YhjA - An Escherichia coli trihemic enzyme with quinol peroxidase activity
| Autor: | Cláudia S. Nóbrega, Bart Devreese, Sofia R. Pauleta |
|---|---|
| Přispěvatelé: | UCIBIO - Applied Molecular Biosciences Unit, DQ - Departamento de Química |
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Heme binding Stereochemistry Genetic Vectors Heme enzyme Respiratory chain Biophysics Gene Expression Heme Quinol peroxidase activity Biochemistry Substrate Specificity 03 medical and health sciences chemistry.chemical_compound Escherichia coli Trihemic bacterial peroxidase Cloning Molecular Binding Sites 030102 biochemistry & molecular biology biology Hydroquinone Cytochrome c peroxidase Escherichia coli Proteins Active site Hydrogen Peroxide Cell Biology Cytochrome-c Peroxidase Hydrogen-Ion Concentration Recombinant Proteins Hydroquinones Kinetics 030104 developmental biology chemistry Peroxidases Oxidative stress Menadiol biology.protein Biocatalysis Oxidation-Reduction Peroxidase Protein Binding |
| Popis: | Belgian Federal Science Policy Office (Belspo) (grant to BD, IAP7/44, iPROS project). co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). 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 KM values using hydroquinone and menadiol (a menaquinol analogue) as electron donors (KM = 0.6 ± 0.2 and 1.8 ± 0.5 mM H2O2, respectively), with similar turnover numbers (kcat = 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. publishersversion published |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|