Acetylene is a much larger substrate than N2; hence, its ability

Acetylene is a much larger substrate than N2; hence, its ability to access the active site might be more affected in the mutants than smaller substrates are (N2 and protons). In strains PW357 (V75I) and PW350 (V75I, V76I) acetylene reduction was about 2.5–5% of wild-type activity and was not affected by N2 (Fig. 3b), suggesting that acetylene has poor access to the active site as a result of the V75I substitution. In an N2 atmosphere, acetylene reduction decreased significantly compared with argon in both the

wild-type strain and the mutant PW253 (V76I) (Fig. 3b), indicating that N2 has good access to the active site in PW253. The two mutants, PW357 and PW350, containing the V75I substitution were unimpaired in H2 production (Fig. 3a) but were greatly impaired in acetylene reduction (Fig. 3b), and were also impaired in 15N2 reduction, showing a rate about 30% of the wild type (Fig. 3c). As suggested by the inhibition of acetylene reduction in strain PW253 (V76I)

by N2 (Fig. 3b), this strain was capable of fixing 15N2 at rates similar to the wild-type strain (Fig. 3c), indicating that the introduction of an isoleucine at amino acid position 76 does not impair access of N2 to the active site. Substitution of isoleucine for valine at the NifD2 α-75 site resulted in a fourfold higher hydrogen production in the presence of N2 compared with the wild type, and H2 production in N2 was nearly as high as H2 production in an argon atmosphere. This result Selleckchem Ceritinib is in agreement with studies on purified enzyme from A. vinelandii in which the specific activity for H2 production in nitrogenase with the comparable V70I substitution in an N2 atmosphere was found to be about 90% of the value determined under argon (Mayer et al., 2002; Barney et al., 2004). There were similar hydrogen production rates for the wild-type enzyme and the V75I substitution Liothyronine Sodium mutant under argon; however, acetylene and dinitrogen reduction activities decreased in

the V75I substitution mutant compared with the wild-type enzyme. The mutation did not increase hydrogen production compared with the wild-type enzyme, suggesting that there is no change in the ability of the mutant enzyme to reduce substrates, but rather simply an increased selectivity for substrates. With purified enzyme from A. vinelandii, the specific activity for acetylene reduction and reduction of N2 to NH3 by the nitrogenase with the V70I substitution was found to be about 6.5% and 9% of the wild-type specific activity, respectively. Whereas the acetylene reduction activity of the A. vinelandii mutant was slightly higher than we observed for the analogous substitution in the Nif2 nitrogenase of A.

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