Thursday, August 6, 2015

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.


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