title: Compound I formation in artichoke (Cynara scolymus L.) peroxidase is modulated by the equilibrium between pentacoordinated and 6-aquo hexacoordinated forms of the heme and by calcium ions.
abstract: Basic artichoke (Cynara scolymus L.) peroxidase (AKP-C), when purified from the plant, has an unusually intense and sharp Soret absorption peak. The resonance Raman spectrum [López-Molina, D., et al. (2003) J. Inorg. Biochem. 94, 243-254] suggested a mixture of pentacoordinate high-spin (5cHS) and 6-aquo hexacoordinate high-spin (6cHS) ferric heme species. The rate constant (k(1)) of compound I formation with hydrogen peroxide (H(2)O(2)) was also lower than expected. Further stopped-flow studies have shown this reaction to be biphasic: a nonsaturating fast phase and a slow phase with complex H(2)O(2) concentration dependence. Addition of calcium ions (Ca(2+)) changed the absorption spectrum, suggesting the formation of a fully 5cHS species with a k(1) more than 5 orders of magnitude greater than that in the absence of Ca(2+) using the chelator ethylenediaminetetraacetic acid. Ca(2+) titrations gave a dissociation constant for a single Ca(2+) of approximately 20 microM. The circular dichroism spectrum of AKP-C was not significantly altered by Ca(2+), indicating that any structural changes will be minor, but removal of Ca(2+) did suppress the alkaline transition between pH 10 and 11. A kinetic analysis of the reaction of Ca(2+)-free AKP-C with H(2)O(2) supports an equilibrium between a slow-reacting 6cHS form and a more rapidly reacting 5cHS species, the presence of which was confirmed in nonaqueous solution. AKP-C, as purified, is a mixture of Ca(2+)-bound 5cHS, 6-aquo 6cHS, and Ca(2+)-free 5cHS species. The possibility that Ca(2+) concentration could control peroxidase activity in the plant is discussed.
authors.affiliation: Grupo de Enzimología (GENZ), Departamento de Bioquímica y Biología Molecular-A, Facultad de Biología, Universidad de Murcia, Murcia, Spain.
authors.forename: Alexander N P
authors.initials: AN
authors.lastname: Hiner
authors.forename: Lara
authors.initials: L
authors.lastname: Sidrach
authors.forename: Soledad
authors.initials: S
authors.lastname: Chazarra
authors.forename: Ramón
authors.initials: R
authors.lastname: Varón
authors.forename: José
authors.initials: J
authors.lastname: Tudela
authors.forename: Francisco
authors.initials: F
authors.lastname: García-Cánovas
authors.forename: José Neptuno
authors.initials: JN
authors.lastname: Rodríguez-López
mesh_terms: D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D031943:Cynara scolymus / Q000201:enzymology*; D004492:Edetic Acid / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D009195:Peroxidase / Q000737:chemistry*; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D014466:Ultraviolet Rays
chemical_list: D006418:Heme; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D009195:Peroxidase; D002118:Calcium