YhjA - An Escherichia coli trihemic enzyme with quinol peroxidase activity

Abstract

The trihemic bacterial cytochrome c peroxidase from Escherichia coli, YhjA, is a membrane-anchored protein with a C-terminal domain homologous to the classical bacterial peroxidases and an additional N-terminal (NT) heme binding domain. Recombinant YhjA is a 50 kDa monomer in solution with three c-type hemes covalently bound. Here is reported the first biochemical and spectroscopic characterization of YhjA and of the NT domain demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P (active site), NT and E hemes were established to be -170 mV, +133 mV and +210 mV, respectively, at pH 7.5. YhjA has quinol peroxidase activity in vitro with optimum activity at pH 7.0 and millimolar range K_M values using hydroquinone and menadiol (a menaquinol analogue) as electron donors (K_M = 0.6 ± 0.2 and 1.8 ± 0.5 mM H₂O₂, respectively), with similar turnover numbers (k_cat = 19 ± 2 and 13 ± 2 s^-1, respectively). YhjA does not require reductive activation for maximum activity, in opposition to classical bacterial peroxidases, as P heme is always high-spin 6-coordinated with a water-derived molecule as distal axial ligand but shares the need for the presence of calcium ions in the kinetic assays. Formation of a ferryl Fe(IV) = O species was observed upon incubation of fully oxidized YhjA with H₂O₂. The data reported improve our understanding of the biochemical properties and catalytic mechanism of YhjA, a three-heme peroxidase that uses the quinol pool to defend the cells against hydrogen peroxide during transient exposure to oxygenated environments.

Keywords: Escherichia coli; Heme enzyme; Oxidative stress; Quinol peroxidase activity; Trihemic bacterial peroxidase.