  Superoxide reductase SOR is a non-heme iron metalloenzyme that detoxifies
superoxide radical in microorganisms. Its active site consists of an unusual
non-heme Fe2+ center in a [His4 Cys1] square pyramidal pentacoordination, with
the axial cysteine ligand proposed to be an essential feature in catalysis. Two
NH peptide groups from isoleucine 118 and histidine 119 establish H-bondings
with the sulfur ligand (Desulfoarculus baarsii SOR numbering). In order to
investigate the catalytic role of these H-bonds, the isoleucine 118 residue of
the SOR from Desulfoarculus baarsii was mutated into alanine, aspartate or
serine residues. Resonance Raman spectroscopy showed that the mutations
specifically induced an increase of the strength of the Fe3+-S(Cys) and
S-C$\beta$(Cys) bonds as well as a change in conformation of the cysteinyl side
chain, which was associated with the alteration of the NH hydrogen bonding to
the sulfur ligand. The effects of the isoleucine mutations on the reactivity of
SOR with $O2\bullet$- were investigated by pulse radiolysis. These studies
showed that the mutations induced a specific increase of the pKa of the first
reaction intermediate, recently proposed to be an $Fe2+-O2\bullet-$ species.
These data were supported by DFT calculations carried out on three models of
the $Fe2+-O2\bullet-$ intermediate, with one, two or no H-bonds on the sulfur
ligand. Our results demonstrated that the hydrogen bonds between the NH
(peptide) and the cysteine ligand tightly control the rate of protonation of
the $Fe2+-O2\bullet-$ reaction intermediate to form an Fe3+-OOH species.
