Hemoglobins are a group of respiratory proteins principally functioning in transport of oxygen and carbon dioxide in red blood cells of all vertebrates and some invertebrates. The blood clamT. granosais one of the few invertebrates that have hemoglobin-containing red hemocytes. In the present research, the peroxidase activity ofT. granosahemoglobins (Tg-Hbs) was characterized and the associated mechanism of action was deciphered via structural comparison with other known peroxidases. We detected that purified Tg-Hbs catalyzed the oxidation of phenolic compounds in the presence of exogenous H₂O₂. Tg-Hbs peroxidase activity reached the maximum at pH 5 and 35°C and was inhibited by Fe²⁺, Cu²⁺, SDS, urea, and sodium azide. Tg-Hbs shared few similarities in amino acid sequence and overall structural characteristics with known peroxidases. However, the predicted structure at their heme pocket was highly similar to that of horseradish peroxidase (HRP) and myeloperoxidase (MPO). This research represented the first systemic characterization of hemoglobin as a peroxidase.

This study investigates the essential role of the heme cavity in the peroxidase and antibacterial activities of homodimeric hemoglobin (Tg-HbI) from the blood clam Tegillarca granosa. After treatment with sodium dodecyl sulfate (SDS), the peroxidase and antibacterial activities of the Tg-HbI were significantly inhibited, with the degree of inhibition correlating positively with the SDS concentration. Fluorescence spectroscopy, UV-Vis spectroscopy, and molecular docking analysis further revealed that SDS interacts with key amino acid residues (e.g., His70 and His102) in the heme cavity of Tg-HbI, causing conformational changes that disrupt the internal hydrophobic interactions, thus inhibiting its function. This study confirms that the antibacterial effect of Tg-HbI is mediated through its peroxidase activity and that the heme cavity plays a critical role in maintaining this activity. These findings lay a foundation for further research on the immune defense functions of hemoglobin and provide new insights into the mechanisms of environmental adaptation in T. granosa.

Ferritin heavy chain (FTH1) is a key subunit of ferritin and serves as a core regulator of iron metabolism, playing an important role in alleviating cellular damage caused by oxidative stress or regulating programmed cell death. This study identified 7 FTH1 homologs (AgFTH1-1 to AgFTH1-7) across the entire genome of Anadara granosa and investigated their expression responses during Vibrio infection. The 7 AgFTH1 genes are arranged in tandem across 6 chromosomes, with AgFTH1-5 and AgFTH1-6 undergoing gene amplification via a local duplication event. Among these homologous genes, 5 genes contain a single conserved ferritin domain (PF00210) and retain key ferroxidase center residues (Glu23, His65). Following Vibrio infection, these 5 genes exhibit downregulated expression, which may increase intracellular free iron and be consistent with ferroptosis-like cell death contributing to pathogen clearance, as suggested by previous studies. AgFTH1-5 contains a signal peptide and exhibits increased expression, suggesting it may regulate extracellular local iron storage. AgFTH1-4 (synaptonemal N-terminal SNARE) and AgFTH1-7 (GTPase domain) lack signal peptides, exhibit atypical structures, and show no significant expression changes under bacterial stress, indicating they may be associated with vesicle trafficking rather than classical iron storage. This study systematically analyzed the genomic features and expression patterns of the FTH1 gene family in A. granosa, laying a foundation for further revealing its role in shellfish immune defense.