Evidence for a central role of lysine 15 of Azotobacter vinelandii nitrogenase iron protein in nucleotide binding and protein conformational changes.
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 microM) and MgATP (571 microM) 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.
Ryle
M J
MJ
Department of Chemistry and Biochemistry, Utah State University, Logan 84322, USA.
Lanzilotta
W N
WN
Mortenson
L E
LE
Watt
G D
GD
Seefeldt
L C
LC
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009591:Nitrogenase / Q000737:chemistry / Q000378:metabolism*; D010088:Oxidoreductases; D011485:Protein Binding; D011487:Protein Conformation
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase; D008239:Lysine