Regulating Multiple Catalytic Function Of Myoglobin By Redesigning The Heme Active Center

Abstract:Hemeproteins perform a large array of biological functions,including electron-transfer,oxygen delivery,catalysis and signaling.More fantastically,the same heme protein may exhibit diverse functions in different situations.To reveal the structure-function relationship of heme proteins,and to provide clues for creating artificial heme proteins with improved functions,we here used myoglobin（Mb）as a model protein and redesigned the heme active center,by introducing one or two distal histidines,and by creating a channel to the heme center with removal of the native distal His64 gate（His to Ala mutation）.By evaluating their nitrite reductase（NIR）activity and peroxidase activity in presence of H₂O₂ using guaiacol and ABTS as a typical substrate,we revealed that a single distal histidine with a suitable position to the heme iron and creation of a water channel to the heme center play a crucial role in nitrite reduction.Moremore,the mutant with two distal histidines and a channel to the heme pocket,L29H/F43H/H64A Mb,exhibits an enhanced peroxidase activity.Moreover,we herein rationally introduced a pair of cysteines（F46C/M55C）into the scaffold of myoglobin（Mb）,mimicking those in native neuroglobin.Molecular modeling suggested that it is possible for Cys46 and Cys55 to form an intramolecular disulfide bond,which was confirmed experimentally by ESI-MS analysis,DTNB reaction and CD spectrum.Remarkably,the formation of an intramolecular disulfide bond of Cys46-Cys55 in F46C/M55C Mb improves the protein stability and enhances peroxidase activity.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 artificial enzymes with improved catalytic performance.