Reduction of the internal disulfide bond between Cys 38 and 83 switches the ligand migration pathway in cytoglobin.

Despite the similar tertiary structure between cytoglobin (Cygb) and myoglobin, several structural features indicate a distinct mechanism of Cygb interactions with exogenous ligands. Here we present a spectroscopic investigation of the dynamics and thermodynamics of structural changes associated with the exogenous ligand migration between the solvent and the heme active site in Cygb with reduced and oxidized Cys 38 and Cys 83 side-chains (Cygb(ox) and Cygb(red), respectively). Photo-acoustic and transient absorption data show that disulfide bond formation alters the ligand migration pathway(s) as evident from the distinct geminate quantum yields (Φgem=0.35 for Cygb(ox) and Φgem=0.63 for Cygb(red)) and rate constants for bimolecular CO rebinding. Moreover, ligand escape from the protein matrix is fast (<40ns) and coupled with an enthalpy change of 18±2kcalmol(-1) in Cygb(red), whereas the disulfide bridge formation promotes a biphasic ligand escape associated with an overall enthalpy change of 9±4kcalmol(-1). These results demonstrate that the disulfide bond connecting helix E and helix B modulates the conformational dynamics in Cygb including the size and energy barrier between the internal hydrophobic sites. Based on the comparison of the thermodynamic profiles for CO photo-dissociation from Cygb, myoglobin, and neuroglobin we propose that in Cygb(red) the photo-dissociated ligand escapes through the hydrophobic tunnel, whereas the CO preferably migrates through the His64 gate in Cygb(ox) suggesting that Cygb's physiological role may vary in response to intracellular redox conditions.