title: 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.
authors.affiliation: School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK. dave.clarke@ed.ac.uk
authors.forename: David J
authors.initials: DJ
authors.lastname: Clarke
authors.forename: C Logan
authors.initials: CL
authors.lastname: Mackay
authors.forename: Dominic J
authors.initials: DJ
authors.lastname: Campopiano
authors.forename: Pat
authors.initials: P
authors.lastname: Langridge-Smith
authors.forename: Alan R
authors.initials: AR
authors.lastname: Brown
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D002846:Chromatography, Affinity; D003573:Cytochrome b Group / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D005293:Ferritins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013879:Thioredoxins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D013879:Thioredoxins; D005293:Ferritins; C018032:bacterioferritin