Abstract
This work compares the effect of photogenerated singlet oxygen (O(2)((1)Delta(g))) (type II mechanism) and free radicals (type I mechanism) on cytochrome c structure and reactivity. Both reactive species were obtained by photoexcitation of methylene blue (MB(+)) in the monomer and dimer forms, respectively. The monomer form is predominant at low dye concentrations (up to 8 microm) or in the presence of an excess of SDS micelles, while dimers are predominant at 0.7 mm SDS. Over a pH range in which cytochrome c is in the native form, O(2) ((1)Delta(g)) and free radicals induced a Soret band blue shift (from 409 to 405 nm), predominantly. EPR measurements revealed that the blue shift of the Soret band was compatible with conversion of the heme iron from its native low spin state to a high spin state with axial symmetry (g approximately 6.0). Soret band bleaching, due to direct attack on the heme group, was only detected under conditions that favored free radical production (MB(+) dimer in SDS micelles) or in the presence of a less structured form of the protein (above pH 9.3). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry of the heme group and the polypeptide chain of cytochrome c with Soret band at 405 nm (cytc405) revealed no alterations in the mass of the cytc405 heme group but oxidative modifications on methionine (Met(65) and Met(80)) and tyrosine (Tyr(74)) residues. Damage of cytc405 tyrosine residue impaired its reduction by diphenylacetaldehyde, but not by beta-mercaptoethanol, which was able to reduce cytc405, generating cytochrome c Fe(II) in the high spin state (spin 2).
Highlights
The association of cytochrome c with lipid bilayers is modulated by the heme iron redox states [1]
The rate of cytochrome c reduction and oxidation was measured over a pH range of 3.0 to 9.0 in which cytochrome c remains in its native conformation and spin state
Within this range of pH, different rates of cytochrome c reduction promoted by MBϩ and the irreversible heme iron re-oxidation produced by O2(1⌬g) were obtained
Summary
The association of cytochrome c with lipid bilayers is modulated by the heme iron redox states [1]. This association changes the cytochrome c spin state [1, 2] and influences its reactivity with aldehydes, ketones [3], and peroxides [4] These reactions produce free radicals and excited states, both able to attack cytochrome c amino acid side chains and heme group. In this regard, the reaction of cytochrome c with t-butyl-hydroperoxide leads to bleaching of the Soret band, with conversion of the hemeprotein from its low spin form with rhombic symmetry to a high spin form with axial and rhombic symmetry [3].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
