Metal Preference Hierarchy in the HDAC8 Active Site: A DFT Study

HDAC8 is a histone deacetylase enzyme that plays a key role in the development of various diseases in humans, including cancers, neurodegenerative diseases, and alcohol use disorder. Although HDAC8 is classified as a Zn²⁺-dependent metalloenzyme, available data regarding the affinity of other biologically relevant ions, such as Fe²⁺, Ni²⁺, Co²⁺, and Mg²⁺, for the HDAC8 enzyme active site remain unclear and contradictory. The mechanism by which these ions compete with Zn²⁺ for the HDAC8 active site is not well understood. In this study, we performed density functional theory (DFT) calculations at the B3LYP/6-31+G(d) level of theory, combined with polarizable continuum model computations (PCM) in water (ε = 78) and methanol (ε = 32). The results show that Zn²⁺ remains the thermodynamically preferred cofactor across all modeled reactions. Although Fe²⁺ and Co²⁺ gain partial stabilization upon increasing coordination number, the associated entropic and desolvation penalties prevent them from outcompeting Zn²⁺ under physiologically relevant conditions. Only a limited number of substitution reactions for Fe²⁺ and Co²⁺ yield ∆G values near thermodynamic neutrality, and only in specific coordination states. In contrast, all modeled Ni²⁺ substitution reactions are unfavorable, and Mg²⁺ is strongly excluded from the HDAC8 active site in all reactions. The resulting metal preference hierarchy-Zn²⁺ > Co²⁺ ≈ Fe²⁺ > Ni²⁺ > Mg²⁺-supports experimental observations and clarifies the intrinsic selectivity of the HDAC8 enzyme towards Zn²⁺. These insights provide a molecular basis for understanding HDAC8 metallo-regulation and may guide the rational design of novel, isoform-specific HDACi with improved binding properties.