Tryptophan Fluorescence Yields and Lifetimes as a Probe of Conformational Changes in Human Glucokinase.

Five variants of glucokinase (ATP-D-hexose-6-phosphotransferase, EC 2.7.1.1) including wild type and single Trp mutants with the Trp residue at positions 65, 99, 167 and 257 were prepared. The fluorescence of Trp in all locations studied showed intensity changes when glucose bound, indicating that conformational change occurs globally over the entire protein. While the fluorescence quantum yield changes upon glucose binding, the enzyme's absorption spectra, emission spectra and fluorescence lifetimes change very little. These results are consistent with the existence of a dark complex for excited state Trp. Addition of glycerol, L-glucose, sucrose, or trehalose increases the binding affinity of glucose to the enzyme and increases fluorescence intensity. The effect of these osmolytes is thought to shift the protein conformation to a condensed, high affinity form. Based upon these results, we consider the nature of quenching of the Trp excited state. Amide groups are known to quench indole fluorescence and amides of the polypeptide chain make interact with excited state Trp in the relatively unstructured, glucose-free enzyme. Also, removal of water around the aromatic ring by addition of glucose substrate or osmolyte may reduce the quenching.

Mutational analysis of allosteric activation and inhibition of glucokinase.

GK (glucokinase) is activated by glucose binding to its substrate site, is inhibited by GKRP (GK regulatory protein) and stimulated by GKAs (GK activator drugs). To explore further the mechanisms of these processes we studied pure recombinant human GK (normal enzyme and a selection of 31 mutants) using steady-state kinetics of the enzyme and TF (tryptophan fluorescence). TF studies of the normal binary GK-glucose complex corroborate recent crystallography studies showing that it exists in a closed conformation greatly different from the open conformation of the ligand-free structure, but indistinguishable from the ternary GK-glucose-GKA complex. GKAs did activate and GKRP did inhibit normal GK, whereas its TF was doubled by glucose saturation. However, the enzyme kinetics, GKRP inhibition, TF enhancement by glucose and responsiveness to GKA of the selected mutants varied greatly. Two predominant response patterns were identified accounting for nearly all mutants: (i) GK mutants with a normal or close to normal response to GKA, normally low basal TF (indicating an open conformation), some variability of kinetic parameters (k(cat), glucose S(0.5), h and ATP K(m)), but usually strong GKRP inhibition (13/31); and (ii) GK mutants that are refractory to GKAs, exhibit relatively high basal TF (indicating structural compaction and partial closure), usually show strongly enhanced catalytic activity primarily due to lowering of the glucose S(0.5), but with reduced or no GKRP inhibition in most cases (14/31). These results and those of previous studies are best explained by envisioning a common allosteric regulator region with spatially non-overlapping GKRP- and GKA-binding sites.

Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium-HCl, urea, and pH.

The stability of bovine lactoperoxidase to denaturation by guanidinium-HCl, urea, or high temperature was examined by differential scanning calorimetry (DSC) and tryptophan fluorescence. The calorimetric scans were observed to be dependent on the heating scan rate, indicating that lactoperoxidase stability at temperatures near Tm is controlled by kinetics. The values for the thermal transition, Tm, at slow heating scan rate were 66.8, 61.1, and 47.2 degrees C in the presence of 0.5, 1, and 2 M guanidinium-HCl, respectively. The extrapolated value for Tm in the absence of guanidinium-HCl is 73.7 degrees C, compared with 70.2 degrees C obtained by experiment; a lower experimental value without a denaturant is consistent with distortion of the thermal profile due to aggregation or other irreversible phenomenon. Values for the heat capacity, Cp, at Tm and Ea for the thermal transition decrease under conditions where Tm is lowered. At a given concentration, urea is less effective than guanidinium-HCl in reducing Tm, but urea reduces Cp relatively more. Both fluorescence and DSC indicate that thermally denatured protein is not random coil. A change in fluorescence around 35 degrees C, which was previously reported for EPR and CD measurements (Boscolo et al. Biochim. Biophys. Acta 1774 (2007) 1164-1172), is not seen by calorimetry, suggesting that a local and not a global change in protein conformation produces this fluorescence change.

Water in the half shell: structure of water, focusing on angular structure and solvation.

Water is a highly polar molecule, consisting of a very electronegative atom, oxygen, bonded to two weakly electropositive hydrogen atoms with two lone pairs of electrons. These features give water remarkable physical properties, some of which are anomalous, such as its lower density in the solid phase compared with the liquid phase. Its ability to serve as both a hydrogen bond donor and hydrogen bond acceptor governs its role as a solvent, a role that is of central interest for biological chemists. In this Account, we focus on water's properties as a solvent. Water dissolves a vast range of solutes with solubilities that range over 10 orders of magnitude. Differences in solubility define the fundamental dichotomy between polar, or hydrophilic, solutes and apolar, or hydrophobic, solutes. This important distinction plays a large part in the structure, stability, and function of biological macromolecules. The strength of hydrogen bonding depends on the H-O...O H-bond angle, and the angular distribution is bimodal. Changes in the width and frequency of infrared spectral lines and in the heat capacity of the solution provide a measure of the changes in the strength and distribution of angles of the hydrogen bonds. Polar solutes and inorganic ions increase the population of bent hydrogen bonds at the expense of the more linear population, while apolar solutes or groups have the opposite effect. We examine how protein denaturants might alter the solvation behavior of water. Urea has very little effect on water's hydrogen bond network, while guanidinium ions promote more linear hydrogen bonds. These results point to fundamental differences in the protein denaturation mechanisms of these molecules. We also suggest a mechanism of action for antifreeze (or thermal hysteresis) proteins: ordering of water around the surface of these proteins prior to freezing appears to interfere with ice formation.

Research and development of glucokinase activators for diabetes therapy: theoretical and practical aspects.

Glucokinase Glucokinase (GK GK ; EC 2.7.1.1.) phosphorylates and regulates glucose metabolism in insulin-producing pancreatic beta-cells, hepatocytes, and certain cells of the endocrine and nervous systems allowing it to play a central role in glucose homeostasis glucose homeostasis . Most importantly, it serves as glucose sensor glucose sensor in pancreatic beta-cells mediating glucose-stimulated insulin biosynthesis and release and it governs the capacity of the liver to convert glucose to glycogen. Activating and inactivating mutations of the glucokinase gene cause autosomal dominant hyperinsulinemic hypoglycemia and hypoinsulinemic hyperglycemia in humans, respectively, illustrating the preeminent role of glucokinase in the regulation of blood glucose and also identifying the enzyme as a potential target for developing antidiabetic drugs antidiabetic drugs . Small molecules called glucokinase activators (GKAs) glucokinase activators (GKAs) which bind to an allosteric activator allosteric activator site of the enzyme have indeed been discovered and hold great promise as new antidiabetic agents. GKAs increase the enzyme's affinity for glucose and also its maximal catalytic rate. Consequently, they stimulate insulin biosynthesis and secretion, enhance hepatic glucose uptake, and augment glucose metabolism and related processes in other glucokinase-expressing cells. Manifestations of these effects, most prominently a lowering of blood glucose, are observed in normal laboratory animals and man but also in animal models of diabetes and patients with type 2 diabetes mellitus (T2DM T2DM ) type 2 diabetes mellitus (T2DM) . These compelling concepts and results sustain a strong R&D effort by many pharmaceutical companies to generate GKAs with characteristics allowing for a novel drug treatment of T2DM.

Water structure in vitro and within Saccharomyces cerevisiae yeast cells under conditions of heat shock.

The OH stretch mode from water and organic hydroxyl groups have strong infrared absorption, the position of the band going to lower frequency with increased H-bonding. This band was used to study water in trehalose and glycerol solutions and in genetically modified yeast cells containing varying amounts of trehalose. Concentration-dependent changes in water structure induced by trehalose and glycerol in solution were detected, consistent with an increase of lower-energy H-bonds and interactions at the expense of higher-energy interactions. This result suggests that these molecules disrupt the water H-bond network in such a way as to strengthen molecule-water interactions while perturbing water-water interactions. The molecule-induced changes in the water H-bond network seen in solution do not translate to observable differences in yeast cells that are trehalose-deficient and trehalose-rich. Although comparison of yeast with low and high trehalose showed no observable effect on intracellular water structure, the structure of water in cells is different from that in bulk water. Cellular water exhibits a larger preference for lower-energy H-bonds or interactions over higher-energy interactions relative to that shown in bulk water. This effect is likely the result of the high concentration of biological molecules present in the cell. The ability of water to interact directly with polar groups on biological molecules may cause the preference seen for lower-energy interactions.

Sugar binding to recombinant wild-type and mutant glucokinase monitored by kinetic measurement and tryptophan fluorescence.

Tryptophan fluorescence was used to study GK (glucokinase), an enzyme that plays a prominent role in glucose homoeostasis which, when inactivated or activated by mutations, causes diabetes mellitus or hypoglycaemia in humans. GK has three tryptophan residues, and binding of D-glucose increases their fluorescence. To assess the contribution of individual tryptophan residues to this effect, we generated GST-GK [GK conjugated to GST (glutathione transferase)] and also pure GK with one, two or three of the tryptophan residues of GK replaced with other amino acids (i.e. W99C, W99R, W167A, W167F, W257F, W99R/W167F, W99R/W257F, W167F/W257F and W99R/W167F/W257F). Enzyme kinetics, binding constants for glucose and several other sugars and fluorescence quantum yields (varphi) were determined and compared with those of wild-type GK retaining its three tryptophan residues. Replacement of all three tryptophan residues resulted in an enzyme that retained all characteristic features of GK, thereby demonstrating the unique usefulness of tryptophan fluorescence as an indicator of GK conformation. Curves of glucose binding to wild-type and mutant GK or GST-GK were hyperbolic, whereas catalysis of wild-type and most mutants exhibited co-operativity with D-glucose. Binding studies showed the following order of affinities for the enzyme variants: N-acetyl-D-glucosamine>D-glucose>D-mannose>D-mannoheptulose>2-deoxy-D-glucose>>L-glucose. GK activators increased sugar binding of most enzymes, but not of the mutants Y214A/V452A and C252Y. Contributions to the fluorescence increase from Trp(99) and Trp(167) were large compared with that from Trp(257) and are probably based on distinct mechanisms. The average quantum efficiency of tryptophan fluorescence in the basal and glucose-bound state was modified by activating (Y214A/V452A) or inactivating (C213R and C252Y) mutations and was interpreted as a manifestation of distinct conformational states.

Mirror image forms of snow flea antifreeze protein prepared by total chemical synthesis have identical antifreeze activities.

The recently discovered glycine-rich snow flea antifreeze protein (sfAFP) has no sequence homology with any known proteins. No experimental structure has been reported for this interesting protein molecule. Here we report the total chemical synthesis of the mirror image forms of sfAFP (i.e., L-sfAFP, the native protein, and D-sfAFP, the native protein's enantiomer). The predicted 81 amino acid residue polypeptide chain of sfAFP contains Cys residues at positions 1, 13, 28, and 43 and was prepared from four synthetic peptide segments by sequential native chemical ligation. After purification, the full-length synthetic polypeptide was folded at 4 degrees C to form the sfAFP protein containing two disulfides. Chemically synthesized sfAFP had the expected antifreeze activity in an ice recrystallization inhibition assay. Mirror image D-sfAFP protein was prepared by the same synthetic strategy, using peptide segments made from d-amino acids, and had an identical but opposite-sign CD spectrum. As expected, D-sfAFP displays the same antifreeze properties as L-sfAFP, because ice presents an achiral surface for sfAFP binding. Facile synthetic access to sfAFP will enable determination of its molecular structure and systematic elucidation of the molecular basis of the antifreeze properties of this unique protein.

X-ray structure of snow flea antifreeze protein determined by racemic crystallization of synthetic protein enantiomers.

Chemical protein synthesis and racemic protein crystallization were used to determine the X-ray structure of the snow flea antifreeze protein (sfAFP). Crystal formation from a racemic solution containing equal amounts of the chemically synthesized proteins d-sfAFP and l-sfAFP occurred much more readily than for l-sfAFP alone. More facile crystal formation also occurred from a quasi-racemic mixture of d-sfAFP and l-Se-sfAFP, a chemical protein analogue that contains an additional -SeCH2- moiety at one residue and thus differs slightly from the true enantiomer. Multiple wavelength anomalous dispersion (MAD) phasing from quasi-racemate crystals was then used to determine the X-ray structure of the sfAFP protein molecule. The resulting model was used to solve by molecular replacement the X-ray structure of l-sfAFP to a resolution of 0.98 A. The l-sfAFP molecule is made up of six antiparallel left-handed PPII helixes, stacked in two sets of three, to form a compact brick-like structure with one hydrophilic face and one hydrophobic face. This is a novel experimental protein structure and closely resembles a structural model proposed for sfAFP. These results illustrate the utility of total chemical synthesis combined with racemic crystallization and X-ray crystallography for determining the unknown structure of a protein.

Coupling of complex aromatic ring vibrations to solvent through hydrogen bonds: effect of varied on-ring and off-ring hydrogen-bonding substitutions.

In this study, we examine the coupling of a complex ring vibration to solvent through hydrogen-bonding interactions. We compare phenylalanine, tyrosine, l-dopa, dopamine, norepinephrine, epinephrine, and hydroxyl-dl-dopa, a group of physiologically important small molecules that vary by single differences in H-bonding substitution. By examination of the temperature dependence of infrared absorptions of these molecules, we show that complex, many-atom vibrations can be coupled to solvent through hydrogen bonds and that the extent of that coupling is dependent on the degree of both on- and off-ring H-bonding substitution. The coupling is seen as a temperature-dependent frequency shift in infrared spectra, but the determination of the physical origin of that shift is based on additional data from temperature-dependent optical experiments and ab initio calculations. The optical experiments show that these small molecules are most sensitive to their immediate H-bonding environment rather than to bulk solvent properties. Ab initio calculations demonstrate H-bond-mediated vibrational coupling for the system of interest and also show that the overall small molecule solvent dependence is determined by a complex interplay of specific interactions and bulk solvation characteristics. Our findings indicate that a full understanding of biomolecule vibrational properties must include consideration of explicit hydrogen-bonding interactions with the surrounding microenvironment.

Changes in water structure induced by the guanidinium cation and implications for protein denaturation.

The effect of the guanidinium cation on the hydrogen bonding strength of water was analyzed using temperature-excursion Fourier transform infrared spectra of the OH stretching vibration in 5% H 2O/95% D 2O solutions containing a range of different guanidine-HCl and guanidine-HBr concentrations. Our findings indicate that the guanidinium cation causes the water H-bonds in solution to become more linear than those found in bulk water, and that it also inhibits the response of the H-bond network to increased temperature. Quantum chemical calculations also reveal that guanidinium affects both the charge distribution on water molecules directly H-bonded to it as well as the OH stretch frequency of H-bonds in which that water molecule is the donor. The implications of our findings to hydrophobic solvation and protein denaturation are discussed.

Effects of Salts of the Hofmeister Series on the Hydrogen Bond Network of Water.

The effect of salts on water behavior has been a topic of interest for many years; however, some recent reports have suggested that ions do not influence the hydrogen bonding behavior of water. Using an effective two-state hydrogen bonding model to interpret the temperature excursion infrared response of the O-H stretch of aqueous salt solutions, we show a strong correlation between salt effects on water hydrogen bonding and the Hofmeister order. These data clearly show that salts do have a measurable impact on the equilibrium hydrogen bonding behavior of water and support models which explain Hofmeister effects on the basis of solute charge density.

Temperature excursion infrared (TEIR) spectroscopy used to study hydrogen bonding between water and biomolecules.

Water is a highly polar molecule that is capable of making four H-bonding linkages. Stability and specificity of folding of water-soluble protein macromolecules are determined by the interplay between water and functional groups of the protein. Yet, under some conditions, water can be replaced with sugar or other polar protic molecules with retention of protein structure. Infrared (IR) spectroscopy allows one to probe groups on the protein that interact with solvent, whether the solvent is water, sugar or glycerol. The basis of the measurement is that IR spectral lines of functional groups involved in H-bonding show characteristic spectral shifts with temperature excursion, reflecting the dipolar nature of the group and its ability to H-bond. For groups involved in H-bonding to water, the stretching mode absorption bands shift to lower frequency, whereas bending mode absorption bands shift to higher frequency as temperature decreases. The results indicate increasing H-bonding and decreasing entropy occurring as a function of temperature, even at cryogenic temperatures. The frequencies of the amide group modes are temperature dependent, showing that as temperature decreases, the amide group H-bonds to water strengthen. These results are relevant to protein stability as a function of temperature. The influence of solvent relaxation is demonstrated for tryptophan fluorescence over the same temperature range where the solvent was examined by infrared spectroscopy.

Hydrogen bonding and the cryoprotective properties of glycerol/water mixtures.

Molecular dynamics simulations and infrared spectroscopy were used to determine the hydrogen bond patterns of glycerol and its mixtures with water. The ability of glycerol/water mixtures to inhibit ice crystallization is linked to the concentration of glycerol and the hydrogen bonding patterns formed by these solutions. At low glycerol concentrations, sufficient amounts of bulk-like water exist, and at low temperature, these solutions demonstrate crystallization. As the glycerol concentration is increased, the bulk-like water pool is eventually depleted. Water in the first hydration shell becomes concentrated around the polar groups of glycerol, and the alkyl groups of glycerol self-associate. Glycerol-glycerol hydrogen bonds become the dominant interaction in the first hydration shell, and the percolation nature of the water network is disturbed. At glycerol concentrations beyond this point, glycerol/water mixtures remain glassy at low temperatures and the glycerol-water hydrogen bond favors a more linear arrangement. High glycerol concentration mixtures mimic the strong hydrogen bonding pattern seen in ice, yet crystallization does not occur. Hydrogen bond patterns are discussed in terms of hydrogen bond angle distributions and average hydrogen bond number. Shift in infrared frequency of related stretch and bend modes is also reviewed.

Temperature dependence of hydrogen bonding and freezing behavior of water in reverse micelles.

The mid-infrared spectra of H2O and D2O confined in Aerosol OT (AOT) reverse micelles at various water/surfactant molar ratios (wo) were measured. Previous descriptions of reverse micellar (RM) water have identified three different hydrogen bonding populations in the water pool. (Onori, G.; Santucci, A. J. Phys. Chem. 1993, 97, 5430-5434.) Fitting of the O-H and O-D stretching vibrational modes to Gaussian components corresponding to these three H-bonding populations was used to determine the temperature dependence of the hydrogen bonding populations and to observe the freezing behavior of the encapsulated water pool. The H-bond network behavior of the RM water pool exhibits a strong dependence on wo and does not approximate that of bulk water until wo = 40. The freezing temperature of RM water was wo-independent. The infrared spectra of frozen RM samples has also led us to suggest a mechanism for the low-temperature phase transition behavior of AOT reverse micelles, a subject of interest for cryoenzymology and low-temperature structural biology.

Carbohydrate intramolecular hydrogen bonding cooperativity and its effect on water structure.

Molecular dynamics (MD) simulations combined with water-water H-bond angle analysis and calculation of solvent accessible surface area and approximate free energy of solvation were used to determine the influence of hydroxyl orientation on solute hydration and surrounding water structure for a group of chemically identical solutes-the aldohexopyranose sugars. Intramolecular hydrogen bond cooperativity was closely associated with changes in water structure surrounding the aldohexopyranose stereoisomers. The OH-4 group played a pivotal role in hydration as it was able to participate in a number of hydrogen bond networks utilizing the OH-6 group. Networks that terminated within the molecule (OH-4 --> OH-6 --> O-5) had relatively more nonpolar-like hydration than those that ended in a free hydroxyl group (OH-6 --> OH-4 --> OH-3). The OH-2 group modulated the strength of OH-4 networks through syndiaxial OH-2/4 intramolecular hydrogen bonding, which stabilized and induced directionality in the network. Other syndiaxial interactions, such as the one between OH-1 and OH-3, only indirectly affected water structure. Water structure surrounding hydrogen bond networks is discussed in terms of water-water hydrogen bond populations. The impact of syndiaxial versus vicinal hydrogen bonds is also reviewed. The results suggest that biological events such as protein-carbohydrate recognition and cryoprotection by carbohydrates may be driven by intramolecular hydrogen bond cooperativity.

Tryptophan interactions with glycerol/water and trehalose/sucrose cryosolvents: infrared and fluorescence spectroscopy and ab initio calculations.

In order to correlate how the solvent affects emission properties of tryptophan, the fluorescence and phosphorescence emission spectra of tryptophan and indole model compounds were compared for solid sugar glass (trehalose/sucrose) matrix and glycerol/water solution and under the same conditions, these matrices were examined by infrared spectroscopy. Temperature was varied from 290 to 12 K. In sugar glass, the fluorescence and phosphorescence emission spectra are constant over this temperature range and the fluorescence remains red shifted; these results are consistent with the static interaction of OH groups with tryptophan in the sugar glass. In sugar glass containing water, the water retains mobility over the entire temperature range as indicated by the HOH infrared bending frequency. The fluorescence of tryptophan in glycerol/water shifts to the blue as temperature decreases and the frequency change of the absorption of the HOH bend mode is larger than in the sugar glass. These results suggest rearrangement of glycerol and water molecules over the entire temperature change. Shifts in the fluorescence emission maximum of indole and tryptophan were relatively larger than shifts for the phosphorescence emission-as expected for the relatively smaller excited triplet state dipole for tryptophan. The fluorescence emission of tryptophan in glycerol/water at low temperature has maxima at 312, 313, and 316 nm at pH 1.4, 7.0, and 10.6, respectively. The spectral shifts are interpreted to be an indication of a charge, or Stark phenomena, effect on the excited state molecule, as supported by ab initio calculations. To check whether the amino acid remains charged over the temperature range, the infrared spectrum of alanine was monitored over the entire range of temperature. The ratio of infrared absorption characteristic of carboxylate/carbonyl was constant in glycerol/water and sugar glass, which indicates that the charge was retained. Tryptophan buried in proteins, namely calcium parvalbumin from cod and aldolase from rabbit, showed temperature profiles of the fluorescence spectra that were largely independent of the solvent (glycerol/water or sugar glass) and temperature whereas the fluorescence and phosphorescence yields were dependent. The results demonstrate how the rich information found in tryptophan luminescence can provide information on the dipolar nature and dynamics of the matrix.

The hydration of amides in helices; a comprehensive picture from molecular dynamics, IR, and NMR.

We examined the hydration of amides of alpha(3)D, a simple, designed three-helix bundle protein. Molecular dynamics calculations show that the amide carbonyls on the surface of the protein tilt away from the helical axis to interact with solvent water, resulting in a lengthening of the hydrogen bonds on this face of the helix. Water molecules are bonded to these carbonyl groups with partial occupancy ( approximately 50%-70%), and their interaction geometries show a large variation in their hydrogen bond lengths and angles on the nsec time scale. This heterogeneity is reflected in the carbonyl stretching vibration (amide I' band) of a group of surface Ala residues. The surface-exposed amides are broad, and shift to lower frequency (reflecting strengthening of the hydrogen bonds) as the temperature is decreased. By contrast, the amide I' bands of the buried (13)C-labeled Leu residues are significantly sharper and their frequencies are consistent with the formation of strong hydrogen bonds, independent of temperature. The rates of hydrogen-deuterium exchange and the proton NMR chemical shifts of the helical amide groups also depend on environment. The partial occupancy of the hydration sites on the surface of helices suggests that the interaction is relatively weak, on the order of thermal energy at room temperature. One unexpected feature that emerged from the dynamics calculations was that a Thr side chain subtly disrupted the helical geometry 4-7 residues N-terminal in sequence, which was reflected in the proton chemical shifts and the rates of amide proton exchange for several amides that engage in a mixed 3(10)/alpha/pi-helical conformation.

Melittin as model system for probing interactions between proteins and cyclodextrins.

Cylcodextrin sugars are cyclic sugars that have a hydrophilic exterior and a hydrophobic center. This enables cyclodextrins to solubilize hydrophobic molecules in aqueous media. Cyclodextrins may inhibit aggregation by intercalating surface aromatic residues and competing with interprotein aromatic clusters (pi-pi interactions). In order to investigate this concept, the interaction of hydroxypropyl-beta-cyclodextrin (HPBCD) with melittin is studied with steady-state and time-resolved fluorescence, fluorescence polarization, circular dichroism, and IR spectroscopy. HPBCD inhibits the aggregation of melittin. This inhibition and the spectroscopic results are consistent with the lone aromatic tryptophan of the peptide being intercalated within HPBCD.

Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase.

Oxygen and other molecules of similar size take part in a variety of protein reactions. Therefore, it is critical to understand how these small molecules penetrate the protein matrix. The protein system studied in this case is horseradish peroxidase (HRP). We have converted the native HRP into a phosphorescent analog by replacing the heme prosthetic group by Pd-mesoporphyrin. Oxygen readily quenches the phosphorescence of Pd porphyrins, and this can be used to characterize oxygen diffusion through the protein matrix. Our measurements indicate that solvent viscosity and pH modulate the accessibility of the heme pocket relative to small molecules. The binding of the substrate benzohydroxamic acid (BHA) to the protein drastically impedes oxygen access to the heme pocket. These results indicate that, first, the penetration of small molecules through the protein matrix is a function of protein dynamics, and second, there are specific pathways for the diffusion of these molecules. The effect of substrate and pH on protein dynamics has been investigated with the use of molecular dynamics calculations. We demonstrate that the model of a "fluctuating entry point," as suggested by Zwanzig (J Chem Phys 1992;97:3587-3589), properly describes the diffusion of oxygen through the protein matrix.