Kinetic characterization of a BCP‐class peroxiredoxin from Escherichia coli

Abstract

Peroxiredoxins (Prx) possess a conserved active site structure centered around the catalytic residue (the “peroxidatic” cysteine) which forms a cysteine sulfenic acid upon reaction with peroxides. This cysteine subsequently forms a disulfide bond with a resolving cysteine (either inter‐ or intramolecular) and/or the reductant during reductive recycling. In Escherichia coli, bacterioferritin‐comigratory protein (BCP) is a peroxiredoxin which catalyzes the reduction of hydrogen peroxide and alkyl hydroperoxides. BCP exhibits a broad specificity for reductants and peroxide substrates, potentially allowing BCP to remain fully active and functional under varying cellular conditions. BCP demonstrated comparable peroxidase activity when acting upon H2O2 and cumene hydroperoxide. During thioredoxin‐dependent peroxidase activity studies conducted by stopped flow spectroscopy, a linear rate increase was observed with increasing reducing substrate along with a turnover rate ~44 times faster than those previously reported. In our studies, BCP exhibited a kcat of 1.31 s−1 and a kcat/Km (for H2O2) of 1.42 x 104 M−1 s−1 using 10 μM thioredoxin as reductant. An exploration of other potential reductants of BCP revealed that Grx1 and Trx2 also act as reductants of BCP in E. coli. However, kcat/Km of BCP for H2O2 when utilizing Trx as an electron donor was calculated to be 2x and 11x higher than those demonstrated by Grx1 and Trx2 respectively. Supported by NIH RO1 GM50389 to LBP.