NMR Study On Oxidative Modification Of Cytochrome C

Cytochrome c is a multi-conformation metalloprotein located in mitochondria.Under normal conditions,the cytochrome c mainly transfers electrons on the respiratory chain.When mitochondria under oxidative stress,cytochrome c could be released from mitochondria into cytoplasmic,thereby combining with downstream substances to trigger apoptosis.Mitochondria inevitably generate reactive oxygen species(ROS)during respiration.When the environment of the organism changes or stimulated,the ROS will increase.At this time,the organism will oxidative stress due to its own defense mechanism.It has been found that cytochrome c undergoes oxidative modification in the presence of ROS,resulting in its release from mitochondria,but the mechanism of oxidative modification is not clear.In this paper,we studied the oxidative modification and the structure transformation of cytochrome c in the presence of different oxidants using Nuclear Magnetic Resonance(NMR)with combination of selectively methionine labeling method.the detail works were shown as follows.1.Using 1H-13C MAXY-HMQC(Maximum-quantum correlation spectroscopy),the methyl signal on free methionine was identified.It was found that the chemical shift of the methyl group of methionine would change significantly with the degree of the oxidation.Therefore,tracking the chemical shift of the methyl group or the change of the signal intensity could quickly determine the degree of oxidation of methionine on the protein.2.The oxidative modification of cytochrome c under different H2O2 concentrations was observed by 13C labeling of the methionine terminal methyl group on Saccharomyces cerevisiae cytochrome c in liquid NMR spectrum and confirmed by circular dichroism experiments.These results showed that Fe in cytochrome c was oxidized at the first,and then the methionine was oxidative modified.The oxidative modification mainly occurred on Met80.Unlike the previous studies by others,the secondary structure of the protein did not change significantly,indicating that Saccharomyces cerevisiae cytochrome c had a certain ability to resist ROS in an oxidative environment.As the accumulation of ROS further increased,the Met80 of the protein which coordinates with iron around heme would be oxidized.3.The oxidative modification of Saccharomyces cerevisiae cytochrome c was studied by chloramine T(CT)combined with liquid NMR and mass spectrometry experiments under alkaline conditions.It was found that CT not only sulfoxides Met80,but also triggered the dimerization and changed the structure of the protein.These results showed that the secondary structure of Saccharomyces cerevisiae cytochrome c was changed greatly after oxidative modification,whereas that of human cytochrome c was stable.Comparing with the results in presence of H2O2,the result indicated that conformation changes of Saccharomyces cerevisiae cytochrome c may lead to the difference of oxidation modification sites and degree,affect the function changes of cytochrome c.In summary,the oxidative modification of cytochrome c was mainly studied by liquid NMR.Comparing the oxidative modification of proteins in neutral and alkaline environments and in different source proteins.It was found that the oxidative modification of cytochrome c was related to its original conformation.This research provides new ideas for further revealing the mechanism of oxidative modification of cytochrome c.