DNA Enzyme Activity Of Myoglobin And Its Derivatives

Heme proteins are important metalloproteins and perform diverse biological functions. The study of protein-DNA interactions will help understanding the structure of proteins and the function of artificial nuclease at a molecular level, which is of biological and significances and has potential application. The study uses myoglobin(Mb) as a representative model, redesigns its heme active center, and fine-tunes its DNA cleavage activity, which includes following chapters:Chapter 1 : As an introduction, it represents the structure and function of myoglobin, the progress in the study of the structure and function of heme proteins, and the interaction of heme proteins and DNA.Chapter 2: We found that the O2 carrier Mb is able to cleave the double stranded DNA upon aerobic dithiothreitol-induced reduction. The additional distal histidine engineered in the heme active center, His29and/or His43, fine-tuned the DNA cleavage activity. Inhibition studies indicated that single oxygen and hydroxyl radicals are responsible for efficient DNA cleavage via an oxidative cleavage mechanism.Spectroscopic studies(UV-vis and EPR) showed that His43 enhanced the cleaving ability whereas His29 inhibited it. These results show that the structure and enzymatic activities of heme proteins can be fine-tuned by the microenvironment of the heme active center with a hydrogen-bonding network.Chapter 3: We designed a mutant protein L29 E Mb, with a distal glutamine in the heme active center, and found that exhibited an excellent DNA cleavage activity in the absence of external reducing agent.Inhibition and T4-ligation studies indicated the first time that L29 E Mb cleaves double stranded DNA into both the linear and circular forms via a hydrolytic cleavage mechanism, which resembles native endonucleases.This study provides deep insight into the distinct mechanisms for DNA cleavage by heme proteins, and lays down a base for creating artificial endonucleases by rational design of heme proteins.Chapter 4: We found that the reconstituted myoglobin in which the heme prosthetic group was substituted by protoporphyrinato zinc(Zn PP)performed high nuclease activity under irradiation at 420-430 nm. The UV-vis spectroscopy study indicated that Zn PP was successfully incorporated into the hydrophobic cavity of apo-Mb. Gel electrophoresis experiments further showed that the Zn-substituted myoglobin(Zn PP-Mb)can effectively cleavage DNA at lower concentration compared to the zinc complex Zn PP alone under irradiation. Inhibition studies indicatedthat the activated species including singlet oxygen, hydroxyl radical and superoxide radical may participate in the cleavage. Additional studies of Zn PP-L29 H Mb suggest that His29 can inhibit the nuclease activity of Zn PP-Mb. Therefore, the studies of photosensitized protein help us to learn the molecular design of protein and drug, which are valuables in the application of proteins in pharmaceutical chemistry.