title: Heme enzymes. Neutron cryo-crystallography captures the protonation state of ferryl heme in a peroxidase.
abstract: Heme enzymes activate oxygen through formation of transient iron-oxo (ferryl) intermediates of the heme iron. A long-standing question has been the nature of the iron-oxygen bond and, in particular, the protonation state. We present neutron structures of the ferric derivative of cytochrome c peroxidase and its ferryl intermediate; these allow direct visualization of protonation states. We demonstrate that the ferryl heme is an Fe(IV)=O species and is not protonated. Comparison of the structures shows that the distal histidine becomes protonated on formation of the ferryl intermediate, which has implications for the understanding of O-O bond cleavage in heme enzymes. The structures highlight the advantages of neutron cryo-crystallography in probing reaction mechanisms and visualizing protonation states in enzyme intermediates.
authors.affiliation: Department of Biochemistry and Henry Wellcome Laboratories for Structural Biology, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK. Institut Laue-Langevin, 71 Avenue des Martyrs, 38000, Grenoble, France.
authors.forename: Cecilia M
authors.initials: CM
authors.lastname: Casadei
authors.affiliation: Department of Chemistry, University of Leicester, University Road, Leicester LE1 7RH, UK.
authors.forename: Andrea
authors.initials: A
authors.lastname: Gumiero
authors.affiliation: Department of Chemistry, University of Leicester, University Road, Leicester LE1 7RH, UK.
authors.forename: Clive L
authors.initials: CL
authors.lastname: Metcalfe
authors.affiliation: Department of Chemistry, University of Leicester, University Road, Leicester LE1 7RH, UK.
authors.forename: Emma J
authors.initials: EJ
authors.lastname: Murphy
authors.affiliation: Department of Biochemistry and Henry Wellcome Laboratories for Structural Biology, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK.
authors.forename: Jaswir
authors.initials: J
authors.lastname: Basran
authors.affiliation: The Photon Science Institute, The University of Manchester, Manchester M13 9PL, UK.
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Concilio
authors.affiliation: Institut Laue-Langevin, 71 Avenue des Martyrs, 38000, Grenoble, France. EPSAM, Keele University, Keele, Staffordshire ST5 5BG, UK.
authors.forename: Susana C M
authors.initials: SC
authors.lastname: Teixeira
authors.affiliation: Jülich Centre for Neutron Science (JCNS), Forschungszentrum Jülich GmbH, Outstation at MLZ, Lichtenbergstraße 1, 85747 Garching, Germany.
authors.forename: Tobias E
authors.initials: TE
authors.lastname: Schrader
authors.affiliation: The Photon Science Institute, The University of Manchester, Manchester M13 9PL, UK.
authors.forename: Alistair J
authors.initials: AJ
authors.lastname: Fielding
authors.affiliation: Heinz Maier-Leibnitz Zentrum (MLZ), Technische Universität München, Lichtenbergstraße 1, D-85748 Garching, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Ostermann
authors.affiliation: Institut Laue-Langevin, 71 Avenue des Martyrs, 38000, Grenoble, France.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Blakeley
authors.affiliation: Department of Chemistry, University of Leicester, University Road, Leicester LE1 7RH, UK. emma.raven@le.ac.uk peter.moody@le.ac.uk.
authors.forename: Emma L
authors.initials: EL
authors.lastname: Raven
authors.affiliation: Department of Biochemistry and Henry Wellcome Laboratories for Structural Biology, University of Leicester, Lancaster Road, Leicester LE1 9HN, UK. emma.raven@le.ac.uk peter.moody@le.ac.uk.
authors.forename: Peter C E
authors.initials: PC
authors.lastname: Moody
mesh_terms: D018360:Crystallography, X-Ray / Q000379:methods; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry; D007501:Iron / Q000737:chemistry*; D033363:Neutron Diffraction; D009502:Neutrons; D010100:Oxygen / Q000737:chemistry; D011522:Protons
chemical_list: D011522:Protons; C066317:ferryl iron; D006418:Heme; D006639:Histidine; D007501:Iron; D003578:Cytochrome-c Peroxidase; D010100:Oxygen