Determination of microscopic rate constants for CO binding and migration in myoglobin encapsulated in silica gels.
ABSTRACT CO rebinding kinetics after nanosecond photolysis of myoglobin encapsulated in wet silica gels exhibits an enhanced geminate phase that allows the determination of the microscopic rate constants and the activation barriers for distinct ligand docking sites inside the protein matrix. Using a maximum entropy method, we demonstrate that the geminate phase can be well-described by a biphasic lifetime distribution, reflecting rebinding from the distal and proximal sites. Microscopic rates and activation barriers were estimated using a four-state model.
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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.PLoS ONE 01/2013; 8(1):e49770. · 3.73 Impact Factor
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ABSTRACT: The presence of cavities and tunnels in the interior of proteins, in conjunction with the structural plasticity arising from the coupling to the thermal fluctuations of the protein scaffold, has profound consequences on the pathways followed by ligands moving through the protein matrix. In this perspective we discuss how quantitative analysis of experimental rebinding kinetics from laser flash photolysis, trapping of unstable conformational states by embedding proteins within the nanopores of silica gels, and molecular simulations can synergistically converge to gain insight into the migration mechanism of ligands. We show how the evaluation of the free energy landscape for ligand diffusion based on the outcome of computational techniques can assist the definition of sound reaction schemes, leading to a comprehensive understanding of the broad range of chemical events and time scales that encompass the transport of small ligands in hemeproteins.Physical Chemistry Chemical Physics 06/2013; · 3.83 Impact Factor
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ABSTRACT: Protoglobin from Methanosarcina acetivorans (MaPgb) is a dimeric globin with peculiar structural properties such as a completely buried haem and two orthogonal tunnels connecting the distal cavity to the solvent. CO binding to and dissociation from MaPgb occur through a biphasic kinetics. We show that the heterogenous kinetics arises from binding to (and dissociation from) two tertiary conformations in ligation-dependent equilibrium. Ligation favours the species with high binding rate (and low dissociation rate). The equilibrium is shifted towards the species with low binding (and high dissociation) rates for the unliganded molecules. A quantitative model is proposed to describe the observed carbonylation kinetics.PLoS ONE 01/2012; 7(3):e33614. · 3.73 Impact Factor