663 - Azotobacter vinelandii Vanadium Nitrogenase: Formaldehyde Is a Product of Catalyzed HCN Reduction, and Excess Ammonia Arises Directly from Catalyzed Azide Reduction

The V nitrogenase is similar to but distinct from the Mo nitrogenase in various ways. This study tests its activity and patterns with cyanide and azide as substrates; previously this had only been tested with the Mo nitrogenase.

What They Saw
As with the Mo version, cyanide inhibited hydrogen production and the overall electron flux through the V nitrogenase, though less than with the Mo nitrogenase; more cyanide had more effect, up to 75% inhibition of hydrogen and electrons at 50 mM cyanide. Methane formation from cyanide increased at first, and then decreased with more cyanide, while ammonia continued to increase. They didn't measure methylamine in most conditions though, so the electron flux numbers might be off. With low cyanide, there was significant methylamine relative to the methane, about 0.66 to 1, higher than the Mo nitrogenase; this ratio seems to increase to 1:1 as methane decreases.

Interestingly, the enzyme also produces formaldehyde from cyanide. It's not clear whether the Mo version does too and it's too hard to detect, or if it just doesn't. It can be tricky, since other components in the reaction react with it, and cyanide inhibits the Mo enzyme a lot more.

Azide inhibited hydrogen production from the V nitrogenase about 50%. Hydrazine was more of a product from azide than it is for the Mo nitrogenase, relative to dinitrogen and ammonia. Overall activity was less than the Mo version, which is typical, but also because azide seems to reduce the total electron flux in this version. The ammonia seems to come from azide directly, not from the dinitrogen produced, because adding hydrogen gas (which specifically inhibits nitrogen reduction) didn't change the values.

They tried seeing what adding carbon monoxide (CO) might do to affect these reactions. With azide, it rescued hydrogen production, though not the electron flux, and CO didn't entirely prevent some azide reduction. With cyanide, the effects were similar, except the electron flux inhibition was relieved too.

What This Means
It seems that these chemicals have similar effects on the V nitrogenase as they do on the Mo version, but not completely the same. I wonder, though, about the CO assays: CO can be a substrate for the V nitrogenase, reduced mostly to ethylene but also some propylene and methane; how did this affect their assays?

Reference:

Fisher, K., Dilworth, M. J. & Newton, W. E. Azotobacter vinelandii Vanadium Nitrogenase: Formaldehyde Is a Product of Catalyzed HCN Reduction, and Excess Ammonia Arises Directly from Catalyzed Azide Reduction. Biochemistry 45, 4190–4198 (2006).

555 - Differential Effects on N2 Binding and Reduction, HD Formation, and Azide Reduction with α-195His- and α-191Gln-Substituted MoFe Proteins of Azotobacter vinelandii Nitrogenase

Similar to 554, this study looked at mutated versions of the Mo nitrogenase in Azotobacter vinelandii, but different reactions this time: interactions with nitrogen gas (hooray), with dihydrogen and dideuterium, and with azide (N₃^-).

What They Saw
Adding nitrogen gas revealed that Asn 195 couldn't fix nitrogen; Gln 195 had a slight ability. However, replacing argon with pure nitrogen reduced hydrogen production by Asn 195 about 30%, though increasing the pressure with additional nitrogen didn't affect things further except maybe to increase the ATP required for each electron transfer, almost double what it is at 100% argon. Nitrogen didn't seem to affect hydrogen from Lys 191 at all.

Adding nitrogen also inhibited acetylene reduction in Asn 195, 28%; and again, did not inhibit Lys 191. For the former, nitrogen seems to inhibit the reaction competitively but reversibly; removing the nitrogen restored the rate almost to what it had been.

They tried adding hydrogen to acetylene reduction assays with Asn 195, enough to raise the pressure to two atmospheres. This didn't affect anything with argon; ethylene and ethane were both produced about the same amount. But when nitrogen was present, hydrogen restored most or all the activity that nitrogen would've inhibited.
They also saw that having 50% deuterium with the rest nitrogen doesn't really result in inhibition of hydrogen production by Asn 195.

When they tried adding sodium azide, this didn't really affect hydrogen production, but the activity reducing it to ammonia or hydrazine (N₂H₄) was much less for all the mutants than for the wild-type, at least 8x less. Adding carbon monoxide to Asn 195 assays abolished any azide reduction activity, but adding hydrogen had no effect. The azide might've reduced electron flux through Asn 195 a little (20%) but CO prevented this reduction too.

What This Means
Even some of the mutants that can't fix nitrogen seem to interact with it to some extent, as evidenced by its inhibiting other reactions. The other findings are pretty interesting too. It all relates to how the mutations affect the activity: in affinity, in substrate fit in the active site, and in electron flux through the whole complex.

Reference:

Fisher, K., Dilworth, M. J. & Newton, W. E. Differential Effects on N₂ Binding and Reduction, HD Formation, and Azide Reduction with α-195^His- and α-191^Gln-Substituted MoFe Proteins of Azotobacter vinelandii Nitrogenase. Biochemistry 39, 15570–15577 (2000).

554 - Azotobacter vinelandii Nitrogenases Containing Altered MoFe Proteins with Substitutions in the FeMo-Cofactor Environment: Effects on the Catalyzed Reduction of Acetylene and Ethylene

This is another study looking at mutating the Mo nitrogenase protein in Azotobacter vinelandii to see how it changes the enzyme's activity. Here's a picture they gave of the active center, with the FeMo cofactor in the middle and the protein surrounding it, with certain amino acids they were targeting:

What They Saw
They targeted conserved amino acids, common to homologs in many organisms, such as Gln 191 and His 195, with three mutations: Lys 191 (seen before in 536), Asn 195, and Gln 195. These proteins were extracted and purified.

Under argon, Gln 195 produced about as much hydrogen as the wild-type, while the others only about half as much (though apparently they only contained half as much FeMo cofactor). Under 10% acetylene, the wild-type put most electrons toward producing ethylene, while the others only devoted 55% at most, the others going to hydrogen (Gln 195) or hydrogen and ethane (others).

The mutations also affected how the protons were added to acetylene, whether in cis or in trans, as determined by using C₂D₂ instead of C₂H₂ and looking at where there was hydrogen or deuterium. The mutants had higher proportions of trans-C₂D₂H₂ compared to the wild-type, except Gln 195 which had lower. This seems related to their ability to reduce acetylene all the way to ethane or not.

Then they tested whether ethylene instead of acetylene, with or without CO or acetylene too, could be a substrate or inhibitor of activity. Having 50% ethylene with the rest argon led to ethane production from all versions, including the wild-type, except not Lys 191. It seemed to inhibit overall flux a little, though for most versions it didn't inhibit as much when CO was present (the exception was Lys 191, which had about 3x less flux with CO present compared to just argon, though in both cases it all went to hydrogen). Interestingly, adding 50% hydrogen with 50% ethylene increased the amount of ethane for all versions. Strangely, though it didn't reduce ethylene to ethane, adding 10% acetylene to the mix with Lys 191 showed some ethane production; so it doesn't reduce ethylene, only acetylene. Acetylene also increased the rate of ethane production with Asn 195.

What This Means
This helps understand the precise reaction that takes place in nitrogenase. It seems like the affinity of the enzyme for the substrate affects how far that substrate is reduced before being replaced by a fresh molecule. Still, it's hard to make real comparisons from in vitro assays, but I don't know that there's a good alternative.

Reference:

Fisher, K., Dilworth, M. J., Kim, C.-H. & Newton, W. E. Azotobacter vinelandii Nitrogenases Containing Altered MoFe Proteins with Substitutions in the FeMo-Cofactor Environment: Effects on the Catalyzed Reduction of Acetylene and Ethylene. Biochemistry 39, 2970–2979 (2000).