Spectroscopic studies on human androglobin regulated by its heme distal Gln12 and IQ motif via interaction with...

Androglobin (Adgb) was discovered as the fifth mammalian globin, yet its structural and functional properties remain incompletely understood. In this study, we overexpressed the heme-binding globin domain of Adgb with stabilizing mutations (C114S/C190S, denoted Adgb-SS) and investigated the role of its heme distal residue Gln12 through the generation of Q12H and Q12Y mutants. The protein structures were predicted using AlphaFold3 in combination with molecular dynamic simulations. Spectroscopic studies showed that the Q12H mutation improved protein stability under conditions of alkaline and oxidative (H₂O₂) stress, while the Q12Y substitution significantly enhanced nitrite reductase (NIR) activity. Moreover, we showed that the NIR activity of Adgb was increased by interaction with calmodulin (CaM) via its IQ motif. The structure of Adgb-CaM complex was also predicted using HDOCK. To further probe the role of the IQ motif, we generated an IQ-deletion mutant (ΔIQ-Adgb), which displayed reduced stability compared to Adgb-SS. Moreover, unlike Adgb, the turnover number (k_cat) of ΔIQ-Adgb was not increased but rather inhibited by CaM, indicating that the IQ motif is essential for CaM-mediated activation. Collectively, these results suggest that the heme distal Gln12 regulates both the protein stability and enzymatic activity of Adgb, while the IQ motif acts as a critical structural element that governs Adgb stability and enables proper regulation by CaM. These findings enhance our understanding of the structure-function relationship of Adgb and elucidate its potential role in fertility-related and other pathologies involving CaM interactions.