Proton NMR Investigation of the [4Fe-4S]1+ Cluster Environment of Nitrogenase Iron Protein from Azotobacter vinelandii: Defining Nucleotide-induced Conformational Changes
This work presents the complete assignment of the isotropically shifted 1H NMR resonances of Azotobacter vinelandii nitrogenase iron protein (Fe protein) to β-CH2 and α-CH protons of the [4Fe- 4S]1+ cluster cysteinyl ligands. Four resonances were observed for the reduced Fe protein with chemical shifts of 49, 23, 17, and 13 ppm. T1 measurements and analysis of relative peak areas coupled with one-dimensional nuclear Overhauser effect (NOE) difference spectra were used to assign the two most downfield-shifted resonances (49 and 23 ppm) to cysteinyl ligand β-CH2 protons and the 17 and 14 ppm resonances to cysteinyl ligand α-CH protons. Temperature dependence studies of the isotropically shifted protons revealed both Curie and anti-Curie behavior. These results, along with previous Mossbauer studies of the Fe protein, allowed the assignment of signal A (49 ppm) to four β-CH2 protons and signal C (17 ppm) to 2 α-CH protons of two cysteinyl ligands bound to a mixed-valence iron pair (Fe3+-Fe2+) of the [4Fe-4S]1+ cluster. Signal B (23 ppm) was assigned to four β-CH2 protons, and signal C (17 ppm) and D (13 ppm) were assigned to two α-CH protons of two cysteinyl ligands bound to a ferrous pair of irons (2Fe2+). The effects of MgATP, MgADP, and Mg-adenosine-β,y-methylene-5'-triphosphatbei nding to the Fe protein on the assigned resonances were established and are discussed in the context of nucleotideinduced changes in the protein environment of the [4Fe-4S] cluster. In addition, conditions are described that prevent the long-standing problem of A. vinelandii Fe protein self-oxidation.
Lanzilotta, William N.; Holz, Richard C.; and Seefeldt, Lance C., "Proton NMR Investigation of the [4Fe-4S]1+ Cluster Environment of Nitrogenase Iron Protein from Azotobacter vinelandii: Defining Nucleotide-induced Conformational Changes" (1995). Chemistry Faculty Research and Publications. 317.