CO Rebinding Kinetics and Molecular Dynamics Simulations Highlight Dynamic Regulation of Internal Cavities in Human Cytoglobin

Abstract

Cytoglobin (Cygb) was recently discovered in the human genome and localized in different tissues. It was suggested to play tissue-specific protective roles, spanning from scavenging of reactive oxygen species in neurons to supplying oxygen to enzymes in fibroblasts. To shed light on the functioning of such versatile machinery, we have studied the processes supporting transport of gaseous heme ligands in Cygb. Carbon monoxide rebinding shows a complex kinetic pattern with several distinct reaction intermediates, reflecting rebinding from temporary docking sites, second order recombination, and formation (and dissociation) of a bis-histidyl heme hexacoordinated reaction intermediate. Ligand exit to the solvent occurs through distinct pathways, some of which exploit temporary docking sites. The remarkable change in energetic barriers, linked to heme bis-histidyl hexacoordination by HisE7, may be responsible for active regulation of the flux of reactants and products to and from the reaction site on the distal side of the heme. A substantial change in both protein dynamics and inner cavities is observed upon transition from the CO-liganded to the pentacoordinated and bis-histidyl hexacoordinated species, which could be exploited as a signalling state. These findings are consistent with the expected versatility of the molecular activity of this protein.

Dynamics of the Heme-binding Bacterial Gas-sensing Dissimilative Nitrate Respiration Regulator (DNR) and Activation Barriers for Ligand Binding and Escape

“…In these globins, CO dynamics between intraprotein cavities have been studied with lower time resolution (Ͼ10 ns). The temperature dependence of the small recombination phases on the submicrosecond time scale (the extent of which appears to correlate with the presence of an internal heme ligand (54)) also indicates virtually barrierless recombination toward the heme pocket and activated migration toward the solvent (55,56), with barriers on the same order of magnitude as found in the present work. Thus, whereas these studies do not cover the initial ligand migration steps, comparison with our work supports that these properties are general features of 6-coordinate heme proteins.…”

Immobilization of Proteins in Silica Gel: Biochemical and Biophysical Properties

“…4) shows a pattern similar to the one observed for Ngb [146,162]. Photodissociated ligands can exit to the solvent following separate pathways, some of which exploit temporary docking sites [162] Hexacoordination by the distal His residue imposes large changes in energetic barriers that were suggested to modulate the flux of reactants to and products from the reaction site at the heme. Ligation appears to strongly affect CO rebinding kinetics to Cygb gels [162].…”

“…Photodissociated ligands can exit to the solvent following separate pathways, some of which exploit temporary docking sites [162] Hexacoordination by the distal His residue imposes large changes in energetic barriers that were suggested to modulate the flux of reactants to and products from the reaction site at the heme. Ligation appears to strongly affect CO rebinding kinetics to Cygb gels [162]. Cygb gels prepared by embedding unliganded Cygb (a bis-histidyl hexacoordinated species) allow easy formation of the CO adduct.…”

“…Comparison between the CO rebinding kinetics to Cygb solutions (filled circles), COCygb gels (open squares, Cygb was embedded in the gel as COCygb) and Cygb+CO gels (open circles, Cygb was embedded in the gel as deoxy Cygb and CO was added after the gel was formed). T = 40°C, 1 atm CO. Data from [162]. matrices [163][164][165][166][167][168][169].…”

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

“…An increase in the geminate quantum yield was observed for Tyr44Phe and Tyr44Phe The observed heterogeneity was attributed to CO rebinding from primary and secondary ligand docking sites, as previously described in chapter 4 . Multiphasic kinetics has also been reported for CO geminate rebinding in Ngb and Cygb encapsulated in silica gels (Abbruzzetti et al, 2009;Gabba et al, 2013), but the fast rate constant reported here was not resolved in those studies.…”