Friday, August 7, 2015

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.


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