducing methylene blue.

They found that carbon monoxide (CO) inhibited tritium uptake, though it was less inhibitory when ATP was present.

What This Means
This supports the idea that nitrogenase produces hydrogen while hydrogenase oxidizes it and reduces electron acceptors such as methylene blue. It's weird that ATP should stimulate that though, and also weird that some fractions had reduction activity but not hydrogen oxidation.

Reference:

Suzuki, T., Maruyama, Y. & Nakamura, M. Hydrogen Uptake and Methylene Blue Reduction Activities of Hydrogenase in Azotobacter agile. Agricultural and Biological Chemistry 43, 2067–2073 (1979).

457 - Hydrogenase and Nitrogen Fixation by Azotobacter

This study looked at hydrogenase in different Azotobacter species (A. vinelandii, A. chroococcum, A. agile whatever that is).

What They Saw
They looked at different kinds and amounts of fixed nitrogen and their effect and different gases in the atmosphere. Many experiments used ammonium phosphate or other forms of ammonium, and they thought maybe the drop in pH seen as ammonium was consumed led to decreased hydrogenase activity, but actually even when they used forms that didn't allow a pH drop, they still saw the same decrease, suggesting that it's the fixed nitrogen itself that leads to decreased activity. Which makes sense.

They found, consistent across species, that ammonium led to the biggest activity decrease, about 60-80%; nitrate as little as 20%; and glutamate hardly at all. I think these cultures were not adapted to these compounds though.

So they tried adapted cultures too. They found that the more fixed nitrogen they added, the less hydrogenase activity they saw. Adaptation didn't matter with ammonium, but cultures adapted to nitrate had more of a decrease in activity. Apparently they didn't test glutamate.

Then they compared cells with various nitrogen sources grown in air or in a hydrogen-oxygen mixture. They didn't test cells without a nitrogen source in this gas mixture though, maybe because they couldn't grow. Anyway, the hydrogenase was always more active in air with no fixed nitrogen than with any kind of fixed nitrogen (as seen before), and with H₂-O₂ the activity seemed even lower, even than with the same fixed nitrogen source in air. Activity was almost zero in nitrate-adapted cells given nitrate. This seems odd; previous studies seemed to show that hydrogen stimulated hydrogenase activity.

What This Means
I'd say other studies showing stimulation by hydrogen were more convincing, but at least this one was consistent showing an adaptation effect and down-regulation in the presence of fixed nitrogen.

Reference:

Lee, S. B. & Wilson, P. W. Hydrogenase and Nitrogen Fixation by Azotobacter. J. Biol. Chem. 151, 377–385 (1943).

454 - Activity of the H2-oxidizing hydrogenase in different N2-fixing bacteria

Despite some studies suggesting that hydrogen stimulates hydrogenase, other data suggested it does not. So the people who generated this data did this study on various species, including Azotobacter vinelandii CA, and claimed that low oxygen stimulated hydrogenase activity.

What They Saw
They grew A. vinelandii with ammonium chloride and measured hydrogenase activity with different electron acceptors (oxygen, methylene blue, etc). As A. vinelandii grew, it used up the dissolved oxygen, and hydrogenase activity went up but then back down after the oxygen was gone (when oxygen or iron cyanide were the electron acceptors, it went to zero; otherwise it didn't go all the way to zero). This was all in the presence of ammonium.

What This Means
Based on other studies, I wouldn't expect much activity from hydrogenase in general when growing with fixed nitrogen. I'm not sure how to interpret these results, especially with electron acceptors other than oxygen, but I guess it would make sense if hydrogenase were somewhat downregulated in low-oxygen conditions, even if other acceptors were present.

Reference:

Pinkwart, M., Bahl, H., Reimer, M., Wölfle, D. & Berndt, H. Activity of the H₂-oxidizing hydrogenase in different N₂-fixing bacteria. FEMS Microbiology Letters 6, 177–181 (1979).

453 - Direct mass-spectrometric determination of the relationship between respiration, hydrogenase and nitrogenase activities in Azotobacter chroococcum

This study looked at hydrogen and its relationship to different enzymatic processes in Azotobacter chroococcum.

What They Saw
The organism was grown in continuous culture with limited oxygen, 5% glucose. Samples were removed and sparged with different mixtures of argon, oxygen, and deuterium. Gases are measured by mass spectrometer.

Their figure is pretty confusing, poorly designed, and poorly described, but as far as I can tell, when they added either 80% argon with 20% oxygen or 70% argon, 20% oxygen, and 10% deuterium, the oxygen goes down to near zero within about 5 minutes either way, at which point hydrogen production starts increasing and deuterium uptake slows down or stops. So it seems like oxygen is required for hydrogenase to work.

They tried again with the addition of some carbon monoxide and acetylene to inhibit hydrogenase. This didn't really change the oxygen consumption, but deuterium consumption was a lot lower. Hydrogen evolution was the same.

What This Means
The deuterium consumption is by hydrogenase, of course, and it seems like oxygen is a necessary electron acceptor for it to function (in the absence of something else). But it seems like the increase in hydrogen evolution is not because hydrogenase stopped working, but rather because nitrogenase started, as oxygen stopped interfering. This is something we've seen before.

Oxygen is important for many things: it provides energy by accepting electrons, powering the nitrogenase and allowing more hydrogen production. It accepts electrons from hydrogenase, enabling hydrogen oxidation. It inhibits nitrogenase, reducing hydrogen production. Pretty confusing.

Reference:

Lespinat, P.-A., Gerster, R. & Berlier, Y. Direct mass-spectrometric determination of the relationship between respiration, hydrogenase and nitrogenase activities in Azotobacter chroococcum. Biochimie 60, 339–341 (1978).

362 - Mutants of Azotobacter chroococcum Defective in Hydrogenase Activity

This study isolated some hydrogenase-negative mutants of Azotobacter chroococcum by chemical mutagenesis and looked at how they behaved.

What They Saw
Almost all of the 16 mutants had almost no hydrogenase activity, as expected. Some had a little, <2% of wild-type. Some more had a little hydrogen-producing activity in the right conditions, usually less than 7% of the wild-type, but one had 40% of wild-type. That one also seemed to have a relatively active soluble hydrogenase (possibly the uptake hydrogenase in soluble form). All of them seemed able to take up nickel.

The one weirdest mutant, MCD-124, showed max activity at a different pH (5.5 instead of 8) and was weird in other ways.

Also, the authors were surprised by the frequency with which they could get hydrogenase mutants. They wondered whether the relevant genes were just more susceptible, or if the growth medium was more favorable to mutants somehow, or if there were just that many necessary genes. But judging from the genome sequence, this isn't quite a sufficient explanation.

Overall, it's hard to know exactly what's going on in this study.

Reference:

Yates, M. G. & Robson, R. L. Mutants of Azotobacter chroococcum Defective in Hydrogenase Activity. J Gen Microbiol 131, 1459–1466 (1985).

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).

308 - Hydrogen-mediated enhancement of hydrogenase expression in Azotobacter vinelandii

This study looked at whether added hydrogen could stimulate hydrogenase activity in Azotobacter vinelandii.

What They Saw
They grew cells with or without ammonium, then added argon or hydrogen to their headspace, and measured whole-cell or purified hydrogenase activity. Oxygen or methylene blue were electron acceptors.

With ammonium, there was a little activity, but adding hydrogen gas increased it about 2.5 to 5 times. As a control, injecting the same amount of argon didn't change anything. In nitrogen-fixing cells, adding hydrogen didn't affect activity.

As with others, activity increased over time in the culture, even corrected by biomass; the hypothesis was that excess carbon inhibits it somehow.

If they added an mRNA or protein synthesis inhibitor (rifampin or chloramphenicol) before adding the hydrogen, activity didn't increase with either case, so it seemed like the regulation was transcriptional.

Also, since the effect was the same with methylene blue (which doesn't require electron transport chain components to act as electron acceptor), it seemed that the regulation was at the hydrogenase directly rather than a related component.

Comparing a couple of Mo nitrogenase-deficient strains (CA11 and CA30) to their parent, they saw that hydrogen didn't affect hydrogenase activity in CA much (in nitrogen-fixing conditions), but it did increase the activity a lot in the mutants. The hydrogenase protein abundance increased too. But in conditions with ammonium, CA and CA11 behaved pretty similar.

Reference:

Prosser, J., Graham, L. & Maier, R. J. Hydrogen-mediated enhancement of hydrogenase expression in Azotobacter vinelandii. J. Bacteriol. 170, 1990–1993 (1988).

307 - The Relationship Between Hydrogenase and Nitrogenase in Azotobacter chroococcum: Effect of Nitrogen Sources on Hydrogenase Activity

This study looked at the influence of different sources of nitrogen on the activity of nitrogenase and hydrogenase in Azotobacter chroococcum.

What They Saw
They grew cells in batch or continuous culture with sodium nitrate, ammonium acetate or chloride, or dinitrogen gas. Cultures were either carbon- or sulfate-limited. Dissolved oxygen was kept above zero. Nitrogenase activity was measured by acetylene reduction and hydrogenase by methylene blue (or by adding H-T with radioactive tritium and measuring radioactivity of resulting water when oxygen was the electron acceptor).

There was about twice as much hydrogenase activity when cells were fixing nitrogen in batch than when they had either ammonium or nitrate. They cite other results in A. chroococcum and A. vinelandii that showed higher activity with nitrate than ammonium though, but still less than when fixing nitrogen. This might be because cells have to adapt to use nitrate, and they'll be fixing nitrogen before that happens. But care is necessary because activity changes over the course of a batch culture, increasing throughout exponential phase even when standardized by protein concentration.

In continuous cultures, they started growing with ammonium, then switched to nitrogen-free, watched what happened with hydrogenase and nitrogenase, and then pulsed a limited amount of ammonium. As expected, nitrogenase activity started up and rose to a plateau, then immediately stopped when ammonium was added, and restarted when it was removed. Hydrogenase activity showed a similar pattern, though more delayed, and it never went all the way to zero.

In sulfate-limited culture, hydrogenase activity was lower even when fixing nitrogen, about the same as with ammonium in carbon limitation, and when going from fixing to non-fixing (with ammonium), the activity declined a bit but then went back up to about the same level. Results were similar going from non-fixing to fixing. So with sulfate limitation, nitrogen source doesn't matter much.

Also with nitrogen-fixing cells in sulfate limitation, when they increased the dilution rate, hydrogenase activity decreased but nitrogenase increased. The decrease wasn't linear, though; it leveled off.

They also tested whether adding hydrogen to the atmosphere of an ammonium-grown culture would influence hydrogenase activity, and it did! Hydrogenase activity doubled. This was not the case with sulfate limitation though, only carbon limitation. They didn't test nitrogen-fixing cells.

What This Means
The continuous culture experiments helped overcome the constantly changing activity in batch cultures.

That hydrogenase activity lags behind nitrogenase activity increase when ammonium runs out could be explained by the last observation: maybe nitrogenase starts producing hydrogen (as it does) and this stimulates hydrogenase activity.

They reasoned from the data that excess carbon might inhibit hydrogenase activity somehow (like catabolite repression). I'm not sure that makes sense, but it seems possible, and does fit with the data from batch cultures, sulfate limitations, and increasing dilution rates. Interesting.

Reference:

Partridge, C. D. P., Walker, C. C., Yates, M. G. & Postgate, J. R. The Relationship Between Hydrogenase and Nitrogenase in Azotobacter chroococcum: Effect of Nitrogen Sources on Hydrogenase Activity. J Gen Microbiol 119, 313–319 (1980).

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).

296 - The Azotobacter chroococcum hydrogenase gene cluster: sequences and genetic analysis of four accessory genes, hup A, hupB, hupY and hupC

So at this point, hupSL (hydrogenase structural genes) and hupDE (accessory genes at the end of the operon) had already been identified. This study found a few more upstream of hupDE.

What They Saw
They sequenced the DNA upstream of hupDE and found four open reading frames, which they called hupABYC. The AB and C were similar to E. coli genes, but the Y wasn't, so they called it Y (for Ynknown, I guess). These were all homologous to A. vinelandii genes though, and in the same order.

Then they tried knocking out each of these. Each knockout was unable to oxidize hydrogen, even in the presence of methylene blue as an electron acceptor.

They also made a fusion of HupL (the structural subunit) and beta-galactosidase, then knocked out hupY or hupB to see if this changed the expression of hupL. Beta-galactosidase activity rose a little bit, like 25% in each, but it didn't seem either was an important regulator.

Reference:

Tibelius, K. H., Du, L., Tito, D. & Stejskal, F. The Azotobacter chroococcum hydrogenase gene cluster: sequences and genetic analysis of four accessory genes, hup A, hupB, hupY and hupC. Gene 127, 53–61 (1993).

295 - Sequences, organization and analysis of the hupZMNOQRTV genes from the Azotobacter chroococcum hydrogenase gene cluster

This study focuses on the hydrogenase genes in Azotobacter chroococcum. They had already found hupSL encoding the structural genes (equivalent to A. vinelandii's hoxKG I guess), and then the accessory genes hupABYCDE further downstream (homologous to A. vinelandii's hypABFCDE). Now, in between, are the hupZMNOQRTV genes, completing the 16-gene operon.

What They Saw
This set of genes seems to be homologous to A. vinelandii's hoxZMLOQRTV string, making the whole operon very similar in both organisms.

As in previous studies, HupZ (and its analog, HoxZ) seems to be an electron carrier in the membrane, possibly a cytochrome. When they knocked it out, they observed similar results to 070: hydrogenase could oxidize hydrogen with methylene blue as an electron acceptor, but not with oxygen, so HupZ seems to be part of the transport chain to oxygen.

Based on comparison with homologs in other organisms, HupM may help the hydrogenase attach to the membrane, or with processing the subunits. It's unclear. The other genes may be involved in processing or metal stuff. HupR may be another electron-carrying protein. Further study is required.

Reference:

Du, L., Tibelius, K. H., Souza, E. M., Garg, R. P. & Yates, M. G. Sequences, organization and analysis of the hupZMNOQRTV genes from the Azotobacter chroococcum hydrogenase gene cluster. Journal of Molecular Biology 243, 549–557 (1994).

159 - Effect of chelating agents on hydrogenase in Azotobacter chroococcum: Evidence that nickel is required for hydrogenase synthesis

This study used chelating (metal-binding) compounds to study the cofactor of hydrogenase in Azotobacter chroococcum.

What They Saw
All the chelators they added (NTA, EDTA, etc.) decreased the hydrogenase activity in batch cultures, though to different extents. NTA was much stronger than EDTA. The effect was not inhibition of already-formed enzyme (since adding chelators to resting cells or extracts didn't affect activity), so it must be from preventing formation of additional enzyme.

They tried adding trace metal salts along with the chelators to see if pure excess of whatever was missing could restore activity. Copper, zinc, and manganese didn't really do anything. Cobalt helped if it was added in fairly large amounts, but the most helpful was nickel. Adding extra iron boosted this effect even more.

Monitoring nickel uptake by adding radioactive nickel, they saw that cyanide completely wiped out uptake (possibly by binding the nickel), but juglone and 2,4-dinitrophenol enhanced it (despite inhibiting respiration). Sodium azide didn't really affect either. The chelators they tested earlier generally seemed to inhibit nickel uptake too, generally in the same proportions as they had inhibited hydrogenase activity.

What This Means
Chelators seem to inhibit hydrogenase, but rather than acting on the enzyme directly, it seems to be by inhibiting its synthesis, and even this mechanism seems to be by inhibiting nickel uptake in most cases, rather than something more direct. Nickel is important for synthesizing the enzyme; it's a part of its essential cofactor. It seems like cobalt might be able to substitute for nickel somewhat though. I wonder if palladium would work too, since it has similar orbital arrangements. But apparently cobalt doesn't help in the absence of chelators or contaminating trace metals, so maybe it only helped here because it distracted the chelators away from nickel (so to speak).

Reference:

Partridge, C. D. & Yates, M. G. Effect of chelating agents on hydrogenase in Azotobacter chroococcum: Evidence that nickel is required for hydrogenase synthesis. Biochem. J. 204, 339–344 (1982).

071 - Two open reading frames (ORFs) identified near the hydrogenase structural genes in Azotobacter vinelandii, the first ORF may encode for a polypeptide similar to rubredoxins

This study looked at the genetics of the two open reading frames (ORFs) near the hydrogenase structural genes (hoxZM).

What They Saw
They sequenced the ORFs and compared to known genes. hoxZ seemed to have most homology with genes encoding proteins called rubredoxin from other species. These are typically small proteins that play roles in electron transport, which makes sense. And that's all.

Reference:

Chen, J. & Mortenson, L. Two open reading frames (ORFs) identified near the hydrogenase structural genes in Azotobacter vinelandii, the first ORF may encode for a polypeptide similar to rubredoxins. Biochim Biophys Acta 1131, 122–124 (1992).

070 - The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase

Here they wanted to look into the hoxZ gene more closely. Previous studies suggested that the product might be involved in electron transport for the hydrogenase.

What They Saw
They grew Azotobacter vinelandii DJ (an easy-to-transform strain) and knocked out hoxZ and hoxKG by transformation and screening for hydrogen production.

Comparing the hoxZ mutant to DJ (positive control) and the hoxKG mutant (negative control), they observed an intermediate rate of hydrogen oxidation, so there seemed to still be some activity. DJ consumed nearly all the hydrogen, and hoxKG consumed very little (the graph showed a decrease but it was apparently because gas leaked out of the vial, so it's a good thing they had good controls!).

Then they tried measuring short-term hydrogen oxidation with different electron acceptors: oxygen or methylene blue. DJ quickly oxidized all the hydrogen while reducing oxygen or methylene blue, as expected. Both mutants didn't show activity with either acceptor at first, despite the difference in the previous assay. But then they added sodium dithionite (a powerful reducer of oxygen) and more methylene blue, and the hoxZ mutant showed up to 80% of the activity of DJ. As far as I can tell, the hoxKG didn't show the same effect when they gave it the same treatment, but they don't say that explicitly. But it seems like the hydrogenase needs to be activated somehow, as by dithionite.

These results were confirmed by observing methylene blue color change too; DJ quickly started oxidizing hydrogen, but the hoxZ mutant did too after a longer lag period.

When they isolated membrane-bound hydrogenase from cells (still embedded in membranes), even DJ needed activation with dithionite. hoxKG mutants had no activity in any case, of course. But hoxZ mutant had more activity in the soluble supernatant portion than DJ did, at least when membranes were isolated aerobically; it seemed like lack of hoxZ led to more soluble enzyme. But it had low activity in general so this conclusion was uncertain. Though membrane-bound activity in general was higher when isolated anaerobically, and they didn't measure soluble activity in that case for some reason. So HoxZ may help stabilize hydrogenase in the presence of oxygen.

The increased presence of detached hydrogenase in the mutant was not confirmed by Western blot, so it seems like an artifact.

What This Means
HoxZ seems to have a role in shuttling electrons between hydrogenase and oxygen, though there may be other components involved in this path. It's possible that when HoxZ is missing, another acceptor can take the electrons, but isn't as good at it.

It also may be involved in activating the enzyme (which requires removing oxygen and providing reduction); somehow hydrogen is not enough for this. And it may help stabilize the hydrogenase to keep oxygen from inactivating it, maybe also using its role as electron transporter.

Reference:

Sayavedra-Soto, L. A. & Arp, D. J. The hoxZ gene of the Azotobacter vinelandii hydrogenase operon is required for activation of hydrogenase. J. Bacteriol. 174, 5295–5301 (1992).