Mutation-induced heme pocket destabilization in myoglobinopathy.

Introduction: Myoglobin (Mb) is a heme-binding protein essential for oxygen storage, transport, and redox regulation, governed by a finely tuned network of electrostatic and hydrogen bonding interactions within the heme pocket. A point mutation His97Tyr leads to a hereditary disease, myoglobinopathy which disrupts heme-propionate interactions and leads to protein aggregation. Previous structural studies relied on a crystallographic model in which a Lys45Arg substitution was introduced to stabilize the heme pocket, masking the effects of the His97Tyr mutation and preventing clear interpretation of its role. Methods: To resolve this, we performed classical molecular dynamics simulations to systematically dissect the individual and combined structural effects of residues 45 and 97 by comparing the His97Tyr mutation in the native Lys45 (wild-type) and the crystallographic Arg45 residue. Results: Our results show that residue 45 plays a dominant role in regulating long-range dynamical networks, with Arg45 producing more spatially coherent correlations than the diffuse dynamics observed in the wild-type Lys45 protein. Structural variability within the heme pocket is primarily driven by redistribution of heme-propionates, where residue 45 acts as the main determinant, while residue 97 contributes an additive effect. Although iron coordination remains preserved across all systems, the wild-type His97Tyr variant exhibits tilting of the heme group within the pocket, indicating increased conformational flexibility. Discussion: Together, these findings suggest that the disease-associated mutation alters heme dynamics and highlights the critical role of sequence context in interpreting mutation-driven changes in myoglobin structure and function.