title: Creation of a Thermally Tolerant Peroxidase.
abstract: An artificial peroxidase with thermal tolerance and high catalytic activity has been successfully prepared by mutagenesis of an electron transfer protein, cytochrome c552 from Thermus thermophilus. The mutant enzymes were rationally designed based on the general peroxidase mechanism and spectroscopic analyses of an active intermediate formed in the catalytic reaction. Stopped flow UV-vis spectroscopy and EPR spectroscopy with a rapid freezing sample technique revealed that the initial double mutant, V49D/M69A, which was designed to reproduce the peroxidase mechanism, formed an active oxo-ferryl heme intermediate with a protein radical predominantly localized on Tyr45 during the catalytic reaction. The magnetic power saturation measurement obtained from EPR studies showed little interaction between the oxo-ferryl heme and the tyrosyl radical. Kinetics studies indicated that the isolated oxo-ferryl heme component in the active intermediate was a possible cause of heme degradation during the reaction with H2O2. Strong interaction between the oxo-ferryl heme and the radical was achieved by replacing Tyr45 with tryptophan (resulting in the Y45W/V49D/M69A mutant), which was similar to a tryptophanyl radical found in active intermediates of some catalase-peroxidases. Compared to the protein radical intermediates of V49D/M69A mutant, those of the Y45W/V49D/M69A mutant showed higher reactivity to an organic substrate than to H2O2. The Y45W/V49D/M69A mutant exhibited improved peroxidase activity and thermal tolerance.
authors: Research Center of Materials Science, Nagoya University, Nagoya, Japan.
authors: Y
authors: Y
authors: Watanabe
authors: Graduate School of Science, Osaka City University, Osaka, Japan. Electronic address: nakajima@sci.osaka-cu.ac.jp.
authors: H
authors: H
authors: Nakajima
mesh_terms: D002384:Catalysis; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D009195:Peroxidase / Q000138:chemical synthesis / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D013696:Temperature; D016963:Thermus thermophilus / Q000737:chemistry / Q000201:enzymology*
chemical_list: D009195:Peroxidase
keywords: Cytochrome c(552); Electron transfer protein; Oxo-ferryl heme; Peroxidase; Thermus thermophilus