Dynamics of dehaloperoxidase-hemoglobin A derived from NMR relaxation spectroscopy and molecular dynamics simulation

Dehaloperoxidase-hemoglobin is the first hemoglobin identified with biologically-relevant oxidative functions, which include peroxidase, peroxygenase and oxidase activities. Herein we report a study of the protein backbone dynamics of DHP using heteronuclear NMR relaxation methods and molecular dynamics (MD) simulations to address the role of protein dynamics in switching from one function to another. The results show that DHP's backbone helical regions and turns have average order parameters of S² = 0.87 ± 0.03 and S² = 0.76 ± 0.08, respectively. Furthermore, DHP is primarily a monomer in solution based on the overall tumbling correlation time τ_m is 9.49 ± 1.65 ns calculated using the prolate diffusion tensor model in the program relax. A number of amino acid residues have significant R_ex using the Lipari-Szabo model-free formalism. These include Lys³, Ile⁶, Leu¹³, Gln¹⁸, Arg³², Ser⁴⁸, Met⁴⁹, Thr⁵⁶, Phe⁶⁰, Arg⁶⁹, Thr⁷¹ Cys⁷³, Ala⁷⁷, Asn⁸¹, Gly⁹⁵, Arg¹⁰⁹, Phe¹¹⁵, Leu¹²⁷ and Met¹³⁶, which may experience slow conformational motions on the microseconds-milliseconds time scale according to the model. Caution should be used when the model contains >4 fitting parameters. The program caver3.0 was used to identify tunnels inside DHP obtained from MD simulation snapshots that are consistent with the importance of the Xe binding site, which is located at the central intersection of the tunnels. These tunnels provide diffusion pathways for small ligands such as O₂, H₂O and H₂O₂ to enter the distal pocket independently of the trajectory of substrates and inhibitors, both of which are aromatic molecules.