Viscosity‐dependent energy barriers and equilibrium conformational fluctuations in oxygen recombination with hemerythrin

Abstract

The recombination kinetics of photo-dissociated oxyhemerythrin (Sipunculus nudus) have been investigated between 298 K and 90 K. Fast geminate recombinations compete with oxygen escape into the solvent, from which a subsequent slower bimolecular rebinding takes place. In phosphate buffer @H 7.7) at 278 K, the fast and slow processes are exponential and have comparable amplitudes. Whereas the oxygen escape rate rapidly decreases upon increasing the viscosity, the inward rate from the solvent is found to be independent of viscosity, up to about 50 CP (50 mPa . s). The data suggest that a Brownian-motion-driven displacement of one or several side-chain residues is implied in oxygen escape from within the protein and also that hemerythrin undergoes a conformational change in the deoxy state.At higher viscosities and lower temperature only the geminate phase is observed and the kinetics progressively depart from an exponential. Below about 130 K, the kinetics resemble those reported in the literature for heme proteins. They are consistent with a temperature-independent non-equilibrium frozen distribution of conformational substates. However, between 190 K and 130 K, the profile of the kinetics is invariant on a log/ log plot and the results simply differ by a translation along the log t axis. It is shown that this property is expected only for a temperature-dependent distribution of substates in a Boltzmann equilibrium. From room temperature, where rebinding is exponential, down to the 'freezing' temperature, the geminate recombinations display a variety of kinetic laws. It can be shown, however, that for a broad class of substate distributions, the initial slope of the kinetic plot follows an Arrhenius relationship. The activation energy is equal to that of the exponential rate constant measured at high temperature. This result establishes the conditions under which protein data obtained from low-temperature kinetics can be extrapolated to physiological temperature.A protein at thermodynamic equilibrium is subject to transient fluctuations. More than ten years ago, it had been pointed out that volume fluctuations 'if concentrated in one area would produce cavities or channels in the proteins sufficient to allow entry of solvent or probe molecules' [l] and the importance of viscosity-dependent structural fluctuations in enzyme catalysis had been emphasized [2]. A great deal of our present knowledge on protein conformational fluctuations comes from chemical relaxation experiments on oxygen-carrying heme proteins. It is well known that hemoglobin or myoglobin undergo important spectroscopic changes upon binding a ligand (generally oxygen or carbon monoxide) and that ligand dissociation can be triggered, with a relatively high yield, by a light pulse. The subsequent rebinding events can be followed accurately using fast optical absorption spectroscopy.A unique property of heme proteins is that their prosthetic group, the heme, exhibits the elementary reaction of ligand binding in the isolated state. In hemoglobin or myo...

CO Rebinding Kinetics to Myoglobin- and R-State-Hemoglobin-Doped Silica Gels in the Presence of Glycerol

“…It is worthwhile to point out that a similar trend of the rate constants k CA and k CS as a function of viscosity was observed previously for oxygen binding to hemerythrin following nanosecond laser flash photolysis. 77 In that case, the authors discussed the observed different viscosity dependence of the rate constants for the rebinding from the protein matrix and from the solution as arising from the different pathways followed by the ligand in the two processes. The authors concluded that hemerythrin undergoes a conformational transition upon ligand binding, which closes an exit channel necessary for ligand escape.…”

Competition with Xenon Elicits Ligand Migration and Escape Pathways in Myoglobin

“…Although the gas pressure decreased approximately threefold upon cooling, reequilibration of the dissolved gases due to pressure change or to change of solubility with temperature is not expected to occur over the duration of the experiments, owing to the very high viscosity of the cold glycerol/water solvent. The solubility of xenon at room temperature was taken as 4.4 mM/atm in water (Rubin et al, 2002), and 1.1 mM/atm in 79% glycerol, assuming a reduction factor of the solubility due to the presence of 79% glycerol similar to that measured for O 2 (Lavalette and Tetreau, 1988).…”

“…It is known that CO is able to detectably escape or enter the protein down to 200 K in 79% glycerol/ water (w/w). Because these processes are essentially regulated by the viscosity damping of fluctuations at the protein surface (Beece et al, 1980;Lavalette and Tetreau, 1988) we may assume that this temperature limit remains approximately the same for xenon and CO. Extrapolation of the Arrhenius plot of the equilibrium constant (Fig.…”

“…Protein motions, especially at the surface, are damped by friction in viscous solvents. The (isothermal) viscosity dependence of ligand escape has been reported long ago not only for Mb (Beece et al, 1980) but also for the non-heme oxygen carrier hemerythrin (Lavalette and Tetreau, 1988;Yedgar et al, 1995). A full description of the individual kinetic rates in myoglobin would require a series of measurements in isoviscous solvents at variable temperature and another series at variable viscosity but constant temperature.…”

Viscosity Dependence of Some Protein and Enzyme Reaction Rates: Seventy-Five Years after Kramers

“…Some other earlier investigations also found reduced overdamping of the isomerization rate of trans -stilbene versus alkane shear viscosity. , With regard to protein and enzyme reactions, numerous studies sought to examine the extent to which the Kramers basic result, set as a rate–friction inverse relationship ( k ∝ 1/η, where the hydrodynamic proportionality of solvent viscosity η and friction coefficient γ is used), can be observed. These studies have generally concluded that the rate–viscosity relationship holds as k ∝ η –n , where the exponent n lies in the 0–1 bound for a variety of reactions, including enzyme kinetics, − protein folding, − end-to-end intrachain looping in small peptides, inter- and intramolecular electron transfer processes, , the photodissociation rate in oxygen complexes of myoglobin and hemerythrin, , ligand binding to myoglobin, and amide hydrogen exchange in some proteins. − A recent single-molecule study also finds a fractional viscosity exponent . Taking all literature data at face values under experimental conditions used, one might suppose that macromolecular reactions are as overdamped as the Kramers equation would, and underdamping in some cases could be due to a host of factors, including characteristic equilibrium thermal fluctuations, low-amplitude motions required for certain protein functions, , colored Gaussian noise, insufficient coupling of the reaction site with the bulk solvent, , and anomalous internal friction.…”