ABSTRACT
The site of substrate binding and reduction for the nitrogenase complex is located on the iron molybdenum cofactor (FeMo-co) which is contained within the a-subunit of the molybdenum iron protein. FeMo co consists of a metal sulfur core composed of an FeS cluster bridged by three inorganic sulfides to a MoFeS cluster. An organic acid, homocitrate, is coordinated to the Mo atom through its 2-carboxy and 2-hydroxy groups. Homocitrate is formed by the condensation of acetyl-CoA and a-ketoglutarate, which is catalyzed by a homocitrate synthase encoded by nifV. By deleting the nifV gene from Azotobacter vinelandii we were able to study the role of homocitrate in nitrogenase catalysis. A poly-histidine tail was incorporated into the C-termini of the a-subunit permitting isolation of the homocitrateless MoFe protein by using metal affinity chromatography. We have found that the addition of a poly-histidine tag does not alter the catalytic behavior of the native enzyme. In NifV- strains of Klebsiella pneumoniae, citrate has been found to replace homocitrate as the organic constituent of FeMo-co. We have found no evidence this is so in A. vinelandii. Gas chromatography mass spectrophotometry studies indicate little or no organic acids are associated with FeMo-co. We examined the catalytic properties of the NifV- MoFe protein In the mutant, H2 evolution is inhibited by the addition of CO, unlike in the wild type. We have found that the NifV- MoFe protein from A. vinelandii is able to catalyze the reduction of acetylene to both ethylene and ethane.
