X-ray Crystal Structures of Reduced Rubrerythrin and Its Azide Adduct: A Structure-Based Mechanism for a Non-Heme Diiron Peroxidase

Rubrerythrin (Rbr) is a 44-kDa homodimeric protein, found in many air-sensitive bacteria and
archaea, which contains a unique combination of a rubredoxin-like [Fe(SCys)4] site and a non-sulfur, oxo/dicarboxylato-bridged diiron site. The diiron site structure resembles those found in O2-activating diiron
enzymes. However, Rbr instead appears to function as a hydrogen peroxide reductase (peroxidase). The
diferrous site in all-ferrous Rbr (Rbrred) shows a much greater reactivity with H2O2 than does the diferric
site in all-ferric Rbr (Rbrox), but only the latter structure has been reported. Here we report the X-ray crystal
structures of the recombinant Rbrred from the sulfate reducing bacterium, Desulfovibrio vulgaris, as well as
its azide adduct (RbrredN3). We have also redetermined the structure of Rbrox to a higher resolution than
previously reported. The structural differences between Rbrox and Rbrred are localized entirely at the diiron
site. The most striking structural change upon reduction of the diferric to the diferrous site of Rbr is a 1.8-Å
movement of one iron away from a unique glutamate carboxylate ligand and toward a trans-disposed
histidine side chain, which replaces the glutamate as a ligand. This movement increases the inter-iron
distance from 3.3 to 4 Å. RbrredN3 shows this same iron movement and His→Glu ligand replacement relative
to Rbrox, and, in addition, an azide coordinated to the diiron site in a cis μ-1,3 fashion, replacing two solvent
ligands in Rbrred. Relative to those in O2-activating enzymes, the bridging carboxylate ligation of the Rbr
diiron site is less flexible upon diferric/diferrous interconversion. The diferrous site is also much more rigid,
symmetrical, and solvent-exposed than those in O2-activating enzymes. On the basis of these unique
structural features, a mechanism is proposed for facile reduction of hydrogen peroxide by Rbr involving a
cis μ-η2 H2O2 diferrous intermediate.