The effects of biliverdin on pressure-induced unfolding of apomyoglobin: The specific role of Zn 2+ ions

Apomyoglobin (apoMb), a model protein in biochemistry, exhibits a strong propensity to bind various ligands, which makes it a good candidate as a carrier of bioactive hydrophobic drugs. The stability of its hydrophobic pocket determines its potential as a carrier of bioactive compounds. High pressure (HP) is a potent tool for studying protein stability, revealing the specific role of hydrophobic cavities in unfolding. We probed the effects of biliverdin (BV) binding and its complex with Zn ions on the structure and HP stability of apoMb. CD spectroscopy and SAXS measurements revealed that BV and BV-Zn complexes make the apoMb structure more compact with higher α-helical content. We performed in situ HP measurements of apoMb intrinsic fluorescence to demonstrate the ability of BV to stabilise apoMb structure at HP conditions. Furthermore, the presence of Zn within the apoMb-BV complex significantly enhances the BV stabilisation effect. In situ visible absorption study of BV chromophore confirmed the ability of Zn to increase the stability of apoMb-BV complex under HP: the onset of complex dissociation is shifted by ∼100 MPa in presence of Zn . By combining HP-fluorescence and HP-visible absorption spectroscopy, our strategy highlights the crucial role of tetrapyrrole-metal complexes in stabilising apoMb hydrophobic pocket.