Effects Of Cysteine On Myoglobin And Neuroglobin In Post Translational Modifications

The structure and function of heme proteins are regulated by diverse post-translational modifications including chemical modification,heme-protein cross-links and intramolecular disulfide bond.In this study,To explore the mechanisms of heme-protein cross-links,we introduced a Cys（K42C）close to the heme 4-vinyl group in sperm whale myoglobin（Mb）and solved its X-ray crystal structure.Interestingly,we found that K42 C Mb can partially form a Cys-heme cross-link（termed K42 C Mb-X）under dithiothreitol-induced reductive conditions in presence of O₂,as suggested by guanidine hydrochloride-induced unfolding and heme extraction studies.Mass spectrometry（MS）studies,together with trypsin digestion studies,further indicated that a thioether bond was formed between Cys42 and the heme 4-vinyl group with an additional mass of 16 Da,likely due to hydroxylation of theα-carbon.A plausible mechanism for the formation of the novel Cys-heme cross-link was then proposed based on MS,kinetic UV-Vis and electron paramagnetic resonance（EPR）studies.Moreover,the Cys-heme cross-link was shown to fine-tune the protein reactivity toward activation of H₂O₂.This study provides valuable insights into the post-translational modification of heme proteins,and also suggests that the Cys-heme cross-link can be induced to form in vitro,making it useful for design of new heme proteins with a non-dissociable heme and improved functions.In addition,we constructed a second intramolecular disulfide bond in human neuroglobin（Ngb）and obtained the mutant protein A15 C Ngb with a higher purity.We performed UV-Vis spectroscopy and Circular Dichroism（CD）spectroscopy for WT Ngb and A15 C Ngb.The Mass spectra（MS）showed that both Cys15 and Cys120 formed an intramolecular disulfide bond in A15 C Ngb.Remarkably,the disulfide bond improves the protein stability,as proved by MS,guanidinehydrochloride-inducedunfoldingandthermal denaturation studies.These results provide valuable information for elucidating the structure-function relationship of heme proteins and these approaches are expected be extended to design of other heme proteins with superior functions.