RESUMEN
We have shown that diphenlacetaldehyde (DPAA) is able to promote mitochondrial DeltaPsi disruption accompanied by damage in mitochondrial DNA, lipids, and proteins [Almeida, A. M.; Bechara, E. J. H.; Vercesi, A. E.; Nantes, I. L. Free Radic. Biol. Med. 27:744-747; 1999]. In this work, DPAA was used as a model of carbonyl reagent for cytochrome c. The results suggest that DPAA is a redox cytochrome c modifier. Conversion of Fe(III) to Fe(II) cytochrome c promoted by DPAA is pH dependent. The second-order rate determined for heme iron reduction (k2) is 698 M(-1) s(-1) and this process occurs with an activation energy of 8.5 +/- 0.8 kcal/mol. Analysis of the pH profile suggests the presence of two ionizable cytochrome c groups (pKa1 = 8.9 and pKa2 = 11.4) related to the electron transfer from DPAA to heme iron. The heats of ionization of the two prototropic groups, pKa1 (DeltaH(ion) = 6.5 kcal/mol, DeltaS(ion) = -29.0 cal/mol.K), and pKa2 (DeltaH(ion) = 5.0 kcal/mol, DeltaS(ion) = -24.0 cal/mol.K), suggest involvement of two tyrosine residues, probably Y67 and Y74, related to DPAA-promoted heme iron reduction. The cytochrome c chemical modification by iodination of tyrosine groups significantly decreased the reduction rate promoted by DPAA, and shifted the pH(opt) value from 10.0 to 9.25. The cytochrome c-promoted DPAA electron abstraction quickly produces the expected enol-derived radical, as indicated by 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) spin trapping EPR measurements. This radical reacts with molecular oxygen, producing a peroxyl intermediate radical that, via a putative dioxetane intermediate, promotes formation of benzophenone as the main final product of this reaction, detected by high-performance liquid chromatography coupled with tandem mass spectrometry.