New insights into NO bonding in Tan sheep myoglobin for meat pigmentation: Spectroscopic and density functional theory investigations

Abstract

Color of meat remains crucial quality attribute that attracts attention of researchers worldwide. Various strategies have been employed to manipulate meat color by generating nitric oxide (NO), which interacts with myoglobin (Mb) to yield the pinkish-red pigment, nitrosomyoglobin (MbFeⅡNO). However, the formation mechanism of MbFeⅡNO warrants further exploration at the molecular-structural level for meat pigmentation. Here, Tan sheep Mb was purified and characterized its primary structure via LC-MS/MS, which found it to be identical to sheep Mb. Circular dichroism spectroscopy revealed 70.05 % α-helix content in the secondary structure of Tan sheep Mb. UV-visible and resonance Raman spectroscopy monitored NO bonding in Tan sheep Mb, revealing that NO displaced H2O of MbFeⅢ, forming MbFeⅢNO, which subsequently transformed into low-spin six-coordination MbFeⅡNO. Additionally, three-dimensional structure of Tan sheep Mb was constructed utilizing Alphafold2, and theoretical simulations and calculations were performed through molecular docking and DFT. Results confirmed the synergistic effect of the strong metal-ligand and hydrogen bonding interactions among the NO, heme iron, and distal sites significantly contributed to the stable formation of MbFeⅡNO. Overall, this study offers a novel perspective on the mechanism of NO binding in Mb, potentially guiding the development of innovative strategies to mitigate meat color deterioration.