A sulfenic acid enzyme intermediate is involved in the catalytic mechanism of peptide methionine sulfoxide reductase from Escherichia coli.

Methionine oxidation into methionine sulfoxide is known to be involved in many pathologies and to exert regulatory effects on proteins. This oxidation can be reversed by a ubiquitous monomeric enzyme, the peptide methionine sulfoxide reductase (MsrA), whose activity in vivo requires the thioredoxin-regenerating system. The proposed chemical mechanism of Escherichia coli MsrA involves three Cys residues (positions 51, 198, and 206). A fourth Cys (position 86) is not important for catalysis. In the absence of a reducing system, 2 mol of methionine are formed per mole of enzyme for wild type and Cys-86 --> Ser mutant MsrA, whereas only 1 mol is formed for mutants in which either Cys-198 or Cys-206 is mutated. Reduction of methionine sulfoxide is shown to proceed through the formation of a sulfenic acid intermediate. This intermediate has been characterized by chemical probes and mass spectrometry analyses. Together, the results support a three-step chemical mechanism in vivo: 1) Cys-51 attacks the sulfur atom of the sulfoxide substrate leading, via a rearrangement, to the formation of a sulfenic acid intermediate on Cys-51 and release of 1 mol of methionine/mol of enzyme; 2) the sulfenic acid is then reduced via a double displacement mechanism involving formation of a disulfide bond between Cys-51 and Cys-198, followed by formation of a disulfide bond between Cys-198 and Cys-206, which liberates Cys-51, and 3) the disulfide bond between Cys-198 and Cys-206 is reduced by thioredoxin-dependent recycling system process.