Increased functional unit flexibility and solvent accessibility favours oxygen capture in molluscan hemocyanin.

Hemocyanins are a class of copper-based oxygen transport proteins, widely studied for their unique oxygen-binding processes and their role in the molluscan immune response. In this study, we utilised computational simulations to investigate the first functional unit (FU-a) of (slipper limpet) hemocyanin, a member of the keyhole limpet hemocyanin family. Using quantum mechanics/molecular mechanics (QM/MM) methods, we designed oxygenated and deoxygenated models of FU-a and conducted molecular dynamics simulations to explore their functional dynamics and oxygen accessibility. We specifically focused on understanding the global and localised dynamics between the two conformational states. By employing principal component analysis (PCA) and modevector analysis, we differentiated the dynamic properties of the deoxygenated and oxygenated states of the hemocyanin. Furthermore, we explored the impact of oxygenation on hydration and tunnel cavity formation. Our results reveal that oxygen entry is mediated by a single bidirectional tunnel, with its permeability tightly regulated by differential histidine-based copper coordination. Importantly, we identified Glu352 as an evolutionary conserved molecular "shutter," whose conformational changes govern the opening and closure of this tunnel. These findings provide insight into the mechanistic regulation of oxygen transport in molluscan hemocyanins, with implications for understanding their functional versatility and potential applications.