title: Autocatalytic formation of green heme: evidence for H2O2-dependent formation of a covalent methionine-heme linkage in ascorbate peroxidase.
abstract: The mammalian heme peroxidases are distinguished from their plant and fungal counterparts by the fact that the heme group is covalently bound to the protein through ester links from glutamate and aspartate residues to the heme 1- and 5-methyl groups and, in the case of myeloperoxidase, through an additional sulfonium link from the Cbeta of the 2-vinyl group to a methionine residue. To duplicate the sulfonium link in myeloperoxidase and to obtain information on its mechanism of formation, we have engineered a methionine residue close to the 2-vinyl group in recombinant pea cytosolic ascorbate peroxidase (rpAPX) by replacement of Ser160 by Met (S160M variant). The S160M variant is isolated from Escherichia coli as apo-protein. Reconstitution of apo-S160M with exogenous heme gives a red protein (S160M(R)) which has UV-visible (lambda(max)/nm = 407, 511, 633) and steady-state kinetic (kcat = 156 +/- 7 s(-1), KM = 102 +/- 15 microM) properties that are analogous to those of rpAPX. The reaction of S160M(R) with H2O2 gives a green protein (S160M(G)). Electronic spectroscopy, mass spectrometry, and HPLC analyses are consistent with the formation of a covalent linkage between the methionine residue and the heme vinyl group in S160M(G). Single-wavelength and photodiode array stopped-flow kinetic analyses identify a transient Compound I species as a reaction intermediate. The results provide the first direct evidence that covalent heme linkage formation occurs as an H2O2-dependent process that involves Compound I formation. A mechanism that is consistent with the data is presented.
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.forename: Michael
authors.initials: M
authors.lastname: Ott
authors.forename: Neesha
authors.initials: N
authors.lastname: Patel
authors.forename: Kuldip
authors.initials: K
authors.lastname: Singh
authors.forename: Sharad C
authors.initials: SC
authors.lastname: Mistry
authors.forename: Harold M
authors.initials: HM
authors.lastname: Goff
authors.forename: Emma Lloyd
authors.initials: EL
authors.lastname: Raven
mesh_terms: D000595:Amino Acid Sequence; D060387:Ascorbate Peroxidases; D002384:Catalysis; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008715:Methionine / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018532:Pisum sativum / Q000201:enzymology; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013056:Spectrophotometry, Ultraviolet
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D006418:Heme; D008715:Methionine; D006861:Hydrogen Peroxide; D010544:Peroxidases; D060387:Ascorbate Peroxidases