Evidence for a Central Role of Lysine 15 of Azotobacter vinelandii Nitrogenase Iron Protein in Nucleotide Binding and Protein Conformational Changes

• Published in: The Journal of biological chemistry Vol. 270; no. 22; pp. 13112 - 13117
• Main Authors: Ryle, Matthew J., Lanzilotta, William N., Mortenson, Leonard E., Watt, Gerald D., Seefeldt, Lance C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.06.1995; American Society for Biochemistry and Molecular Biology
• Subjects: adenosindifosfato; adenosine diphosphate; Adenosine Diphosphate - metabolism; adenosine triphosphate; Adenosine Triphosphate - metabolism; adenosintrifosfato; Amino Acid Sequence; Azotobacter vinelandii; Azotobacter vinelandii - enzymology; fer; fijacion del nitrogeno; fixation de l' azote; hierro; iron; lisina; lysine; Lysine - metabolism; magnesio; magnesium; Molecular Sequence Data; mutacion; mutation; nitrogen fixation; nitrogenasa; nitrogenase; Nitrogenase - chemistry; Nitrogenase - metabolism; Oxidoreductases; Protein Binding; Protein Conformation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.22.13112

Biological nitrogen fixation catalyzed by purified nitrogenase requires the hydrolysis of a minimum of 16 MgATP for each N2 reduced. In the present study, we demonstrate a central function for Lys-15 of Azotobacter vinelandii nitrogenase iron protein (FeP) in the interaction of nucleotides with nitrogenase. Changing Lys-15 of the FeP to Arg resulted in an FeP with a dramatically reduced affinity for both MgATP and MgADP. From equilibrium column binding experiments at different nucleotide concentrations, apparent dissociation constants (Kd) for wild type FeP binding of MgADP (143 μM) and MgATP (571 μM) were determined. Over the same nucleotide concentration ranges, the K15R FeP showed no significant affinity for either nucleotide. This contrasts sharply with previous results with an FeP in which Lys-15 was changed to Gln (K15Q) where it was found that the K15Q FeP bound MgADP with the same affinity as wild type FeP and MgATP with a slightly reduced affinity. Analysis of K15R FeP by EPR, circular dichroism (CD), and microcoulometry 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 the protein conformational changes normally detected by these techniques. These results are integrated into a model for how MgATP and MgADP bind and induce conformational changes within the FeP.