Conformation Changes Of Saccharomyces Cerevisiae Cytochrome C In Mitochondria

Cytochrome c(cyt c)is a soluble heme protein with a molecular weight of about 12.5 kDa,it can adopt a variety of different spatial conformations under different conditions.Under normal conditions,cyt c is located in mitochondria,and its main function is to transfer electrons in the process of oxidative phosphorylation.However,under the condition of apoptosis,cyt c can translocate into the cytoplasm.Because the abnormalities of function caused by the conformational changes of cyt c are often related to specific diseases,understanding the process of the changes of cyt c in its in situ environment will help to better clarify its mechanism of functional transformation.In this dissertation,we mainly studied the structure of cyt c in situ by nuclear magnetic resonance(NMR)and observed its conformational and functional changes in living mitochondria.The main research contents are as follows:1.Conformational changes of cyt c after interaction with cardiolipin(CL).CL is an important component of the mitochondrial membrane.Its interaction with cyt c is considered to be a key step in the occurrence of apoptosis.However,whether this interaction affects cyt c structure is controversial.We studied the conformational changes of the protein by tracking the changes of modified methylation of Lys72 of saccharomyces cerevisiae iso-1 cyt c and the conformational changes of cyt c and CL after the interaction by NMR.It was found that the conformation would change after the combination of cyt c and CL,which was related to the concentration of CL.At lower CL concentration,the Fe-Met80 bond in cyt c natural structure breaks and is replaced into Fe-His and five-coordination conformations without axial ligand.With the continuous increase of CL contents,the Fe-His conformation will be further transformed into a conformation five-coordinated.Therefore,our results show that the interaction with CL can induce the conformation of cyt c to change gradually,and then obtain higher peroxidase activity,which maybe an important premise for its subsequent role in apoptosis.2.Oxidative modification and conformational changes of cyt c under reactive oxygen species(ROS).Mitochondria are an important place for material metabolism and the main source of ROS.We developed an NMR method to detect the oxidative modification of cyt c,and observed the oxidative modification of cyt c induced by ROS.The degree of oxidative modification of cyt c increases with the increase of concentration of H2O2,and its conformation also changes.The expansion degree of its structure increases with the increase of oxidative modification degree and gradually changes to the five-coordination conformation,which means that cyt c will obtain higher peroxidase activity,which is conducive to removal of ROS.Therefore,the oxidative modification of cyt c under the action of ROS and then changing its conformation may be an important mechanism for the transformation of its function from electron carrier to ROS scavenger.3.Based on the above research,we studied the in situ conformation of cyt c in the extracted living yeast mitochondria.We found that in living mitochondria,except for a small amount of free cyt c,most of cyt c was mainly bound to CL on the inner membrane of mitochondria through electrostatic interaction,and its structure was completely different from the so-called "natural conformation" in dilute solution.It existed in mitochondria in a partially expanded conformation.We further monitored the NMR spectra of cyt c in living mitochondria in real-time and found that more and more cyt c existed in the free state in mitochondria over time.These cyt c may be caused by the increase of ROS in mitochondria.ROS can induce the carbonylation modification of lysine on the surface of cyt c,resulting in the reduction of positive charge on the protein surface,which weakens the interaction between cyt c and CL on the mitochondrial membrane and causes the release of cyt c from the mitochondrial inner membrane.The above studies show that the carbonylation modification of cyt c lysine can trigger its release from the mitochondrial membrane,so this modification may be one of the mechanisms of cyt c release in the process of apoptosis.4.The release of cyt c from mitochondria is an important step of apoptosis,and whether and how the complex environment of cytoplasm will affect its conformation has not been confirmed.Therefore,we further studied the conformational changes of cyt c in the cytoplasm of yeast cell.We found that the cytosol has a great influence on the conformation of cyt c.Cyt c has at least four different oxidized conformations and one reduced conformation in the cytosol.Moreover,with the delay of time,the changes of environment in the cytosol can lead to the conversion of cyt c between different conformations,which may be related to the resistance of cyt c to oxidative stress.In general,the structure and changes of cyt c in living mitochondria were studied by NMR.It was found that the structure of cyt c was not only different from dilute solution but also changed with the change of environment.When ROS and other environmental changes occur,cyt c can undergo carbonylation modification of surface lysine,which may lead to the release of cyt c from the inner membrane of mitochondria.These studies enrich the understanding of the function of cyt c in cells and provide a structural basis for further revealing the mechanism of apoptosis induced by cyt c.