Disruption of a potential disulfide bond of Cys65-Cys141 on the structure and stability of globin X from zebrafish.

Globin X is a newly discovered member of the globin family, while its structure and function are not fully understood. In this study, we performed protein modelling studies using Alphafold3 and molecular dynamics simulations, which suggested that the protein adopts a typical globin fold, with the formation of a potential disulfide bond of Cys65 and Cys141. To elucidate the role of this unique disulfide in protein structure and stability, we constructed a double mutant of C65S/C141S by mutating the two cysteine residues to serine. As suggested by protein mass, ultraviolet-visible (UV-Vis) and circular dichroism (CD) spectroscopy analyses, the potential disulfide bond has minimal effect on the overall protein structure, but its absence reduces the protein stability. Electron paramagnetic resonance (EPR) analysis also revealed an increase in the proportion of high-spin state heme iron, which accelerates the rate of heme degradation in reaction with HO. This study highlights the critical role of the Cys65-Cys141 in maintaining the stability of globin X and the bis-His heme coordination state, providing insights into the structure-function relationship of the newly discovered globin.