Tyrosine Hydrogen-bond Network Influence On Structure And Function Of Myoglobin

Protein design has been demonstrated to be powerful not only in illustrating the structure and function relationship of native enzymes,but also in creating functional artificial enzymes.Peoxidase rational design and de novo design have received much attention during the last few decades,and various approaches have been successfully developed,including using non-heme metal ions,unnatural amino acids,or heme mimics to modify the heme active site for functional fine-tuning.Hydrogen-bond?H-bond?network,specifically Tyr-associated H-bond network,plays key roles in regulating the structure and function of proteins.In this thesis,To explore an approach for fine-tuning the structure and function in design of artificial heme proteins,we herein used myoglobin?Mb?as a model protein and introduced a Tyr residue in the secondary sphere of the heme active site at two different positions?107 and 138?.We performed X-ray crystallography,UV-Vis spectroscopy,stopped-flow kinetics,and electron paramagnetic resonance?EPR?studies for the two single mutants,I107Y Mb and F138Y Mb,and compared to that of wide-type?WT?Mb under the same conditions.The results showed that both Tyr107 and Tyr138 form a distinct H-bond network involving water molecule and neighboring residues,which was found to facilitate ligand binding to the heme iron?in I107Y Mb case?and enhance the protein stability?in F138Y Mb case?,respectively.Moreover,the Tyr107-associated H-bond network was shown to fine-tune both H₂O₂ binding and activation.With two cases demonstrated for Mb,this study suggests that Tyr-associated H-bond network has distinct roles in regulating the protein structure,property and function,depending on its location in the protein scaffold.Therefore,it is possible for design of a Tyr-associated H-bond network in general to create other artificial heme proteins with improved properties and functions.And we found F43Y Mb,with Tyr43 forming a novel cross-link to the heme 4-vinyl group,exhibit dramatically enhanced dehaloperoxidase activity compared to that of wild-type Mb?¹15-fold?,which is beyond the catalytic efficiency of native dehaloperoxidase from Amphitrite ornata?⁹-fold?,indicating a potential application for bioremediation.