title: Stabilization of cytochrome b5by a conserved tyrosine in the secondary sphere of heme active site: A spectroscopic and computational study.
abstract: 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 Tyr30 associated H-bond network and hydrophobic interaction. We performed thermal-induced unfolding studies for the two mutants, Y30F Cyt b5and 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 b5under 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 modeling studies, which revealed that the hydrophobic interaction in the local region of Y30F Cyt b5was well-remained, whereas Y30H Cyt b5formed an H-bond network. These observations suggest that the conserved Tyr30 in Cyt b5is 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 demonstrated here for Cyt b5, it may be of practical importance for design of artificial heme proteins by engineering a Tyr in the secondary sphere with improved properties and functions.
authors.affiliation: School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China.
authors.forename: Shan
authors.initials: S
authors.lastname: Hu
authors.affiliation: School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China.
authors.forename: Bo
authors.initials: B
authors.lastname: He
authors.affiliation: School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China.
authors.forename: Xiao-Juan
authors.initials: XJ
authors.lastname: Wang
authors.affiliation: Laboratory of Protein Structure and Function, University of South China, Hengyang 421001, China.
authors.forename: Shu-Qin
authors.initials: SQ
authors.lastname: Gao
authors.affiliation: Laboratory of Protein Structure and Function, University of South China, Hengyang 421001, China.
authors.forename: Ge-Bo
authors.initials: GB
authors.lastname: Wen
authors.affiliation: School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China; Laboratory of Protein Structure and Function, University of South China, Hengyang 421001, China. Electronic address: linlinying@hotmail.com.
authors.forename: Ying-Wu
authors.initials: YW
authors.lastname: Lin
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D002942:Circular Dichroism; D003198:Computer Simulation; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D015786:Cytochromes b5 / Q000737:chemistry*; D006418:Heme / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry; D011489:Protein Denaturation; D055550:Protein Stability; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship; D013696:Temperature; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D050505:Mutant Proteins; D014443:Tyrosine; D006418:Heme; D015786:Cytochromes b5
keywords: Cyt b(5); Heme proteins; Heme transfer; Secondary sphere; Unfolding