Thermodynamics of oxygenation-linked proton and lactate binding govern the temperature sensitivity of O2 binding in crustacean (Carcinus maenas) hemocyanin.

With the aim of understanding the molecular underpinnings of the enormous variation in the temperature sensitivity of hemocyanin-O2 affinity encountered in crustaceans, we measured O2 binding to Carcinus maenas hemocyanin at two temperatures, varying pH values and in the absence and presence of lactate ions in order to assess the contributions of oxygenation-linked binding of protons (the Bohr effect) and lactate ions to the overall enthalpies of oxygenation (DeltaH'). The hemocyanin binds maximally 0.35 lactate ions per functional subunit. Lactate (which accumulates under hypoxic conditions) increases O2 affinity by preferentially raising the association equilibrium constant of the hemocyanin in the low-affinity Tense state (KT), without significantly affecting that of the high-affinity Relaxed state (KR). In the absence of lactate, the variation in the temperature sensitivity observed with decreasing pH tallies neatly with changes in the nature and magnitude of the Bohr effect. Accordingly, the normal, absent and reverse Bohr effects observed under alkaline, neutral and acid conditions, respectively, reflect endothermic proton dissociation, absence of proton binding and exothermic proton association, respectively, upon oxygen binding. Oxygenation-linked lactate binding is exothermic, highly pH dependent and peaks near pH 7.6, where it contributes approximately -30 kJ mol(-1) to the overall heat of oxygenation. This predictably increases the temperature sensitivity of O2 affinity, potentially hampering O2 loading in warm, hypoxic habitats. The data demonstrate governing roles for lactate and proton ions in determining the temperature sensitivity of hemocyanin-O2 affinity in crustaceans.