title: Quantitative EPR of an S = 7/2 system in thionine-oxidized MoFe proteins of nitrogenase. A redefinition of the P-cluster concept.
abstract: Thionine-oxidized nitrogenase MoFe proteins from Azotobacter vinelandii. Azotobacter chroococcum and Klebsiella pneumoniae exhibit excited-state EPR signals with g = 10.4, 5.8 and 5.5 with a maximal amplitude in the temperature range of 20-50 K. The magnitude of these effective g values, combined with the temperature dependence of the peak area at g = 10.4 from 12 K to 86 K, are consistent with an S = 7/2 system with spin Hamiltonian parameters D = -3.7 +/- 0.7 cm-1, [E] = 0.16 +/- 0.01 cm-1 and g = 2.00. This interpretation predicts nine additional effective g values some of which have been detected as broad features of low intensity at g approximately 10, approximately 2.5 and approximately 1.8. The S = 7/2 EPR is ascribed to the multi-iron exchange-coupled entities known as the P clusters. Quantification relative to the S = 3/2 EPR signal from dithionite-reduced MoFe protein indicates a stoichiometry of one P cluster per FeMo cofactor. Two possible interpretations for these observations, together with data from the literature, are proposed. In the first model there are two P clusters per tetrameric MoFe protein. Each P cluster encompasses approximately 8Fe ions and releases a total of three electrons on oxidation with excess thionine. In the second model the conventional view of four P clusters, each containing approximately 4Fe, is retained. This alternative requires that following one-electron oxidation, the P clusters factorize into two populations, Pa and Pb, only one of which is further oxidized with thionine resulting in the S = 7/2 system. Both models require eight-electron oxidation of tetrameric MoFe protein to reach the S = 7/2 state.
authors.affiliation: Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
authors.forename: W R
authors.initials: WR
authors.lastname: Hagen
authors.forename: H
authors.initials: H
authors.lastname: Wassink
authors.forename: R R
authors.initials: RR
authors.lastname: Eady
authors.forename: B E
authors.initials: BE
authors.lastname: Smith
authors.forename: H
authors.initials: H
authors.lastname: Haaker
mesh_terms: D001395:Azotobacter; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins; D007711:Klebsiella pneumoniae; D008983:Molybdoferredoxin; D009591:Nitrogenase; D010640:Phenothiazines
chemical_list: D005288:Ferredoxins; D008983:Molybdoferredoxin; D010640:Phenothiazines; D009591:Nitrogenase; C009469:thionine