Key features differentiating the coordination environment of the two irons in the mixed-valent (Fe2+,Fe3+) diiron site of Desulfovibrio vulgaris rubrerythrin (Rbrmv) were determined by continuous wave (CW) and pulsed ENDOR spectroscopy at 35GHz. 14N ENDOR evidence indicates that a nitrogen is bound only to the Fe2+ ion of the mixed-valent site. Assuming that this nitrogen is from His131Nδ, the same one that furnishes an iron ligand in the crystal structure of the diferric site, the ENDOR data allow us to specify the Fe2+ and Fe3+ positions within the molecular reference frame. In addition, the 1,2H ENDOR on Rbrmv indicates the presence of a solvent-derived aqua/hydroxo ligand bound either terminally or in a bridging mode to Fe3+ in the mixed-valent site. The relatively large g anisotropy of Rbrmv and weak antiferromagnetic coupling, J ∼ -8 cm-1 (in the 2JS1•S2 formalism), between the irons is more consistent with a bridging than terminal hydroxo ligand. γ-Irradiation was used to cryoreduce Rbr at 77 K, thereby producing a mixed-valent diiron site [(Rbrox)mv] that retains the structure of the diferric site. The EPR spectrum of (Rbrox)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 (Rbrox)mv thus supports the 14N ENDOR-assigned His131 ligation to Fe2+ and assignment of the solvent-derived ligand observed in the 1,2H ENDOR to a hydroxo bridge between the irons of the mixed-valent diiron site.
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