Thesis presented January 27, 2005
Abstract:
superoxide reductase (SOR) is a newly discovered enzymatic activity by which some anaerobic or microaerophilic organisms eliminate superoxide, 0
2-. SOR catalyzes the mono - electronic reduction of 02- to form H202 exclusively :
02.- + 1 e- + 2 H+ -> H202
The active site of SOR consists of a non-heme Fe2+ center in an unusual [His4 Cys1] square pyramidal pentacoordination. A conserved glutamate residue becomes the sixth
Fe3+ ligand in the active site for the SOR oxidized form. There are three classes of SOR which are distinguished by the presence or absence of a rubredoxin -like iron center in the N-terminal domain.
Our resonance Raman spectroscopic studies on representative SOR proteins from each class revealed structural variations between each class at the level of the active site. Studies o f site-directed mutants around the cystein ligand verified that its conformation and it electron density donation are both sensitive to the cystein environment.
An
Fe3+-OOH species is proposed to be an intermediate in the SOR catalytic cycle. During our work we have characterized a meta-stable non-protonated
Fe3+-η2-02 species in the active site after reaction with excess
H202. Mutation of the conserved glutamate residue to alanine resulted in the stabilization of the
Fe3+-η2-02 species. This indicated that this glutamate side chain in the wild type SOR facilitates
H202 release. The residue may also protect the enzyme against oxidative damage.
We have also identified a
Fe3+-OH species which is responsible for previously observed pH-dependent changes in the UV-visible absorption spectrum of the SOR active site.
180/
160 isotopic exchange experiments showed that the HO- species originates from the water solvent. Thus the second proton donor required for the
H202 release after superoxide reduction originates from the water surrounding the solvent-exposed active site.
Finally, we have shown that the conserved lysine residue adjacent to the conserve d glutamate reside was in interaction with Fe3+-OH and Fe3+-η2-02 species resulting in the weakening of the Fe-0 bonds as seen by resonance Raman. Thus, this lysine residue may influence
H202 release during the catalytic cycle.
Keywords:
Superoxide stress, non-heme iron center, resonance Raman spectroscopy, electron density donation sulfur, Fe3+-η2-O2 and Fe3+-OH species
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