title: EPR and ENDOR evidence for a 1-His, hydroxo-bridged mixed-valent diiron site in Desulfovibrio vulgaris rubrerythrin.
abstract: Key features differentiating the coordination environment of the two irons in the mixed-valent (Fe(2+),Fe(3+)) diiron site of Desulfovibrio vulgaris rubrerythrin (Rbr(mv)) were determined by continuous wave (CW) and pulsed ENDOR spectroscopy at 35GHz. (14)N ENDOR evidence indicates that a nitrogen is bound only to the Fe(2+) ion of the mixed-valent site. Assuming that this nitrogen is from His131Ndelta, the same one that furnishes an iron ligand in the crystal structure of the diferric site, the ENDOR data allow us to specify the Fe(2+) and Fe(3+) positions within the molecular reference frame. In addition, the (1,2)H ENDOR on Rbr(mv) indicates the presence of a solvent-derived aqua/hydroxo ligand bound either terminally or in a bridging mode to Fe(3+) in the mixed-valent site. The relatively large g anisotropy of Rbr(mv) and weak antiferromagnetic coupling, J approximately -8 cm(-)(1) (in the 2JS(1)*S(2) formalism), between the irons is more consistent with a bridging than terminal hydroxo ligand. gamma-Irradiation was used to cryoreduce Rbr at 77 K, thereby producing a mixed-valent diiron site [(Rbr(ox))(mv)] that retains the structure of the diferric site. The EPR spectrum of (Rbr(ox))(mv) was nearly identical to that of the as-isolated or chemically reduced samples. This near identity implies that the structure of the mixed-valent Rbr diiron site is essentially identical to that of the diferric site, except for protonation of the oxo bridge, which apparently occurred via a proton jump from hydrogen-bonded solvent at 77 K. The EPR spectrum of (Rbr(ox))(mv) thus supports the (14)N ENDOR-assigned His131 ligation to Fe(2+) and assignment of the solvent-derived ligand observed in the (1,2)H ENDOR to a hydroxo bridge between the irons of the mixed-valent diiron site.
authors.affiliation: Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
authors.forename: Stoyan K
authors.initials: SK
authors.lastname: Smoukov
authors.forename: Roman M
authors.initials: RM
authors.lastname: Davydov
authors.forename: Peter E
authors.initials: PE
authors.lastname: Doan
authors.forename: Bradley
authors.initials: B
authors.lastname: Sturgeon
authors.forename: Irene Y
authors.initials: IY
authors.lastname: Kung
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
authors.forename: Donald M
authors.initials: DM
authors.lastname: Kurtz
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D016969:Desulfovibrio vulgaris / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D005288:Ferredoxins / Q000737:chemistry*; D006422:Hemerythrin; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D007501:Iron / Q000737:chemistry; D008024:Ligands; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000737:chemistry; D012416:Rubredoxins; D012997:Solvents
chemical_list: D001426:Bacterial Proteins; D005288:Ferredoxins; D006422:Hemerythrin; D008024:Ligands; D011994:Recombinant Proteins; D012416:Rubredoxins; D012997:Solvents; C055857:rubrerythrins; D006639:Histidine; D007501:Iron