Kinetic and Thermodynamic Features Reveal That Escherichia coli BCP Is an Unusually Versatile Peroxiredoxin

In Escherichia coli, bacterioferritin comigratory protein (BCP) is a peroxiredoxin (Prx) that catalyzes the reduction of H2O2 and organic hydroperoxides. This protein, along with plant PrxQ, is a founding member of one of the least studied subfamilies of Prxs. Recent structural data have suggested that proteins in the BCP/PrxQ group can exist as monomers or dimers; we report here that, by analytical ultracentrifugation, both oxidized and reduced E. coli BCP behave as monomers in solution at concentrations as high as 200 it M. Unexpectedly, thioredoxin (Trx1)-dependent peroxidase assays conducted by stopped-flow spectroscopy demonstrated that V-max,V-app increases with increasing Trx1 concentrations, indicating a nonsaturable interaction (K-m > 100 mu M). At a physiologically reasonable Trx1 concentration of 10 mu M, the apparent K-m value for H2O2 is similar to 80 mu M, and overall, the V-max/K-m for H2O2, which remains constant at the various Trx1 concentrations (consistent with a ping-pong mechanism), is similar to 1.3 x 10(4) M-1 s(-1). Our kinetic analyses demonstrated that BCP can utilize a variety of reducing substrates, including Trx1, Trx2, Grxa, and Grx3. BCP exhibited a high redox potential of -145.9 +/- 3.2 mV, the highest to date observed for a Prx. Moreover, BCP exhibited a broad peroxide specificity, with comparable rates for H2O2 and cumene hydroperoidde. We determined a pK(a) of similar to 5.8 for the peroxidatic cysteine (Cys45) using both spectroscopic and activity titration data. These findings support an important role for BCP in interacting with multiple substrates and remaining active under highly oxidizing cellular conditions, potentially serving as a defense enzyme of last resort.