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).

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).

518 - Cloning, sequencing and characterization of the [NiFe]hydrogenase-encoding structural genes (hoxK and hoxG) from Azotobacter vinelandii

To continue our journey back in time through the discovery of the uptake hydrogenase genetics in Azotobacter vinelandii, this study describes the sequencing of the hydrogenase structural genes, hoxKG.

What They Saw
They probed A. vinelandii DNA with a chunk from A. chroococcum with its hydrogenase genes, and sequenced a fragment they found. This fragment contained three full open reading frames (ORFs) and possibly the beginning of a fourth. The first two encode the structural subunits, HoxK and HoxG (for hydrogen oxidation), based on comparison to sequences in other organisms.

HoxK seems to undergo some processing, since the final protein is smaller than that predicted by the gene sequence; there might be a signal sequence that localizes the protein to the membrane or something. The structural ORFs overlap each other a little, indicating that they're probably transcribed as a unit (in the same operon) which helps the proportions come out right.

The third ORF, ORF3 (which we know now is hoxZ) was also similar to other organisms, and seemed to be a membrane protein of some sort. Possibly part of the hydrogenase electron transport chain.

Reference:

Menon, A. L., Stults, L. W., Robson, R. L. & Mortenson, L. E. Cloning, sequencing and characterization of the [NiFe]hydrogenase-encoding structural genes (hoxK and hoxG) from Azotobacter vinelandii. Gene 96, 67–74 (1990).

052 - Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii

This study looks at the hydrogenase-related genes in Azotobacter vinelandii in between the structural genes and the hyp genes.

What They Saw
They already knew about the structural genes, hoxKG. So they got some overlapping fragments containing those and sequences downstream of them, and sequenced them. They found 5 new open reading frames (ORFs), ORF3-7. They named these hoxZMLOQ. There was also part of an eighth at the end. All of them were homologous to hydrogenase genes in other organisms.

Then they knocked out each gene by inserting kanamycin or kan+lacZ cassettes, though probably some of these insertions affected other genes (polar effects) so it wasn't possible to study each gene individually. Each insertion abolished hydrogen oxidation. All 5 new ORFs seem to be involved in subunit processing, as the unprocessed form was present in higher proportion (or alone) in their knockouts.

The knockouts didn't seem to be impaired in growth, even when fixing nitrogen, though they might show more effect in certain conditions (like carbon limitation).

HoxZ seems to be a membrane protein, possibly a cytochrome, so part of the electron transport chain. HoxL seems like it might be involved in metal-binding somehow.

What This Means
Knowing more of the sequence of the operon helps to study it. The whole thing, hox and hyp, is pretty big, kinda surprising for an enzyme that doesn't seem to help the organism in some conditions. Probably in nature, the likelihood of encountering carbon-limited circumstances is much higher, so having an uptake hydrogenase is important.

Reference:

Menon, A. L., Mortenson, L. E. & Robson, R. L. Nucleotide sequences and genetic analysis of hydrogen oxidation (hox) genes in Azotobacter vinelandii. J Bacteriol 174, 4549–4557 (1992).

051 - The hypE Gene Completes the Gene Cluster for H2-oxidation in Azotobacter vinelandii

As a followup to 050, this study completed the hyp operon by sequencing hypE. They suspected its existence because other sequenced operons always had a hypE after the hypD, so they purified a fragment from Azotobacter vinelandii that hybridized hypD and contained some downstream region.

What They Saw
The open reading frame (ORF) in the downstream region had good homology to hypE genes in other organisms. A few bases of hypD and hypE genes overlap, suggesting that they're translated together so there should be equal amounts present so they can work together equally.

They knocked out hypE by inserting a lacZ and kanamycin resistance cassette, which also let them detect expression (the product of lacZ can break down certain compounds to make color, so color intensity can be measured as a proxy for enzyme activity). It seemed like transcription of all the hox and hyp genes was in the same direction, and knocking out hypE meant that A. vinelandii couldn't oxidize hydrogen anymore.

They also noticed that there was a higher proportion of unprocessed hydrogenase structural component, so HypE might be involved in processing; this seems true in other organisms. It also affects the localization of hydrogenase, soluble or membrane-bound. It may affect pre-protein folding.

What This Means
This study completes the discovery of the whole uptake hydrogenase gene set in A. vinelandii. It's pretty similar to the gene sets in other organisms, including A. chroococcum, and sequencing A. vinelandii's genome confirmed the results.

Reference:

Garg, R. P., Menon, A. L., Jacobs, K., Robson, R. M. & Robson, R. L. The hypE Gene Completes the Gene Cluster for H₂-oxidation in Azotobacter vinelandii. J. Mol. Biol. 236, 390–396 (1994).

225 - O2-repression of nitrogenase synthesis in Azotobacter chroococcum

Klebsiella pneumoniae only fixes nitrogen when oxygen levels are very low (or absent), so oxygen represses nitrogenase synthesis. This study looks at whether the same is true in Azotobacter chroococcum; it seems unlikely, since this organism is known for its very aerobic nitrogen fixation; however, it is possible to stress this organism with oxygen such that it may shut down nitrogenase, at least temporarily.

What Robson Saw
He grew A. chroococcum in chemostats with Burk medium with mannitol, with or without ammonium for nitrogen, and stressed them with oxygen either by moving ammonium-grown cells to ammonium-free medium or by suddenly increasing aeration in nitrogen-fixing cells. He looked at levels of nitrogenase proteins (by labeling with radioactive sulfur isotopes) and nitrogenase activity.

At initial low oxygen levels, activity increased and radioisotype-labeled protein levels were relatively high (indicating high levels of nitrogenase protein synthesis). Upon oxygen shock, activity went to zero and protein synthesis levels dropped a lot. When the stress was relieved, both measures increased again.

With ammonium removed and then re-added, things were similar: nitrogenase activity went up and synthesis gradually increased to a plateau, but decreased when more ammonium was added. It did take a relatively longer time for nitrogenase activity to pick up after ammonium was removed, about 80 minutes.

Along with nitrogenase, flavodoxin and the small protein that protects nitrogenase from oxygen by temporarily inactivating it both showed up in radiolabeling. The former matched nitrogenase synthesis patterns, but the latter was fairly constant.

What This Means
It seems that oxygen stress can repress synthesis of nitrogenase even in Azotobacter, though radiolabeling might not be the best method for studying this question. This adds another layer to Azotobacter's protection of its enzymes from oxygen.

Reference:

Robson, R. L. O₂-repression of nitrogenase synthesis in Azotobacter chroococcum. FEMS Microbiology Letters 5, 259–262 (1979).

213 - Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum

When respiratory protection fails in Azotobacter, it can temporarily inactivate its nitrogenase to protect it, by association with another protein, called FeSII or Shethna. This study purifies this whole complex (nitrogenase and FeSII) and investigates its characteristics in A. chroococcum.

What They Saw
The more pure the nitrogenase, the less protection from oxygen inactivation they observed. But while crude extract had the most protection, more pure forms were pretty similar until the protective FeSII protein was absent, in which case the nitrogenase was rapidly inactivated. Magnesium ions (or possibly other divalent ions) were also necessary for this stabilization.

This protective protein was 14 kDa, orange in color, and had 2 Fe and 2 S atoms, so a 2Fe-2S center (thus the name). This version seems smaller than the A. vinelandii version though, which is 23 kDa. In stable complexes, the three components (dinitrogenase, dinitrogenase reductase, and FeSII) were present in about 1:1:1 ratios.

What This Means
This FeSII (with Mg ions) appears to be sufficient to protect the nitrogenase complex from oxygen, stabilizing it even outside of the cellular environment. This stability is perhaps not complete though, since crude extracts did show more activity after exposure to oxygen.

Reference:

Robson, R. L. Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum. Biochem J 181, 569–575 (1979).

099 - Oxygen and Hydrogen in Biological Nitrogen Fixation

Oxygen is pretty toxic to nitrogen fixation enzymes, so organisms or the people studying them need to take steps to protect them. They lose more than half their activity within minutes exposed to air. The dinitrogenase reductase is more sensitive than the dinitrogenase itself, at least the Mo version. Some can retain some activity even up to an hour in air. But the FeMo-cofactor, when extracted, is even more sensitive than the dinitrogenase reductase. Overall, it seems that the metal-sulfur centers are the most sensitive parts.

And yet, there are nitrogen-fixing species that are obligate aerobes, or even oxygenic. How do they do it?

Azotobacter has been shown to increase its respiration while its growth efficiency decreases as oxygen increases, seeming to waste the oxygen: this has been called "respiratory protection." The mechanism for this is not simple though; it involves carefully regulated shifts in respiratory components throughout the whole catabolic system.

Azotobacter also has the ability to reversibly inactivate its nitrogenase if respiratory protection is not possible (such as in carbon-limited conditions, or upon a sudden increase in oxygen). This seems to depend on FeSII protein (aka Shethna), though it is suggested that there may be other mechanisms.

When oxygen is too high and cells' supply of fixed nitrogen runs out, production of nitrogenase may be regulated (no sense making an enzyme when it can't function). This regulation may be done by the products of nifAL genes.

Azotobacter also produces gummy alginate which might have a role in protection from oxygen, but non-gummy strains (such as CA) have been isolated that don't seem especially oxygen-sensitive. I wonder if they have higher rates of respiration though, or if they might be more sensitive in carbon-limited conditions.

Nitrogenase also produces hydrogen gas, whether or not it's reducing anything else. This reaction seems separate from the nitrogen fixation reaction, since some things can inhibit the latter without inhibiting the former. Acetylene seems to inhibit hydrogen production though. Nitrogen can't compete with hydrogen for electrons completely, even with pure pressurized nitrogen; the enzyme always produces at least 1 mol hydrogen for each mol nitrogen gas fixed.

Of course, this hydrogen usually doesn't just escape; Azotobacter and other diazotrophs recapture it with their uptake hydrogenase. The exact purpose this serves is not clear though.

Reference:

Robson, R. L. & Postgate, J. R. Oxygen and Hydrogen in Biological Nitrogen Fixation. Ann Rev Microbiol 34, 183–207 (1980).