Hydrogen production in nitrogenase mutants in Anabaena variabilis.

Nitrogenase produces hydrogen as a normal byproduct of the reduction of dinitrogen to ammonia. The Nif2 nitrogenase in Anabaena variabilis is an alternative Mo-nitrogenase and is expressed in vegetative cells grown with fructose under strictly anaerobic conditions. We report here that the V75I substitution in the alpha-subunit of Nif2 showed greatly impaired acetylene reduction and reduced levels of (15)N(2) fixation but had similar hydrogen production rates as the wild-type enzyme under argon. Another mutant containing a substitution in the alpha-subunit, V76I, would result in a decrease in the size of the putative gas channel of nitrogenase and, thus, was hypothesized to affect substrate selectivity of nitrogenase. However, this substitution had no effect on the enzyme selectivity, suggesting that access by gases to the active site through this putative gas channel is not limited by the increased size of the amino acid side chain in the alpha-subunit, V76I substitution.