The [4Fe-4S] cluster domain of the nitrogenase iron protein facilitates conformational changes required for the cooperative binding of two nucleotides.
MgATP binding and hydrolysis are central to all reduction reactions catalyzed by nitrogenase. The iron (Fe) protein component of nitrogenase is a homodimeric protein with a bridging [4Fe-4S] cluster and two nucleotide binding sites, one on each subunit. This work presents evidence that the [4Fe-4S] cluster domain of the nitrogenase Fe protein functions as a hinge region between the two nucleotide binding domains, participating in the cooperative binding of two nucleotides. Alanine residues at position 98 (located near the [4Fe-4S] cluster) of the Azotobacter vinelandii Fe protein were changed by means of site-directed mutagenesis to Val (V) and Gly (G), and the resulting altered proteins were purified and characterized. While the wild-type and A98G Fe proteins were found to bind two nucleotides (MgATP or MgADP) with strong cooperativity (Hill coefficient of 2), the A98V Fe protein was found to bind one nucleotide with no apparent cooperativity. The binding of two nucleotides to the wild-type Fe protein is known to induce protein conformational changes which are reflected as changes in the properties of the [4Fe-4S] cluster, including a change in the redox potential of the [4Fe-4S] cluster of -120 mV for MgATP binding (-300 to -420 mV) and of -160 mV for MgADP binding (-300 to -460 mV). The binding of one nucleotide to the A98V Fe protein was found to result in only half the lowering of the redox potential, with MgATP binding resulting in a -80 mV change (-280 to -360 mV) and MgADP binding resulting in a -50 mV change (-280 to -330 mV). Results from 1H NMR, EPR, and CD spectra, along with Fe chelation rates, were all consistent with the binding of a single nucleotide to the A98V Fe protein inducing a partial conformational change. Finally, the A98V Fe protein with one nucleotide bound, still bound to the molybdenum-iron protein but did not support MgATP hydrolysis, electron transfer, or substrate reduction. A model is discussed in which the [4Fe-4S] cluster domain can be viewed as a hinge region between the two nucleotide binding domains which facilitates conformational rearrangements required for the cooperative binding of a second nucleotide.
Ryle
M J
MJ
Department of Chemistry and Biochemistry, Utah State University, Logan 84322, USA.
Seefeldt
L C
LC
D000114:Acetylene / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000201:enzymology*; D002942:Circular Dichroism; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D005030:Ethylenes / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D009591:Nitrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D017433:Protein Structure, Secondary
D005030:Ethylenes; D007506:Iron-Sulfur Proteins; D009711:Nucleotides; D000255:Adenosine Triphosphate; C036216:ethylene; D009591:Nitrogenase; D000114:Acetylene