Tyr30 And C-type Heme Impact On The Structure And Function Of Cytochrome B₅

Heme proteins perform a large array of biological functions,with the heme group bound non-covalently or covalently.To probe the stabilization role of conserved tyrosine residue in the secondary sphere of heme site in heme proteins,we herein used cytochrome b₅?Cyt b₅?as a model protein,and mutated Tyr30 to Phe or His by removal of Tyr30associated H-bond network and hydrophobic interaction,respectively.We performed thermal-induced unfolding studies for the two mutants,Y30F Cyt b₅ and Y30H Cyt b₅,as monitored by both UV-Vis and CD spectroscopy,as well as heme transfer studies from these proteins to apo-myoglobin,with wild-type Cyt b₅ under the same conditions for comparison.The reduced stability of both mutants indicates that both the H-bonding and hydrophobic interactions associated with Tyr30 contribute to the protein stability.Moreover,we performed molecular modelling studies,which revealed that the hydrophobic interaction in the local region of Y30F Cyt b₅ was well-remained,whereas Y30H Cyt b₅ formed a unique H-bond network.These observations suggest that the conserved Tyr30 in Cyt b₅ is not replaceable due to the presence of both the H-bond network and hydrophobic interaction in the secondary sphere of the heme active site.As the case demonstrated here for Cyt b₅,it is of practical importance for design of artificial heme proteins by engineering a Tyr in the secondary sphere with improved properties and functions.Design of artificial metalloenzymes has achieved tremendous progress,while few designs can achieve catalytic performances comparable to that of native enzymes.Moreover,the structure and function of artificial metalloenzymes in non-native states was rarely explored.In this study,we found that a c-type cytochrome b₅?Cyt b₅?,N57C/S71C Cyt b₅,with heme covalently attached to the protein matrix via two Cys-heme linkages,adopts a non-native state with an open heme site upon Gdn·HCl-induced unfolding,which facilitates H₂O₂ activation and substrate binding.Stopped-flow kinetic studies further revealed that c-type Cyt b₅ in the non-native state exhibits an impressive peroxidase activity comparable to that of native peroxidases,such as the most efficient horseradish peroxidase.This study presents an alternative approach to designing functional artificial metalloenzymes by exploring the enzymatic functions in non-native states.