Neuroglobin Is Capable Of Self-oxidation Of Methionine64 Introduced At The Heme Axial Position And Effect Of Distal Disulfide Bond On Stability

Heme protein is a class of a kind of metalloproteins with the main characteristics of protoporphyrin IX?heme?prosthetic group.Neuroglobin?Ngb?,as a new type of hemoglobin discovered in the early 20th century,has many novel and unknown functions,which make it a good template for studying the structure and function transformation of hemoglobin.We took human neuroglobin?H-Ngb?as the research object to simulate the six-coordinate structure?Met80/His18?of cytochrome c?Cyt c?heme.Mutant protein H64M Ngb was obtained by mutating heme axially64-position histidine?His64?into methionine?Met?.When the molecular weight of the protein was determined by mass spectrometry,we found three peaks in the protein,the molecular weight showing an increase of 16 Da and 32 Da.The trypsinase digestion result of mutant protein demonstrated that the autoxidation site was heme axial Met64.We proposed the mechanism of self-oxidation in H64M Ngb.This study also proves that H64M Ngb has a new function of activating oxygen.Its autoxidation phenomenon is similar to that of Met80 before cyt c induces cell apoptosis.The self-oxidation phenomenon is similar to that of Met80 before cyt c induces apoptosis,which not only enriches the diversity of neuroglobin function,but also further illustrates the importance of Ngb in biological organisms.In order to protect the free Cys120 and to enhance the protein stability,we herein developed a strategy by design of an additional disulfide bond between Cys120 and Cys15 via A15C mutation.Molecular modeling,UV-Vis,ESI-MS,CD and Stopped-flow were used to characterize A15C Ngb and WT Ngb.And the Mass spectrometry confirmed that Cys15-Cys120 formed an intramolecular disulfide bonds;Gdn·HCl-induced unfolding,thermal stability and pH stability were used to evaluate the stability of the mutant protein A15C Ngb.The results demonstrate that the stability of the constructed A15C Ngb was significantly higher than that of WT Ngb.In addition,it was confirmed by Stopped-flow and UV-vis that A15C Ngb still possesses the basic function of binding small molecule ligands such as O₂ and CO.This study not only suggests a curial role of the artificial disulfide in protein stabilization,but also lays the groundwork for further investigation of the structure and function of Ngb,as well as for artificial heme protein design,based on the scaffold of A15C Ngb with an enhanced stability.