Interrogating the molecular details of the peroxiredoxin activity of the Escherichia coli bacterioferritin comigratory protein using high-resolution mass spectrometry.

Abstract

Bacterioferritin comigratory protein (BCP) is a bacterial thioredoxin-dependent thiol peroxidase that reduces a variety of peroxide substrates. Using high-resolution Fourier transform ion cyclotron resonance mass spectrometry coupled with top-down fragmentation techniques, we have analyzed the mechanistic details of hydrogen peroxide reduction by E. coli BCP. We show here that catalysis occurs via an atypical two-cysteine peroxiredoxin pathway. A transient sulfenic acid is initially formed on Cys-45, before resolution by the formation of an intramolecular disulfide bond between Cys-45 and Cys-50. This oxidized BCP intermediate is shown to be a substrate for reduction by thioredoxin, completing the catalytic cycle. Although we invoke Cys-50 in the catalytic cycle of Escherichia coli bacterioferritin comigratory protein (BCP), a previous study had shown that this residue was not absolutely required for peroxiredoxin activity. In order to explain these apparently conflicting phenomena, we analyzed the reaction of a C50S BCP mutant with peroxide. We show that this mutant BCP enzyme adopts a different and novel mechanistic pathway. The C50S BCP mutant reacts with peroxide to form a sulfenic acid on Cys-45, in the same manner as wild-type BCP. However, the nascent intermediate is then resolved by reaction with Cys-45 from a second BCP molecule, resulting in a dimeric intermediate containing an intermolecular disulfide bond. We further show that this novel resolving complex is a substrate for reduction by thioredoxin. The importance of our results in furthering the understanding of catalysis within BCP family is discussed.