title: Distinct mechanisms for DNA cleavage by myoglobin with a designed heme active center.
abstract: Heme proteins perform diverse biological functions, of which myoglobin (Mb) is a representative protein. In this study, the O2 carrier Mb was shown to cleave double stranded DNA upon aerobic dithiothreitol-induced reduction, which is fine-tuned by an additional distal histidine, His29 or His43, engineered in the heme active center. Spectroscopic (UV-vis and EPR) and inhibition studies suggested that free radicals including singlet oxygen and hydroxyl radical are responsible for efficient DNA cleavage via an oxidative cleavage mechanism. On the other hand, L29E Mb, with a distinct heme active center involving three water molecules in the met form, was found to exhibit an excellent DNA cleavage activity that was not depending on O2. Inhibition and ligation studies demonstrated for the first time that L29E Mb cleaves double stranded DNA into both the nicked circular and linear forms via a hydrolytic cleavage mechanism, which resembles native endonucleases. This study provides valuable insights into the distinct mechanisms for DNA cleavage by heme proteins, and lays down a base for creating artificial DNA endonucleases by rational design of heme proteins. Moreover, this study suggests that the diverse functions of heme proteins can be fine-tuned by rational design of the heme active center with a hydrogen-bonding network.
authors.affiliation: School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China. Electronic address: alaike1983@163.com.
authors.forename: Ke-Jie
authors.initials: KJ
authors.lastname: Du
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: School of Chemistry and Chemical Engineering, University of South China, Hengyang 421001, China.
authors.forename: Bo
authors.initials: B
authors.lastname: He
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: Department of Chemistry & Institute of Biomedical Science, Fudan University, Shanghai, 200433, China.
authors.forename: Xiangshi
authors.initials: X
authors.lastname: Tan
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: D004247:DNA / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004587:Electrophoresis, Agar Gel; D006418:Heme / Q000737:chemistry*; D006868:Hydrolysis; D009211:Myoglobin / Q000737:chemistry*
chemical_list: D009211:Myoglobin; D006418:Heme; D004247:DNA
keywords: DNA cleavage; Heme proteins; Hydrogen-bonding network; Mechanism; Protein design