EVIDENCE FOR A CENTRAL ROLE OF LYSINE-15 OF AZOTOBACTER-VINELANDII NITROGENASE IRON PROTEIN IN NUCLEOTIDE FINDING AND PROTEIN CONFORMATIONAL-CHANGES

Biological nitrogen fixation catalyzed by purified nitrogenase require s the hydrolysis of a minimum of 16 MgATP for each N-2 reduced. In the present study, we demonstrate a central function for Lys-1B of Azotob acter vinelandii nitrogenase iron protein (FeP) in the interaction of nucleotides with nitrogenase. Changing Lys-1B of the FeP to Arg result ed in an FeP with a dramatically reduced affinity for both MgATP and M gADP. From equilibrium column binding experiments at different nucleot ide concentrations, apparent dissociation constants (K-d) for wild typ e FeP binding of MgADP (143 mu M) and MgATP (571 mu M) were determined . Over the same nucleotide concentration ranges, the K15R FeP showed n o significant affinity for either nucleotide. This contrasts sharply w ith previous results with an FeP in which Lys-1B was changed to Gln (K 15Q) where it was found that the K15Q FeP bound MgADP with the same af finity as wild type FeP and MgATP with a slightly reduced affinity. An alysis of K15R FeP by EPR, circular dichroism (CD), and microcoulometr y revealed that the [4Fe-4S] cluster was unaffected by the amino acid change and that addition of either MgADP or MgATP did not result in th e protein conformational changes normally detected by these techniques . These results are integrated into a model for how MgATP and MgADP bi nd and induce conformational changes within the FeP.