Hydrogen bonding to the cysteine ligand of superoxide reductase: acid--base control of the reaction intermediates

TL;DR

This study investigates how hydrogen bonds to the cysteine ligand in superoxide reductase influence its catalytic mechanism, showing that these bonds control the protonation rate of key reaction intermediates through mutational and spectroscopic analyses.

Contribution

It reveals the role of peptide NH hydrogen bonds in modulating the reactivity and protonation of the Fe2+-O2•- intermediate in superoxide reductase, a novel insight into enzyme catalysis.

Findings

Mutations alter hydrogen bonding and bond strength in the active site.

Hydrogen bonds regulate the pKa of the reaction intermediate.

Protonation rate of the intermediate is tightly controlled by these hydrogen bonds.

Abstract

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.