Modifications in heme iron of free and vesicle bound cytochrome c by tert-butyl hydroperoxide: A magnetic circular dichroism and electron paramagnetic resonance investigation

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 cy tochrome c with tert-butyl hydroperoxide (tert-BuOOn) by magnetic circular dichroism (MCD) and direct electron paramagnetic resonance (EPR) in the pre sence and absence of different liposomes. Direct low-temperature (11 degree s K) EPR analysis of the cytochrome c heme iron on exposure to tert-BuOOH s hows a gradual (180 s) conversion of the low-spin form to a high-spin Fe(II I) species of rhombic symmetry (g = 4.3), with disappearance of a prior per oxyl radical signal (g(o) = 2.014). The conversion to high spin precedes So rer band bleaching, observable by UV/Vis spectroscopy and by magnetic circu lar dichroism (MCD) at room temperature, that indicates loss of iron coordi nation by the porphyrin ring. The presence of cardiolipin-containing liposo mes delayed formation of the peroxyl radical and conversion to high-spin ir on, while dicetylphosphate (DCP) liposomes accelerated these changes. Corre spondingly, bleaching of cytochrome c by tert-BuOOH at room temperature was accelerated by several negatively charged liposome preparations, and inhib ited by mitochondrial-mimetic phosphatidylcholine\ phosphatidylethanolamine \cardiolipin (PCPECL) liposomes. Concomitant with bleaching, spin-trapping measurements with 5,5-dimethyl-1-pyroline-N-oxide showed that while the rel ative 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 expo sure of cytochrome c to tert-BuOOH is a change to a high-spin Fe(III) speci es, and together with MCD measurements show that unsaturated cardiolipin-co ntaining lipid membranes influence the interaction of tert-BuOOH with cytoc hrome c heme iron, to alter radical production and decrease damage to the c ytochrome. (C) 2000 Elsevier Science Inc.