title: Structural and spectroscopic characterisation of a heme peroxidase from sorghum.
abstract: A cationic class III peroxidase from Sorghum bicolor was purified to homogeneity. The enzyme contains a high-spin heme, as evidenced by UV-visible spectroscopy and EPR. Steady state oxidation of guaiacol was demonstrated and the enzyme was shown to have higher activity in the presence of calcium ions. A Fe(III)/Fe(II) reduction potential of -266 mV vs NHE was determined. Stopped-flow experiments with H2O2 showed formation of a typical peroxidase Compound I species, which converts to Compound II in the presence of calcium. A crystal structure of the enzyme is reported, the first for a sorghum peroxidase. The structure reveals an active site that is analogous to those for other class I heme peroxidase, and a substrate binding site (assigned as arising from binding of indole-3-acetic acid) at the γ-heme edge. Metal binding sites are observed in the structure on the distal (assigned as a Na(+) ion) and proximal (assigned as a Ca(2+)) sides of the heme, which is consistent with the Ca(2+)-dependence of the steady state and pre-steady state kinetics. It is probably the case that the structural integrity (and, thus, the catalytic activity) of the sorghum enzyme is dependent on metal ion incorporation at these positions.
authors.affiliation: Department of Microbiology, University of Nigeria, Nsukka, Nigeria.
authors.forename: Chukwudi I
authors.initials: CI
authors.lastname: Nnamchi
authors.affiliation: Department of Chemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK.
authors.forename: Gary
authors.initials: G
authors.lastname: Parkin
authors.affiliation: Department of Chemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK.
authors.forename: Igor
authors.initials: I
authors.lastname: Efimov
authors.affiliation: Department of Molecular and Cell Biology, Henry Wellcome Laboratory for Structural Biology, University of Leicester, Lancaster Road, Leicester, LE1 9HN, UK.
authors.forename: Jaswir
authors.initials: J
authors.lastname: Basran
authors.affiliation: Department of Molecular and Cell Biology, Henry Wellcome Laboratory for Structural Biology, University of Leicester, Lancaster Road, Leicester, LE1 9HN, UK.
authors.forename: Hanna
authors.initials: H
authors.lastname: Kwon
authors.affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, UK.
authors.forename: Dimitri A
authors.initials: DA
authors.lastname: Svistunenko
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, York, YO10 5DD, UK.
authors.forename: Jon
authors.initials: J
authors.lastname: Agirre
authors.affiliation: Department of Microbiology, University of Nigeria, Nsukka, Nigeria.
authors.forename: Bartholomew N
authors.initials: BN
authors.lastname: Okolo
authors.affiliation: Department of Microbiology, University of Nigeria, Nsukka, Nigeria.
authors.forename: Anene
authors.initials: A
authors.lastname: Moneke
authors.affiliation: Department of Biochemistry, University of Nigeria, Nsukka, Nigeria.
authors.forename: Bennett C
authors.initials: BC
authors.lastname: Nwanguma
authors.affiliation: Department of Molecular and Cell Biology, Henry Wellcome Laboratory for Structural Biology, University of Leicester, Lancaster Road, Leicester, LE1 9HN, UK.
authors.forename: Peter C E
authors.initials: PC
authors.lastname: Moody
authors.affiliation: Department of Chemistry, University of Leicester, University Road, Leicester, LE1 7RH, UK. emma.raven@le.ac.uk.
authors.forename: Emma L
authors.initials: EL
authors.lastname: Raven
mesh_terms: D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry*; D007700:Kinetics; D010544:Peroxidases / Q000737:chemistry*; D045868:Sorghum / Q000737:chemistry*
chemical_list: D006418:Heme; D010544:Peroxidases
keywords: Calcium; Heme; Peroxidase; Porphyrin; Sorghum; X-ray crystallography