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The mechanism underlying allostery in hemoglobin (Hb) is still not completely understood. Various models describing the action of allosteric effectors on Hb function have been published in the literature. It has also been reported that some allosteric effectors—such as chloride ions, inositol hexaphosphate, 2,3-diphospho-glycerate and bezafibrate—considerably lower the oxygen affinity of Hb. In this context, an important question is the extent to which these changes influence the conformational dynamics of the protein. Earlier, we elaborated a challenging method based on phosphorescence quenching, which makes characterizing protein-internal dynamics possible in the ms time range. The experimental technique involves phosphorescence lifetime measurements in thermal equilibrium at varied temperatures from 10 K up to 273 K, based on the signal of Zn-protoporphyrin substituted for the heme in the β-subunits of Hb. The thermal activation of protein dynamics was observed by the enhancement of phosphorescence quenching attributed to O2 diffusion. It was shown that the thermal activation of protein matrix dynamics was clearly distinguishable from the dynamic activation of the aqueous solvent, and was therefore highly specific for the protein. In the present work, the same method was used to study the changes in the parameters of the dynamic activation of human HbA induced by binding allosteric effectors. We interpreted the phenomenon as phase transition between two states. The fitting of this model to lifetime data yielded the change of energy and entropy in the activation process and the quenching rate in the dynamically activated state. The fitted parameters were particularly sensitive to the presence of allosteric effectors and could be interpreted in line with results from earlier experimental studies. The results suggest that allosteric effectors are tightly coupled to the dynamics of the whole protein, and thus underline the importance of global dynamics in the regulation of Hb function. [ABSTRACT FROM AUTHOR]

The studies related to the lysophosphatidic acid (LPA), an in vivo occurring lysophospholipid mediator which promoted amyloid formation under physiological conditions through a complex mechanism is presented. The results obtained and the observed increase in blood LPA levels in dialysis patients suggested that the interaction of LPA with [beta]2m might contribute to the pathomechanism of dialysis-related amyloidosis.

Low-temperature absorption and depolarization ratio dispersion is measured. The strong B(sub 2)(sub g) gives quantum mixed spin state characteristics of class III peroxidases.

Fluorescence site selection spectra of Zn-substituted cytochrome c and Zn-mesoporphyrin were measured as a function of excitation energy and temperature within 5–60 K. The emission energies determined for Zn-cytochrome c were comparable with those of Zn-mesoporphyrin and mesoporphyrin in polar glassy matrices and with resonance Raman spectra of heme proteins. In the site selection spectra of both Zn-cytochrome c and Zn-mesoporphyrin in polar organic glass an increase in the intensity of zero phonon emission lines was observed with an increase of temperature between 5 and 30 K. The irregular spectral changes were interpreted on the basis of the temperature dependence of the linewidth as well as possible hole-burning phenomena. [ABSTRACT FROM AUTHOR]

The general question by what mechanism an “effector” molecule and the hemes of hemoglobin interact over widely separated intramolecular distances to change the oxygen affinity has been extensively investigated, and still has remained of central interest. In the present work we were interested in clarifying the general role of the protein matrix and its dynamics in the regulation of human adult hemoglobin (HbA). We used a spectroscopy approach that yields the compressibility (κ) of the protein matrix around the hemes of the subunits in HbA and studied how the binding of heterotropic allosteric effectors modify this parameter. κ is directly related to the variance of volume fluctuation, therefore it characterizes the molecular dynamics of the protein structure. For the experiments the heme groups either in the α or in the β subunits of HbA were replaced by fluorescent Zn-protoporphyrinIX, and series of fluorescence line narrowed spectra were measured at varied pressures. The evaluation of the spectra yielded the compressibility that showed significant dynamic asymmetry between the subunits: κ of the α subunit was 0.17±0.05/GPa, while for the β subunit it was much higher, 0.36±0.07/GPa. The heterotropic effectors, chloride ions, inositol hexaphosphate and bezafibrate did not cause significant changes in κ of the α subunits, while in the β subunits the effectors lead to a significant reduction down to 0.15±0.04/GPa. We relate our results to structural data, to results of recent functional studies and to those of molecular dynamics simulations, and find good agreements. The observed asymmetry in the flexibility suggests a distinct role of the subunits in the regulation of Hb that results in the observed changes of the oxygen binding capability. [ABSTRACT FROM AUTHOR]

3-Phosphogycerate kinase (PGK) is a two domain enzyme, with a binding site of the 1,3-bisphosphoglycerate on the N-domain and of the ADP on the C-domain. To transfer a phosphate group the enzyme has to undergo a hinge bending motion from open to closed conformation to bring the substrates to close proximity. Molecular dynamics simulation was used to elucidate the effect of ligand binding onto the domain motions of this enzyme. The simulation results of the apo form indicate a hinge bending motion in the ns timescale while the time period of the hinge bending motion of the complex form is clearly over the 20 ns simulation time. The apo form exhibits several hinge points that contribute to the hinge bending motion while upon binding the ligands, the hinge bending becomes strictly restrained with one dominant hinge point in the vicinity of the substrates. At the same time, ligand binding results in an enhanced correlation of internal domain motions. Proteins 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

dUTP pyrophosphatase, a preventive DNA repair enzyme, contributes to maintain the appropriate cellular dUTP/dTTP ratio by catalyzing dUTP hydrolysis. dUTPase is essential for viability in bacteria and eukaryotes alike. Identification of species-specific antagonists of bacterial dUTPases is expected to contribute to the development of novel antimicrobial agents. As a first general step, design of dUTPase inhibitors should be based on modifications of the substrate dUTP phosphate chain, as modifications in either base or sugar moieties strongly impair ligand binding. Based on structural differences between bacterial and human dUTPases, derivatization of dUTP-analogous compounds will be required as a second step to invoke species-specific character. Studies performed with dUTP analogues also offer insights into substrate binding characteristics of this important and structurally peculiar enzyme. In this study, α,β-methylene-dUDP was synthesized and its complex with dUTPase was characterized. Enzymatic phosphorylation of this substrate analogue by pyruvate kinase was not possible in contrast to the successful enzymatic phosphorylation of α,β-imino-dUDP. One explanation for this finding is that the different bond angles and the presence of the methylene group may preclude formation of a catalytically competent complex with the kinase. Crystal structure of E. coli dUTPase:α,β-methylene-dUDP and E. coli dUTPase:dUDP:Mn complexes were determined and analyzed in comparison with previous data. Results show that the 'trans' α-phosphate conformation of α,β-methylene-dUDP differs from the catalytically competent 'gauche' α-phosphate conformation of the imino analogue and the oxo substrate, manifested in the shifted position of the α-phosphorus by more than 3 Å. The three-dimensional structures determined in this work show that the binding of the methylene analogue with the α-phosphorus in the 'gauche' conformation would result in steric clash of the methylene group with the protein atoms. In addition, the metal ion cofactor was not bound in the crystal of the complex with the methylene analogue while it was clearly visible as coordinated to dUDP, arguing that the altered phosphate chain conformation also perturbs metal ion complexation. Isothermal calorimetry titrations indicate that the binding affinity of α,β-methylene-dUDP toward dUTPase is drastically decreased when compared with that of dUDP. In conclusion, the present data suggest that while α,β-methylene-dUDP seems to be practically nonhydrolyzable, it is not a strong binding inhibitor of dUTPase probably due to the altered binding mode of the phosphate chain. Results indicate that in some cases methylene analogues may not faithfully reflect the competent substrate ligand properties, especially if the methylene hydrogens are in steric conflict with the protein. Proteins 2008. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

There are proteins that are built of two structural domains and are deposited full-length in amyloid plaques formed in various diseases. In spite of the known differences in the mechanisms of folding of single- and multidomain proteins, no published studies can be found that address the role of the domain-domain interactions during misfolding and amyloid formation. By the discovery of the role of domain-domain interactions, here we provide important insight in the submolecular mechanism of amyloid formation. A model system based on yeast phosphoglycerate kinase was designed. This system includes the wild-type yeast phosphoglycerate kinase and single-tryptophan mutants of the individual N and C terminal domains and the complete protein. Electron microscopic measurements proved that amyloid fibrils grow from all mutants under identical conditions as for the wild-type protein. Misfolding and amyloid formation was followed in stopped-flow and manual mixing experiments on the 1 ms to 4 days timescale. Tryptophan fluorescence was used for selective detection of conformational changes accompanying the formation of the amyloidogenic intermediates and the growth of amyloid fibrils. The interactions between the polypeptide chains of the two domains direct the misfolding process from the early steps to the amyloid formation, and influence the final structure. The kinetics of misfolding is different for the individual domains, pointing to the significance of the amino acid sequence. Misfolding of the domains within the complete protein is synchronized indicating that domain-domain interactions direct the misfolding and amyloid formation mechanism. Proteins 2006. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: We performed a docking study followed by a 500-ps molecular dynamics simulation of R-state human adult hemoglobin (HbA) complexed to different heterotropic effectors [2,3-diphosphoglycerate (DPG), inositol hexaphosphate (IHP), and 2-[4-[(3,5-dichlorophenylcarbamoyl)-]methyl]-phenoxy]-2-methylpropionic acid (RSR13)) to propose a molecular basis for recently reported interactions of effectors with oxygenated hemoglobin. The simulations were carried out with counterions and explicit solvation. As reported for T-state HbA, the effector binding sites are also located in the central cavity of the R-state and differ depending on effector anionic character. DPG and IHP bind between the α-subunits and the RSR13 site spans the α1-, α2- and β2-subunits. The generated models provide the first report of the molecular details of R-state HbA bound to heterotropic effectors. [Copyright &y& Elsevier]

Abstract: Certain denatured proteins function as cofactors in the activation of plasminogen by tissue-type plasminogen activator. The present study approached the structural requirements for the cofactor activity of a model protein (human serum albumin). Heat denaturation of 100–230μM albumin (80°C and 60–90min) reproducibly yielded aggregates with radius in the range of 10–150nm. The major determinant of the cofactor potency was the size of the aggregates. The increase of particle size correlated with the cofactor activity, and there was a minimal requirement for the size of the cofactor (about 10nm radius). Similar to other proteins, the molecular aggregates with cofactor function contained a significant amount of antiparallel intermolecular β-sheets. Plasmin pre-digestion increased the cofactor efficiency (related to C-terminal lysine exposure) and did not affect profoundly the structure of the aggregates, suggesting a long-lasting and even a self-augmenting cofactor function of the denatured protein. [Copyright &y& Elsevier]

Abstract: Background: The disordered Tubulin Polymerization Promoting Protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system. In this paper the role of dimerization in its microtubule-related functions is established, and an approach is proposed to evaluate thermodynamic constants for multiple equilibrium systems from ITC measurements. Methods: For structural studies size exclusion chromatography, SDS-PAGE, chemical cross-linking, circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry were used; the functional effect was analyzed by tubulin polymerization assay. Numerical simulation of the multiple equilibrium was performed with Mathematica software. Results: The dimerization of TPPP/p25 is promoted by elevation of the protein concentration and by GTP addition. The dimeric form displaying enhanced tubulin polymerization promoting activity is stabilized by disulfide bond or chemical cross-linking. The GTP binding to the dimeric form (Kd-GTP =200μM) is tighter with one order of magnitude than to the monomeric one leading to the enrichment of the dimers. A mathematical model elaborated for the multiple equilibrium of the TPPP/p25-GTP system was validated by fitting the GTP-dependent changes of ellipticity and fluorescence signal in the course of TPPP/p25 titrations. The evaluation of the equilibrium constants rendered it possible to determine the thermodynamic parameters of the association of different TPPP/p25 forms with GTP from ITC measurements. Conclusions/General Significance: The dimerization of TPPP/p25 with favorable physiological functional potency is proposed to play significant role in the fine tuning of TPPP/p25-mediated microtubule assembly; the unfolded monomers might be involved in the formation of pathological inclusions characteristic for Parkinson''s disease and other synucleinopathies. [Copyright &y& Elsevier]

Abstract: l-nucleoside analogues are now largely used as antiviral drugs for the treatment of viral infections like HBV, HCV and HIV. However, in order to be fully active, they need to be phosphorylated by cellular or viral kinases. Human 3-phosphogycerate kinase (hPGK) was shown to catalyze the last step of activation of l-enantiomers and thus constitutes an attractive target for theoretical predictions of its phosphorylation efficiency. Molecular dynamics simulations were carried out with four different nucleotides (d-/l-ADP and d-/l-CDP) in complex with hPGK and 1,3-bisphospho-d-glycerate (bPG). The binding affinities of CDPs (both enantiomers) for hPGK were found very weak while d- and l-ADP were better substrates. Interestingly, the binding affinity of the bPG substrate was found to be lower in presence of d-ADP than l-ADP which indicates a potential antagonistic effect on one substrate to the other. A detailed analysis of the simulations unravels important dynamic conditions for efficient phosphorylation. Indeed, as previously described for the natural substrate, the hinge bending motion of the domains upon substrates binding should be more correlated and directional. Interestingly, the unforeseen finding was the larger dynamics freedoms observed for the substrates that was favored by the protein atoms flexibility around the nucleobase binding site. [Copyright &y& Elsevier]

Abstract: The structure and activity of native horseradish peroxidase C (HRP) is stabilized by two bound Ca2+ ions. Earlier studies suggested a critical role of one of the bound Ca2+ ions but with conflicting conclusions concerning their respective importance. In this work we compare the native and totally Ca2+-depleted forms of the enzyme using pH-, pressure-, viscosity- and temperature-dependent UV absorption, CD, H/D exchange-FTIR spectroscopy and by binding the substrate benzohydroxamic acid (BHA). We report that Ca2+-depletion does not change the alpha helical content of the protein, but strongly modifies the tertiary structure and dynamics to yield a homogeneously loosened molten globule-like structure. We relate observed tertiary changes in the heme pocket to changes in the dipole orientation and coordination of a distal water molecule. Deprotonation of distal His42, linked to Asp43, itself coordinated to the distal Ca2+, perturbs a H-bonding network connecting this Ca2+ to the heme crevice that involves the distal water. The measured effects of Ca2 + depletion can be interpreted as supporting a structural role for the distal Ca2+ and for its enhanced significance in finetuning the protein structure to optimize enzyme activity. [Copyright &y& Elsevier]

Abstract: The activation energy and activation volume of the spectral blue shift subsequent to protochlorophyllide phototransformation (called Shibata shift in intact leaves) were studied in prolamellar body (PLB) and prothylakoid-(PT)-enriched membrane fractions prepared from dark-grown wheat (Triticum aestivum, L.) leaves. The measurements were done at 20, 30 and 40 °C and at various pressure values. The activation energy values were 181±8 kJ mol−1 and 188±6 kJ mol−1 for the PLBs and the PTs, respectively. The pressure stabilized the structure of the NADPH:protochlorophyllide oxidoreductase (POR) macrodomains; it prevented or slowed down the blue shift. There were no significant differences between the activation volumes of PLBs and PTs at 30 or 40 °C giving values around 100–125 ml mol−1 which correspond to changes in the tertiary structure of proteins but also resemble the volume changes occurring during the disaggregation of protein dimers or oligomers, or during dissociation of peripheral membrane proteins from membranes. The small differences in the activation parameters of PLBs and PTs indicate that molecular rearrangements inside the POR macrodomains are the primary reasons of the fluorescence blue shift; however, their lipid microenvironment must be also important in the initialization of the shift. [Copyright &y& Elsevier]

The contribution of heterotropic effectors to hemoglobin allostery is still not completely understood. With the recently proposed global allostery model, this question acquires crucial significance, because it relates tertiary conformational changes to effector binding in both the R- and T-states. In this context, an important question is how far the induced conformational changes propagate from the binding site(s) of the allosteric effectors. We present a study in which we monitored the interdimeric interface when the effectors such as Cl-, 2,3-diphosphoglycerate, inositol hexaphosphate, and bezafibrate were bound. We studied oxy-Hb and a hybrid form (αFeO2)2-(βZn)2 as the T-state analogue by monitoring heme absorption and Trp intrinsic fluorescence under hydrostatic pressure. We observed a pressure-dependent change in the intrinsic fluorescence, which we attribute to a pressure-induced tetramer to dimer transition with characteristic pressures in the 70–200-megapascal range. The transition is sensitive to the binding of allosteric effectors. We fitted the data with a simple model for the tetramer-dimer transition and determined the dissociation constants at atmospheric pressure. In the R-state, we observed a stabilizing effect by the allosteric effectors, although in the T-analogue a stronger destabilizing effect was seen. The order of efficiency was the same in both states, but with the opposite trend as inositol hexaphosphate > 2,3-diphosphoglycerate > Cl-. We detected intrinsic fluorescence from bound bezafibrate that introduced uncertainty in the comparison with other effectors. The results support the global allostery model by showing that conformational changes propagate from the effector binding site to the interdimeric interfaces in both quaternary states. [ABSTRACT FROM AUTHOR]

Abstract: The single Cys residue in the C-terminal domain of bovine eye lens α-crystallin was covalently labelled with 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone. This novel SH-reactive two-band ratiometric fluorescent dye is characterized by excited state intramolecular proton transfer reaction yielding two highly emissive N* and T* bands separated by more than 100nm. Their relative intensities are known to be highly sensitive to the H-bonding ability of the environment. Properties of the environment of the dye attached to the protein were studied under native-like conditions and at a range of elevated temperatures that are known to facilitate α-crystallin chaperone-like activity. We observe that on heating, the environment of the dye becomes more flexible and the H-bonding of the dye with the protein vicinity decreases. The spectroscopic properties observed on heating were partially restored after cooling, but the initial values were not reached on the time scale of our experiments (up to 3h). This suggests that the changes of the dye microenvironment are connected with the rearrangements of α-crystallin quaternary structure. Since there is only one Cys residue in αA subunit of α-crystallin (whereas αB subunit contains no Cys), we attributed the observed temperature-induced changes of the dye''s microenvironment to the particular site within α-crystallin molecule. [Copyright &y& Elsevier]

The 90 kDa heat shock protein, Hsp90, is an abundant molecular chaperone participating in the cytoprotection of eukaryotic cells. Here we analyzed the involvement of Hsp90 in the maintenance of cellular integrity using partial cell lysis as a measure. Inhibition of Hsp90 by geldanamycin, radicicol, cisplatin, and novobiocin induced a significant acceleration of detergent- and hypotonic shock-induced cell lysis. The concentration and time dependence of cell lysis acceleration was in agreement with the Hsp90 inhibition characteristics of the N-terminal inhibitors, geldanamycin and radicicol. Glutathione and other reducing agents partially blocked geldanamycin-induced acceleration of cell lysis but were largely ineffective with other inhibitors. Indeed, geldanamycin treatment led to superoxide production and a change in membrane fluidity. When Hsp90 content was diminished using anti-Hsp90 hammerhead ribozymes, an accelerated cell lysis was also observed. Hsp90 inhibition-induced cell lysis was more pronounced in eukaryotic (yeast, mouse red blood, and human T-lymphoma) cells than in bacteria. Our results indicate that besides the geldanamycin-induced superoxide production, and a consequent increase in cell lysis, inhibition or lack of Hsp90 alone can also comproraise cellular integrity. Moreover, cell lysis after hypoxia and complement attack was also enhanced by any type of Hsp90 inhibition used, which shows that the maintenance of cellular integrity by Hsp90 is important in physiologically relevant lyric conditions of tumor cells. [ABSTRACT FROM AUTHOR]

The photochemical activity of NADPH:protochlorophyllide oxidoreductase (POR) was studied in etiolated wheat (Triticum aestivum, L., cult. MV17) leaf homogenates. The kinetics of the transformation of protochlorophyllide into chlorophyllide was detected by fluorescence intensity changes at 690 nm (formation of chlorophyllide) and 655 nm (decay of protochlorophyllide) at 20 °C, excited at 440 nm while the pressure was varied between 0.1 and 400 MPa. Both kinetics could be fitted by two exponentials and the reaction rates were pressure-dependent. A model was suggested based on the comparison of the two kinetics. Reaction rates of the processes occurring during the prototransformation were determined in function of pressure. The evaluation yielded the activation volume as 1.7 ml mol−1, which corresponds with the formation of one H-bond/molecule. [Copyright &y& Elsevier]

Protein function is governed by the underlying conformational dynamics of the molecule. The experimental and theoretical work leading to contemporary understanding of enzyme dynamics was mostly restricted to the large-scale movements of single-domain proteins. Collective movements resulting from a regulatory interplay between protein domains is often crucial for enzymatic activity. It is not clear, however, how our knowledge could be extended to describe collective near-equilibrium motions of multidomain enzymes. We examined the effect of domain interactions on the low temperature near equilibrium dynamics of the native state, using phosphoglycerate kinase as model protein. We measured thermal activation of tryptophan phosphorescence quenching to explore millisecond-range protein motions. The two protein domains of phosphoglycerate kinase correspond to two dynamic units, but interdomain interactions link the motion of the two domains. The effect of the interdomain interactions on the activation of motions in the individual domains is asymmetric. As the temperature of the frozen protein is increased from the cryogenic, motions of the N domain are activated first. This is a partial activation, however, and the full dynamics of the domain becomes activated only after the activation of the C domain., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Application of porphyrins as photosensitizers is based on their light-triggered generation of reactive oxygen species (ROS) that may cause oxidative tissue damage and ultimately kill cells. Cellular membranes are the action grounds of many sensitizers due to their hydrophobic or amphiphilic character as well as the location of many of the targets attacked by ROS. Hence, the binding ability and location of porphyrins in liposomes as simple models of cellular membranes are of outstanding interest. Here we compare mesoporphyrin IX dimethyl ester (MPE) and its nonesterified form, mesoporphyrin IX dihydrochloride (MPCl). Monocomponent small unilamellar vesicles formed of various saturated phosphatidylcholines with incorporated mesoporphyrins were investigated. We determined the binding parameters and the inhomogeneous distribution functions (IDFs) by different fluorescence techniques. We found in general that the binding ability of MPE is considerably greater than that of MPCl. In the case of MPCl, the IDFs suggest that only one of the two binding site types identified earlier for MPE ("site II") exists; the other one ("site I") vanishes while a new one appears ("site III"). We can confirm that "site I" is located between the two lipid layers, "site II" is situated between the hydrocarbon chains, while the location of the novel "site III" is along the outer part of the hydrocarbon chains partially inserted between the lipid head groups.

Tubulin polymerization promoting protein/p25 (TPPP/p25) modulates the dynamics and stability of the microtubule system and plays crucial role in the myelination of oligodendrocytes. Here we showed by CD, fluorescence, and NMR spectroscopies that Zn(2+) is the first ligand that induces considerable rearrangement of the disordered TPPP/p25. Zinc finger motif (His(2)Cys(2)) (His(61)-Cys(83)) was identified within the flexible region of TPPP/p25 straddled by extended unstructured N- and C-terminal regions. The specific binding of the Zn(2+) to TPPP/p25 induced the formation of molten globule but not that of a well-defined tertiary structure. The Zn(2+)-induced partially folded structure accommodating the zinc binding motif is localized at the single Trp(76)-containing region as demonstrated by fluorescence resonance energy transfer and quenching experiments. We showed that the Zn(2+)-induced change in the TPPP/p25 structure modified its interaction with tubulin and GTP coupled with functional consequences: the TPPP/p25-promoted tubulin polymerization was increased while the TPPP/p25-catalyzed GTPase activity was decreased as detected by turbidimetry and by malachite green phosphate release/(31)P NMR assays, respectively. The finding that the Zn(2+) of the bivalent cations can uniquely influence physiological relavant interactions significantly contributes to our understanding of the role of Zn(2+)-related TPPP/p25 processes in the differentiation/myelination of oligodendrocytes possessing a high-affinity Zn(2+) uptake mechanism.

Conformational dynamics of proteins is of fundamental importance in their physiological functions. The exact mechanisms and determinants of protein motions, including the regulatory interplay between protein and solvent motions, are not yet fully understood. In the present work, the thermal activation of phosphorescence quenching was measured in oxygen-saturated aqueous protein solutions to explore protein dynamics in the millisecond range. The sample was brought to cryogenic temperatures in a fast cooling process to avoid the bulk crystallization of ice. The phosphorescence quenching effect was followed by the phosphorescence lifetime of either Zn-protoporphyrin substituting the heme in the β-subunits of human hemoglobin (Zn-HbA) or tryptophan residues of Zn-HbA and human myoglobin (Mb), measured in thermal equilibrium at temperatures varied from 8 to 273 K. The quenching effect was attributed primarily to the activation of collisions with O(2) molecules made possible by the activated millisecond time-scale dynamics of the matrix around the chromophores. We find that, in the studied temperature range, the activation of protein global dynamics facilitating oxygen diffusion takes place at clearly separated lower temperatures and independently from bulk solvent dynamics and that the energy and entropy differences between the studied frozen and thermally activated states are specific for the protein., (© 2011 American Chemical Society)