Impact of the Internal Disulfide Bond on Structural Dynamics in Vertebrates Hexacoordinate Hemeoglobins

Abstract

There are two new additions to the vertebrate globins family, neuroglobin and cytoglobin, which exhibit bis-hystidyl hexacoordination. Mainly expressed in neurons, neuroglobin (Ngb) has a neuro-protective role under hypoxic-ischemic insults; whereas cytoglobin (Cygb) expression is no tissue-specific and this protein was proposed to protect cells against oxidative stress. To understand the impact of the disulfide bond on structural dynamics in Ngb and Cygb, we determined kinetics and thermodynamics for CO photo-release and rebinding to both proteins in the presence and absence of the disulfide bond (Ngbred and Cygbred). Photoacoustic calorimetry (PAC) data indicate that CO photo-dissociation from Ngb and Ngbred is monophasic (τ<50 ns) leading to an endothermic enthalpy change (ΔH∼20 kcal mol−1) and the volume change is ∼3 times larger for hNgb than hNgbred (ΔV=4.4±0.3 mL mol−1). Interestingly, biphasic kinetics were observed for CO migration from the heme pocket of Cygb with τ1<50 ns and an additional τ2∼200 ns at 20 oC. Cygbred produces monophasic kinetics with τ<50 ns suggesting that the disulfide bond strongly modulates the ligand migration pathway. CO photo-release from Cygb first leads to a volume expansion (ΦΔV1= 4.2 ± 1.9 mL mol−1) and endothermic enthalpy change (ΦΔH1= 23.3 ± 9.1 kcal mol−1), followed by a smaller volume expansion (ΦΔV2= 3.7 ± 1.5 mL mol−1) and exothermic enthalpy change (ΦΔH2= −11.8 ± 7.0 kcal mol−1). Upon disruption of the disulfide bond, CO photo-dissociation from Cygbred produces a prompt endothermic enthalpy change (ΦΔH= 6.3 ± 0.4 kcal mol−1) and a small volume expansion (ΦΔV= 1.8 ± 0.8 mL mol−1). These results suggest different mechanisms of ligand migration among vertebrate hexacoordinate hemoglobins as well as a larger impact of the disulfide bond on mechanism of ligand migration in Cygb than in Ngb.