Effect of pressure on the formation and deoxygenation kinetics of oxymyoglobin. Mechanistic information from a volume profile analysis

Abstract

The effect of pressure on the formation and deoxygenation kinetics of oxymyoglobin was studied by using temperature-jump and stopped-flow techniques, respectively. The corresponding volumes of activation are +5.2 ± 0.5 and +23.3 ± 1.8 cm3 mol'1, which result in a reaction volume of -18.1 ± 2.3 cm3 mol'1 for the system Mb + 02 == Mb02. The latter was also measured directly from the pressure dependence of the equilibrium constant and resulted in a reaction volume of -19.3 ± 1.5 cm3 mol'1. A volume profile analysis indicated that bond breakage during deoxygenation proceeds according to a dissociative mechanism with almost complete Mb-G2 bond cleavage in the transition state. This suggests that almost no bond formation with the heme center occurs in the transition state during the oxygenation of myoglobin. The volume increase

Time Resolved Thermodynamics Associated with Diatomic Ligand Dissociation from Globins

“…The small enthalpy change measured for the 250 ns relaxation (ΔH = -8.9 ± 8.0 kcal mol -1 ) includes the enthalpy change for O 2 solvation (ΔH solv = -2.9 kcal mol -1 (Mills et al, 1979)) and the enthalpy change associated with H 2 O binding to the heme binding pocket (ΔH solv = -7 kcal mol -1 (Vetromile, et al, 2011) indicating that the structural relaxation coupled to the ligand escape from the protein is entropy driven. The overall enthalpy change for O 2 dissociation from Mb was determined to be 11.6 ± 8.5 kcal mol -1 and this value is in agreement with the value of 10 kcal mol -1 reported previously (Projahn et al, 1990). The overall reaction volume change determined here (ΔV overall = +2.5 mL mol -1 ) i s s o m e w h a t l a r g e r t h a n t h e r e a c t i o n v o l u m e c h a n g e d e t e r m i n e d f r o m t h e measurement of the equilibrium constant as a function of pressure (ΔV= -2.9 mL mol -1 ) (Hasinoff, 1974) and significantly smaller than the reaction volume change determined as a difference between the activation volume for oxygen binding and dissociation from Mb that was reported to be 18 mL mol -1 (Projahn et al, 1990).…”

“…The overall enthalpy change for O 2 dissociation from Mb was determined to be 11.6 ± 8.5 kcal mol -1 and this value is in agreement with the value of 10 kcal mol -1 reported previously (Projahn et al, 1990). The overall reaction volume change determined here (ΔV overall = +2.5 mL mol -1 ) i s s o m e w h a t l a r g e r t h a n t h e r e a c t i o n v o l u m e c h a n g e d e t e r m i n e d f r o m t h e measurement of the equilibrium constant as a function of pressure (ΔV= -2.9 mL mol -1 ) (Hasinoff, 1974) and significantly smaller than the reaction volume change determined as a difference between the activation volume for oxygen binding and dissociation from Mb that was reported to be 18 mL mol -1 (Projahn et al, 1990). Unlike photoacoustic studies that allow for reaction volume determination at ambient pressure, the high pressure measurements of equilibrium constant and/or rate constants (to determine activation volumes) may cause a pressure induced protein denaturation and/or structural changes, which may influence the magnitude of reaction volume changes in high pressure studies.…”

“…The (Projahn et al, 1990) and V H2O = 15 mL mol -1 (the partial molar volume of water scaled to the occupancy of water molecule hydrogen bound to distal histidine) (Belogortseva et al, 2007), we estimate that the O 2 release from Mb results in a structural volume change (V doxyMb -V O2-Mb ) of -7.5 mL mol -1 . This value is very similar to that reported previously for CO escape from Mb (ΔV structural = V doxyMb -V CO-Mb = -6 mL mol -1 ) (Vetromile, et al, 2011) demonstrating that the overall structural changes accompanying the ligand bound to ligand free transition in Mb are very similar for both ligands.…”

“…Using V CO = 35 mL mol -1 (Projahn et al, 1990) and V H2O = 9 mL mol -1 (partial molar volume of water scaled by the average occupancy of the Hb chain), we estimate that upon release of one CO molecule per Hb, the protein undergoes a small contraction of -7 mL mol -1 . The small volume change observed here is consistent with the minor structural changes due to deligation of Hb in the R-state as observed in the X-ray structure that are predominantly www.intechopen.com localized in the the -chain and include reposition of the F-helix and shift of the EF and CD corner (Wilson et al, 1996).…”

Conformational Dynamics Associated with Ligand Binding to Vertebrate Hexa-coordinate Hemoglobins

“…where V°O 2 is the partial molar volume of O 2 (28 mL mol -1 ) (Projahn et al, 1990), V°O 2Ngb is the partial molar volume of oxy-Ngb and V°N gb is the partial molar volume of penta-coordinate deoxy Ngb.…”