title: The diheme cytochrome c peroxidase from Shewanella oneidensis requires reductive activation.
abstract: We report the characterization of the diheme cytochrome c peroxidase (CcP) from Shewanella oneidensis (So) using UV-visible absorbance, electron paramagnetic resonance spectroscopy, and Michaelis-Menten kinetics. While sequence alignment with other bacterial diheme cytochrome c peroxidases suggests that So CcP may be active in the as-isolated state, we find that So CcP requires reductive activation for full activity, similar to the case for the canonical Pseudomonas type of bacterial CcP enzyme. Peroxide turnover initiated with oxidized So CcP shows a distinct lag phase, which we interpret as reductive activation in situ. A simple kinetic model is sufficient to recapitulate the lag-phase behavior of the progress curves and separate the contributions of reductive activation and peroxide turnover. The rates of catalysis and activation differ between MBP fusion and tag-free So CcP and also depend on the identity of the electron donor. Combined with Michaelis-Menten analysis, these data suggest that So CcP can accommodate electron donor binding in several possible orientations and that the presence of the MBP tag affects the availability of certain binding sites. To further investigate the structural basis of reductive activation in So CcP, we introduced mutations into two different regions of the protein that have been suggested to be important for reductive activation in homologous bacterial CcPs. Mutations in a flexible loop region neighboring the low-potential heme significantly increased the activation rate, confirming the importance of flexible loop regions of the protein in converting the inactive, as-isolated enzyme into the activated form.
authors.affiliation: Department of Chemistry, Boston University, Boston, Massachusetts 02215, United States.
authors.forename: Gökçe Su
authors.initials: GS
authors.lastname: Pulcu
authors.forename: Katherine E
authors.initials: KE
authors.lastname: Frato
authors.forename: Rupal
authors.initials: R
authors.lastname: Gupta
authors.forename: Hao-Ru
authors.initials: HR
authors.lastname: Hsu
authors.forename: George A
authors.initials: GA
authors.lastname: Levine
authors.forename: Michael P
authors.initials: MP
authors.lastname: Hendrich
authors.forename: Sean J
authors.initials: SJ
authors.lastname: Elliott
mesh_terms: D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics; D004789:Enzyme Activation / Q000235:genetics; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000493:pharmacokinetics; D020592:Shewanella / Q000201:enzymology* / Q000235:genetics
chemical_list: D058619:Maltose-Binding Proteins; D010088:Oxidoreductases; D003578:Cytochrome-c Peroxidase; C090099:diheme cytochrome c