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 clam T. granosa is one of the few invertebrates that have hemoglobin-containing red hemocytes. In the present research, the peroxidase activity of T. granosa hemoglobins (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.

Beyond its role as an oxygen transport protein, the homodimer hemoglobin of blood clam Tegillarca granosa (Tg-HbI) has been found to possess antibacterial activity. However, the mechanism of antibacterial activity of Tg-HbI remain to be investigated. In this study, we investigated the effects of Cu2+ on the structure, peroxidase activity, and antibacterial ability of Tg-HbI. Tg-HbI was significantly inactivated by Cu2+ in a non-competitive inhibition manner, following first-order reaction kinetics. The Spectroscopy results showed that Cu2+ changed the iron porphyrin ring and the coordination of heme with proximal histidine of Tg-HbI, and increased the hydrophobicity of heme pocket. We found that proline could stabilize the heme pocket structure of Tg-HbI, hence, protect peroxidase activity and antimicrobial activity of Tg-HbI against damage by Cu2+. Our results suggest that Cu2+ inhibits the peroxidase and antibacterial activity of Tg-HbI by destroying its heme pocket structure and Tg-HbI probably plays an antibacterial role through its peroxidase activity. This result could provide insights into the antibacterial mechanism of Tg-HbI.