title: Crystal structure of the pristine peroxidase ferryl center and its relevance to proton-coupled electron transfer.
abstract: The reaction of peroxides with peroxidases oxidizes the heme iron from Fe(III) to Fe(IV)=O and a porphyrin or aromatic side chain to a cationic radical. X-ray-generated hydrated electrons rapidly reduce Fe(IV), thereby requiring very short exposures using many crystals, and, even then, some reduction cannot be avoided. The new generation of X-ray free electron lasers capable of generating intense X-rays on the tenths of femtosecond time scale enables structure determination with no reduction or X-ray damage. Here, we report the 1.5-Å crystal structure of cytochrome c peroxidase (CCP) compound I (CmpI) using data obtained with the Stanford Linear Coherent Light Source (LCLS). This structure is consistent with previous structures. Of particular importance is the active site water structure that can mediate the proton transfer reactions required for both CmpI formation and reduction of Fe(IV)=O to Fe(III)-OH. The structures indicate that a water molecule is ideally positioned to shuttle protons between an iron-linked oxygen and the active site catalytic His. We therefore have carried out both computational and kinetic studies to probe the reduction of Fe(IV)=O. Kinetic solvent isotope experiments show that the transfer of a single proton is critical in the peroxidase rate-limiting step, which is very likely the proton-coupled reduction of Fe(IV)=O to Fe(III)-OH. We also find that the pKa of the catalytic His substantially increases in CmpI, indicating that this active site His is the source of the proton required in the reduction of Fe(IV)=O to Fe(IV)-OH.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900;
authors.forename: Georges
authors.initials: G
authors.lastname: Chreifi
authors.affiliation: Macromolecular Crystallographic Group, The Stanford Synchrotron Radiation Lightsource, National Accelerator Laboratory, Stanford University, Stanford, CA 94309;
authors.forename: Elizabeth L
authors.initials: EL
authors.lastname: Baxter
authors.affiliation: Macromolecular Crystallographic Group, The Stanford Synchrotron Radiation Lightsource, National Accelerator Laboratory, Stanford University, Stanford, CA 94309;
authors.forename: Tzanko
authors.initials: T
authors.lastname: Doukov
authors.affiliation: Macromolecular Crystallographic Group, The Stanford Synchrotron Radiation Lightsource, National Accelerator Laboratory, Stanford University, Stanford, CA 94309;
authors.forename: Aina E
authors.initials: AE
authors.lastname: Cohen
authors.affiliation: Macromolecular Crystallographic Group, The Stanford Synchrotron Radiation Lightsource, National Accelerator Laboratory, Stanford University, Stanford, CA 94309;
authors.forename: Scott E
authors.initials: SE
authors.lastname: McPhillips
authors.affiliation: Macromolecular Crystallographic Group, The Stanford Synchrotron Radiation Lightsource, National Accelerator Laboratory, Stanford University, Stanford, CA 94309;
authors.forename: Jinhu
authors.initials: J
authors.lastname: Song
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900;
authors.forename: Yergalem T
authors.initials: YT
authors.lastname: Meharenna
authors.affiliation: Macromolecular Crystallographic Group, The Stanford Synchrotron Radiation Lightsource, National Accelerator Laboratory, Stanford University, Stanford, CA 94309; soltis@slac.stanford.edu poulos@uci.edu.
authors.forename: S Michael
authors.initials: SM
authors.lastname: Soltis
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900; Department of Pharmaceutical Sciences, University of California, Irvine, CA 92697-3900; Department of Chemistry, University of California, Irvine, CA 92697-3900 soltis@slac.stanford.edu poulos@uci.edu.
authors.forename: Thomas L
authors.initials: TL
authors.lastname: Poulos
mesh_terms: D018360:Crystallography, X-Ray; D004583:Electrons; D005290:Ferric Compounds / Q000737:chemistry*; D010544:Peroxidases / Q000737:chemistry*; D011487:Protein Conformation; D011522:Protons
chemical_list: D005290:Ferric Compounds; D011522:Protons; D010544:Peroxidases
keywords: XFEL; compound I; femtosecond crystallography; peroxidase