EPR and Mössbauer studies of nucleotide-bound nitrogenase iron protein from Azotobacter vinelandii.
We have recently shown (Lindahl, P. A., Day, E. P., Kent, T. A., Orme-Johnson, W. H., and Münck, E. (1985) J. Biol. Chem. 260, 11160-11173) that the [4Fe-4S]1+ cluster of the native Fe protein can exist in two forms characterized by different cluster spin: an S = 1/2 state exhibiting a g = 1.94 type EPR signal and an S = 3/2 state yielding signals at g approximately 5.8 and 5.1. We have now extended our study of the Fe protein to include the MgATP- and MgADP-bound forms. The [4Fe-4S]1+ cluster of the nucleotide-bound Fe protein exists in a similar S = 1/2, S = 3/2 spin mixture. The S = 3/2 cluster exhibits a broad EPR signal at g approximately 4.8. In spectra of the MgATP-bound protein, we have also observed a g = 4.3 signal from an S = 5/2 state (D = 1 - 3 cm-1, E/D approximately 0.32). Various experiments strongly suggest that this signal does not originate from adventitiously bound Fe3+. The g = 4.3 signal may arise from approximately 2% of the [4Fe-4S]1+ clusters when MgATP is protein-bound. We have also discovered substoichiometric amounts of what appears to be ADP in some nominally nucleotide-free Fe protein samples. MgATP binds to Fe protein in the presence of perturbing solvents, resulting in EPR spectra similar to those of MgATP-bound samples in aqueous solutions; MgADP binding, on the other hand, results in signals more typical of the solvent state. Spectra of samples frozen during turnover of the nitrogenase system exhibit a mixture of spin states. Characterization of the Fe protein EPR signature described here should aid future mechanistic and nucleotide-binding studies.
Lindahl
P A
PA
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Gorelick
N J
NJ
Münck
E
E
Orme-Johnson
W H
WH
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001395:Azotobacter / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D009591:Nitrogenase / Q000378:metabolism*; D010088:Oxidoreductases; D013057:Spectrum Analysis / Q000379:methods
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D009591:Nitrogenase; C029130:nitrogenase reductase