title: Modifications in heme iron of free and vesicle bound cytochrome c by tert-butyl hydroperoxide: a magnetic circular dichroism and electron paramagnetic resonance investigation.
abstract: To characterize changes to the heme and the influence of membrane lipids in the reaction of cytochrome c with peroxides, we studied the reaction of cytochrome c with tert-butyl hydroperoxide (tert-BuOOH) by magnetic circular dichroism (MCD) and direct electron paramagnetic resonance (EPR) in the presence and absence of different liposomes. Direct low-temperature (11 degrees K) EPR analysis of the cytochrome c heme iron on exposure to tert-BuOOH shows a gradual (180 s) conversion of the low-spin form to a high-spin Fe(III) species of rhombic symmetry (g = 4.3), with disappearance of a prior peroxyl radical signal (g(o) = 2.014). The conversion to high spin precedes Soret band bleaching, observable by UV/Vis spectroscopy and by magnetic circular dichroism (MCD) at room temperature, that indicates loss of iron coordination by the porphyrin ring. The presence of cardiolipin-containing liposomes delayed formation of the peroxyl radical and conversion to high-spin iron, while dicetylphosphate (DCP) liposomes accelerated these changes. Correspondingly, bleaching of cytochrome c by tert-BuOOH at room temperature was accelerated by several negatively charged liposome preparations, and inhibited by mitochondrial-mimetic phosphatidylcholinephosphatidylethanolaminecardiolipin (PCPECL) liposomes. Concomitant with bleaching, spin-trapping measurements with 5,5-dimethyl-1-pyroline-N-oxide showed that while the relative production of peroxyl, alkoxyl, and alkyl radicals was unaffected by DCP liposomes, PCPECL liposomes decreased the spin-trapped alkoxyl radical signal by 50%. The EPR results show that the primary initial change on exposure of cytochrome c to tert-BuOOH is a change to a high-spin Fe(III) species, and together with MCD measurements show that unsaturated cardiolipin-containing lipid membranes influence the interaction of tert-BuOOH with cytochrome c heme iron, to alter radical production and decrease damage to the cytochrome.
authors.affiliation: Centro Interdisciplinar de Investigação Bioquímica (CIIB), Universidade de Mogi das Cruzes (UMC), São Paulo, Brazil.
authors.forename: I L
authors.initials: IL
authors.lastname: Nantes
authors.forename: A
authors.initials: A
authors.lastname: Faljoni-Alário
authors.forename: O R
authors.initials: OR
authors.lastname: Nascimento
authors.forename: B
authors.initials: B
authors.lastname: Bandy
authors.forename: R
authors.initials: R
authors.lastname: Gatti
authors.forename: E J
authors.initials: EJ
authors.lastname: Bechara
mesh_terms: D000818:Animals; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D008081:Liposomes; D008563:Membrane Lipids / Q000378:metabolism; D010084:Oxidation-Reduction; D010545:Peroxides / Q000378:metabolism; D020122:tert-Butylhydroperoxide / Q000494:pharmacology*
chemical_list: D003574:Cytochrome c Group; D005609:Free Radicals; D008081:Liposomes; D008563:Membrane Lipids; D010545:Peroxides; C049375:perhydroxyl radical; D006418:Heme; D020122:tert-Butylhydroperoxide; D007501:Iron