Assessing the role of the active-site cysteine ligand in the superoxide reductase from Desulfoarculus baarsii.

Superoxide reductase is a novel class of non-heme iron proteins that catalyzes the one-electron reduction of O(2)(.) to H(2)O(2), providing an antioxidant defense in some bacteria. Its active site consists of an unusual non-heme Fe(2+) center in a [His(4) Cys(1)] square pyramidal pentacoordination. In this class of enzyme, the cysteine axial ligand has been hypothesized to be an essential feature in the reactivity of the enzyme. Previous Fourier transform infrared spectroscopy studies on the enzyme from Desulfoarculus baarsii revealed that a protonated carboxylate group, proposed to be the side chain of Glu(114), is in interaction with the cysteine ligand. In this work, using pulse radiolysis, Fourier transform infrared, and resonance Raman spectroscopies, we have investigated to what extent the presence of this Glu(114) carboxylic lateral chain affects the strength of the S-Fe bond and the reaction of the iron active site with superoxide. The E114A mutant shows significantly modified pulse radiolysis kinetics for the protonation process of the first reaction intermediate. Resonance Raman spectroscopy demonstrates that the E114A mutation results in both a strengthening of the S-Fe bond and an increase in the extent of freeze-trapping of a Fe-peroxo species after treatment with H(2)O(2) by a specific strengthening of the Fe-O bond. A fine tuning of the strength of the S-Fe bond by the presence of Glu(114) appears to be an essential factor for both the strength of the Fe-O bond and the pK(a) value of the Fe(3+)-peroxo intermediate species to form the reaction product H(2)O(2).