Time-Resolved Thermodynamics and Transient Kinetics for Oxygen Photorelease from Hemoglobin:Effector Complexes

Abstract

Photoacoustic calorimetry and transient absorption have been used to characterize thermodynamic and kinetic parameters for oxygen photorelease from human hemoglobin (Hb) complexed with allosteric effectors in terms of time-resolved volume and enthalpy changes, quantum yields for bimolecular rebinding, and rate constants for oxygen rebinding. Under stripped conditions we observe a significant temperature dependence of enthalpy and volume changes associated with oxygen photorelease. Below 16° C an endothermic enthalpy and volume expansion (ΔH = 272 ± 60 kcal mol−1, ΔV = 19.5 ± 4.5 mL mol−1) were determined for O2 photorelease, while above 16° C the reaction becomes exothermic and a volume contraction is observed (ΔH = −252 ± 79 kcal mol−1, ΔV = −58.3 ± 16.5 mL mol−1). Similar temperature dependence of ΔV/ΔH was observed in phosphate buffer at ionic strength of up to 0.2 M. Moreover, the reaction volume and enthalpy changes observed in the presence of 500 mM NaCl (I = 0.5 M) are significantly smaller (ΔH = −27 ± 8 kcal mol−1, ΔV = 7.9 ± 0.7 mL mol−1) and the temperature dependence is eliminated, suggesting the significant contribution of electrostriction to the observed reaction parameters. L35 also eliminated the temperature dependence without affecting volume and enthalpy changes associated with O2 photorelease, effectively extending the low temperature trend. L35 is known to bind strongly in the central cavity of Hb in the α1β1 and α2β2 clefts, and may exhibit this effect by modulating inter-subunit interactions. IHP acted similarly but decreased related enthalpy changes. In addition, allosteric effectors and phosphate buffers were found to increase the quantum yield for bimolecular rebinding of oxygen, and rate constants for oxygen rebinding to Hb:L35 and Hb:IHP complexes were lower than those for the stripped protein.