A thermodynamic-kinetic analysis of the cytochrome P-450 heme pocket.

Spin state changes in the iron center of cytochrome P-450 during the catalytic cycle suggest alterations in the heme environment that insure proper substrate binding, an increase in redox potential, the formation of an active Fe-O complex, and the attack on the substrate. We used the spin state changes of the iron following physico-chemical perturbations, as an intrinsic probe of discrete changes around the heme, or of larger ones in the protein conformation. These environmental perturbations included temperature, solvent, substrate, and ionic environment. Aqueous and hydro-organic buffers provide complementary data and interpretations; the mixed solvent accommodates temperatures suitable for direct reaction rate measurements and amplified low to high spin transition. The results suggest that the group determining the heme spin state is influenced by the electrostatic potential created by several negative charges near the heme; the modulation of the spin state by various factors reflects the modulation of the electrostatic potential and of the internal paH value. Conformational changes of the whole protein are also indicated by the large entropy terms and their variation with experimental conditions.     

The pH dependence of the hydrolysis of benzoyl-L-arginine ethyl ester in cooled mixed solvents.

Tables of protonic activity (paH) of a number of buffers, determined in mixed solvents and at subzero temperatures, are reported for the following media: water-1,2-propanediol, water-glycerol, and water-dimethylsulfoxide (50:50, in volume). These data with those previously reported allowed us to study enzymic reactions under these conditions. The paH dependence of the tryptic hydrolysis of benzoyl-L-arginine ethyl ester has been studied in the presence of organic solvents (methanol, ethylene glycol, 1,2-propanediol, glycerol, and dimethylsulfoxide, all 50% by volume) between 20 and -20 degrees. The results have allowed us to show the validity of our paH scales in mixed solvents. The paH profiles obtained under these conditions are similar to those observed in pure water at 20 degrees. They are shifted nevertheless by both solvent and temperature. Such shifts are interpreted in terms of the effects of solvents and temperature on pKES on the basis of the conclusions drawn from a study of the effect of these variables on small dissociable molecules. The results obtained under these conditions of solvents and temperature are consistent with the presence at the active site of the enzyme of a histidine residue, and thus provide, concerning the solvent effect, a direct verification of the method of Findlay et al. (Findlay, D., Mathias, A. P., and Rabin, B. R. (1962) Biochem. J. 85, 139-144). On the other hand, the large temperature interval provided by the low temperature procedure, allows us to vary significantly the pK of ionizable groups of the enzymes and thus makes possible their identification, on the basis of their enthalpy of ionization.     

Temperature-dependent conformation change in spin-labeled hemoglobin

Hemoglobin was spin labeled at β-93(F9)-cysteine with N-oxy-2,2,6,6-tetramelhylpiperidinylmaleimide. The inward shift of the high-field hyperfine line (ΔH_XXX) position in the ESR spectra of the Spin label was measured aS a function of temperature. One can expect that an abrupt change in the microenvironment around the tightly bound spin label will be reflected in the function ΔH_XXX(T) as a discontinuity (break point). This was shown for aquo-, azido-. nitro- and oxyhemoglobin derivatives. The presented results suggest that the microenvironment around the tightly hound spin label in those methemoglobin derivatives that exhibit the mixed-spin state of the heme iron is prone to an abrupt change above a certain ligand-specific temperature. The change in microenvironment of the spin label is probably due to a temperature-dependent change in the tertiary structure of the protein.     

Requirement of translocated lysosomal V1 H(+)-ATPase for activation of membrane acid sphingomyelinase and raft clustering in coronary endothelial cells

Acid sphingomyelinase (ASM) mediates the formation of membrane raft (MR) redox signalosomes in a process that depends on a local acid microenvironment in coronary arterial endothelial cells (CAECs). However, it is not known how this local acid microenvironment is formed and maintained. The present study hypothesized that lysosomal V1 H(+)-ATPase provides a hospitable acid microenvironment for activation of ASM when lysosomes traffic and fuse into the cell membrane. Confocal microscopy showed that local pH change significantly affected MRs, with more fluorescent patches under low pH. Correspondingly, the ASM product, ceramide, increased locally in the cell membrane. Electron spin resonance assay showed that local pH increase significantly inhibited NADPH oxidase-mediated production of O(2)(-.) in CAECs. Direct confocal microscopy demonstrated that Fas ligand resulted in localized areas of decreased pH around CAEC membranes. The inhibitors of both lysosomal fusion and H(+)-ATPase apparently attenuated FasL-caused pH decrease. V1 H(+)-ATPase accumulation and activity on cell membranes were substantially suppressed by the inhibitors of lysosomal fusion or H(+)-ATPase. These results provide the first direct evidence that translocated lysosomal V1 H(+)-ATPase critically contributes to the formation of local acid microenvironment to facilitate activation of ASM and consequent MR aggregation, forming MR redox signalosomes and mediating redox signaling in CAECs.     

Autoreactive antibodies are present in sheep with Johne's disease and cross-react with Mycobacterium avium subsp. paratuberculosis antigens

Mycobacterial infection has been linked to the generation of autoantibodies, including anti-neutrophil cytoplasmic autoantibodies (ANCA), in clinical studies and small animal models. In an attempt to identify antibodies that react with both self and mycobacterial antigens in naturally infected ruminants, we generated a phage display library comprising single chain antibody fragments (scFv) from sheep with Johne's disease (JD). The library was screened simultaneously against ovine small intestinal tissue and Mycobacterium avium subsp. paratuberculosis (MAP). A clone (termed AutoH1) reacted strongly with host tissue and MAP, recognizing a proteinase-susceptible 32 kDa determinant in ovine gut tissues and lymphatics, and in blood granulocytes but not mononuclear cells. In granulocytes, binding was to cytoplasmic granules and cell membranes; in MAP, AutoH1 bound bacterial cell wall determinants. We further identified a synthetic peptide sequence recognized by AutoH1, using this to generate a carrier:peptide fusion protein (paH1). Sera of normal and JD sheep were screened for AutoH1-like autoantibody activity; 7/11 JD animals showed autoreactivity that could be blocked by paH1, while 0/21 normal animals showed no such serological reactivity. It is possible that the severe pathology observed in ruminant JD may have an autoimmune component, possibly due to ANCA-type binding; this remains to be further investigated.     

Neisseria gonorrhoeae membrane microenvironment studied by spin-label electron spin resonance: comparison of colony types.

Spin-label electron spin resonance was used to characterize the microenvironment around spin probes which localize (i) in membranes, (ii) at the membrane surface, or (iii) in the cytoplasm of living Neisseria gonorrhoeae. Four colony types (T1, T2, T3, and T4) of gonococci were compared on the basis of the electron spin resonance parameters 2T parallel to, S (order parameter), and tau c (microviscosity). The concentration of spin label used had little or no effect on viability. T1 and T2 gonococci were found to have a more restricted environment for molecular motion of a membrane surface spin label than did T3 and T4. The membrane fluidity, as measured by a membrane lipid spin label, of T4 (S = 0.571) was significantly greater than that of T1 or T3 (S = 0.580). This difference was detected at 37 degrees C, at 25 degrees C, in agar-grown bacteria, and in exponential-phase cells. Studies using spin labels which probe different levels of the membrane indicated the presence of a membrane flexibility gradient. Cytoplasmic spin-label studies indicated that the cytoplasm of all gonococcal colony types was three to five times more viscous than water.     

A comparative study of the lipid phase in native and circulating multiple-modified low-density lipoproteins of human blood by the use of the spin probe method

Different approaches based on the spin probe method were used to compare the physical state of the surface lipid monolayer in subfractions of low-density lipoproteins: in native low-density lipoproteins constituting the bulk of human blood low-density lipoproteins and in circulating multiple-modified low-density lipoproteins whose portion is minor in healthy persons but significantly increases in atherosclerotic patients. The data obtained in in vitro experiments suggest that circulating multiple-modified low-density lipoproteins possess atherogenic properties. The order parameter S, rotational correlation time tau, and hydrophobicity parameter h were calculated from electron spin resonance spectra of a series of spin probes whose paramagnetic groups are located at different depths of the lipid monolayer. These parameters characterize the molecular packing, fluidity, and polarity in the microenvironment of paramagnetic groups. The kinetics of the reduction of paramagnetic groups by ascorbate and oxidation by hypochlorite were obtained for the spin probe whose paramagnetic group is located deeply in the lipid monolayer at the level of the terminal segments of phospholipid acyl chains. No difference between native low-density lipoproteins and circulating multiple-modified low-density lipoproteins was revealed in respect of the physical properties of the lipid domain of surface proteolipid layer, as sampled by spin probes.     

Interactions between phospholipids and barbiturates.

The effects of a number of barbiturates on the temperature of the lipid phase transition have been studied using chlorophyll a as a fluorescence probe. The barbiturates cause a reduction in the temperature of the phase transitions of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidylethanolamine, the effects being greatest at lower pH values where more of the barbiturate is present in the uncharged form. There was no significant interaction between the barbiturates and dipalmitoyl phosphatidylserine. These and other observations on the actions of local anaesthetics are used to develop a model for local anaesthesia. It is suggested that the sodium channel is surrounded by an annulus of lipid in the gel state, this rigid microenvironment preventing the sodium channel relaxing from its active configuration to an inactive one. Local anaesthetics, which reduce the temperature of lipid phase transitions, trigger a change of the annular lipid from the gel to the liquid-crystalline state, with a consequent relaxation of the sodium channel to an inactive configuration, in which the sodium current is reduced or blocked.     

Cytochrome P-450 of bovine adrenal mitochondria. Ligand binding to two forms resolved by EPR spectroscopy.

The binding of cholest-5-ene-3beta,20alpha-diol (20alpha-hydroxycholesterol), 11-deoxycorticosterone, and aminoglutethimide to cytochrome P-450 in bovine adrenal mitochondria was measured by changes in optical spectra at room temperature and by EPR spectra at 14 K. The two methods provided nearly identical quantitation of these interactions with cytochrome P-450. Two distinct high spin forms of cytochrome P-450 were revealed by EPR spectra. The predominant high spin species (g = 8.2) was decreased by addition of 20alpha-hydroxycholesterol and elevated pH but was increased by addition of cholesterol. The minor high spin species (g = 8.1) was incrreased by addition of deoxycorticosterone but decreased by low concentrations of metyrapone. The two forms were evidently not in equilibrium and have been assigned to distinct forms of cytochrome P-450 involved in, respectively, cholesterol side chain cleavage (P-450scc) and steroid 11beta hydroxylation (P-450(11)beta). The high spin states are derived from complexes of these P-450 cytochromes with endogenous substrates, which are, respectively, cholesterol and deoxycorticoids. A high to low spin transition was observed when these complexes were turned over by initiating hydroxylation with malate. The contributions of cytochromes P-450(11)beta and P-450scc to the low spin spectrum were also resolved by similar means. At least 20% of P-450scc is in the low spin state while about 90% of P-450(11)beta is low spin in isolated beef adrenal mitochondria. Low spin complexes of cytochrome P-450scc with 20alpha-hydroxycholesterol and 3beta-hydroxypregn-5-ene-20-one (pregnenolone) gave distinct EPR spectra. Aminoglutethimide interacted with the total cytochrome P-450 content of the bovine adrenal mitochondria forming low spin complexes. Both optical and EPR data indicated binding to two forms of cytochrome P-450. These results suggest a detailed correlation between the spin state and absorbance changes seen at room temperature, illustrate that EPR allows the distinction of two principal forms of P-450, and suggest that there is no appreciable change in the spin state of either cytochrome between 14 K and 300 K.     

Conformation and spin state in methemoglobin.

The properties of human methemoglobin have been investigated under a wide variety of conditions to determine its conformation and to test for evidence of the T state conformation which has been proposed by Perutz to exist in the presence of high spin ligands and inositol hexaphosphate (IHP). Subunit dissociation was measured as a criterion for the T state since marked differences in the tetramer-dimer equilibrium exist for oxyhemoglobin (R state) and deoxyhemoglobin (T state). In the absence of IHP, complexes of methemoglobin with both high spin ligands (water, fluoride) or low spin ligands (azide, cyanide) show extensive dissociation in 2,2-bis(hydroxymethyl)-2,2',2"-nitriloethanol buffers, pH 6, 0.1 M NaCl, with values of the tetramer-dimer dissociation constant (K4,2) near 10-5 M. The addition of IHP lowers K4,2 to a value near 10-5 M for all forms of methemoglobin. Combination of IHP with methemoglobin promotes a conformational change, but the change is apparently independence of spin state. The conformation acquired in the presence of IHP is not identical with the T state (K4,2 similar to 10-12 M) and can also occur with hemoglobin in the ferrous form, as revealed by a substantial reduction in K4,2 for CO-hemoglobin upon addition of IHP. Subunit dissociation has also been measured using the haptoglobin reaction, since haptoglobin binds only to hemoglobin dimers. The haptoglobin experiments give results that are qualitatively in agreement with the conclusions reached by ultracentrifuge measurements. Similar results are also obtained by estimating the degree of dissociation on the basis of the material which aggregates following mixing with dithionite. The effect of IHP on azide-binding kinetics with methemoglobin has also been examined. Changes in reactivity is observed upon addition of IHP, but the principal effect is observed upon addition of IHP, but the principal effect is an enhancement of the rate of reaction of the beta chains. Changes in the reactivity of the beta93 sulfhydryl group of methemoglobin also accompany addition of IHP, but in a manner which is largely independent of the spin state of the iron. Similar changes are again found with CO-hemoglobin upon addition of IHP. The rate of binding of bromthymol blue also shows some changes upon addition of IHP, but the changes are more pronounced for deoxyhemoglobin than for methemoglobin. Since the results obtained did not appear to indicate a significant role for spin state in the changes observed, additional studies were undertaken using EPR spectroscopy.     

Membrane hydrocarbon thickness modulates the dynamics of a membrane transport protein

Nitroxide spin labels were incorporated into selected sites within the β-barrel of the bacterial outer-membrane transport protein BtuB by site-directed mutagenesis, followed by chemical modification with a methanethiosufonate spin label. The electron paramagnetic resonance lineshapes of the spin-labeled side chain (R1) from these sites are highly variable, and have spectral parameters that reflect secondary structure and local steric constraints. In addition, these lineshape parameters correlate with crystallographic structure factors for Cα carbons, suggesting that the motion of the spin label is modulated by both the local modes of motion of the spin label and the local dynamics of the protein backbone. Experiments performed as a function of lipid composition and sample temperature indicate that nitroxide spin labels on the exterior surface of BtuB, which face the membrane hydrocarbon, are not strongly influenced by the phase state of the bulk lipids. However, these spectra are modulated by membrane hydrocarbon thickness. Specifically, the values of the scaled mobility parameter for the R1 lineshapes are inversely proportional to the hydrocarbon thickness. These data suggest that protein dynamics and structure in BtuB are directly coupled to membrane hydrophobic thickness.     

Fate of the hypertrophic chondrocyte: microenvironmental perspectives on apoptosis and survival in the epiphyseal growth plate

The goal of this review is to examine the fate of the hypertrophic chondrocyte in the epiphyseal growth plate and consider the impact of the cartilage microenvironment on cell survival and apoptosis. Early investigations pointed to a direct role of the hypertrophic chondrocyte in osteogenesis. The terminally differentiated cells were considered to undergo a dramatic change in shape, size, and phenotype, and assume the characteristics of an osteoblast. While some studies have supported the notion of transdifferentiation, much of the evidence in favor of reprogramming epiphyseal chondrocytes is circumstantial and based on microscopic evaluation of cells that are present at the chondro-osseous junction. Although these investigations provided a novel perspective on endochondral bone formation, they were flawed by the failure to consider the importance of stem cells in osseous tissue formation. Subsequent studies indicated that many, if not all, of the cells of the cartilage plate die through the induction of apoptosis. With respect to agents that mediate apoptosis, at the chondro-osseous junction, solubilization of mineral and hydrolysis of organic matrix constituents by septoclasts generates high local concentrations of ions, peptides, and glycans, and secreted matrix metalloproteins. Individually, and in combination, a number of these agents serve as potent chondrocyte apoptogens. We present a new concept: hypertrophic cells die through the induction of autophagy. In the cartilage microenvironment, combinations of local factors cause chondrocytes to express an initial survival phenotype and oxidize their own structural macromolecules to generate ATP. While delaying death, autophagy leads to a state in which cells are further sensitized to changes in the local microenvironment. One such change is similar to ischemia reperfusion injury, a condition that leads to tissue damage and cell death. In the growth cartilage, an immediate effect of this type of injury is sensitization to local apoptogens. These two concepts (type II programmed cell death and ischemia reperfusion injury) emphasize the importance of the local microenvironment, in particular pO(2), in directing chondrocyte survival and apoptosis.     

Spin labeling studies of wheat germ calmodulin in solution.

Electron paramagnetic resonance was used to investigate the physical state of plant calmodulin in solution. Wheat germ calmodulin contains a single cysteine residue (Cys-27) on the first of four calcium binding loops. In this study the nitroxide spin label 2,2,6,6-tetramethyl-4-maleimidopiperidine-1-oxyl (MAL-6) was covalently attached to Cys-27 to produce a Ca(2+)-sensitive, biologically-active, labeled protein. The rotational correlation time of the spin label, a measure of its rotational mobility and reflective of the physical state of this region of the protein, was calculated under various conditions. Relative to control, changes in the physical state of the protein reflected by increased motion of the spin label were observed at high pH, low ionic strength and upon addition of Ca2+. These results extend knowledge of the structure of the protein, previously known from solid state and biochemical studies, to calmodulin in solution.     

Transition between iron spin states in myoglobin: Roentgen absorption

The spin transition of Fe in the active center of a myoglobin molecule, stimulated by a temperature variation, was studied by the theoretical multiple scattering analysis of experimental X-ray absorption data. The spin transition was followed by the movement of the Fe ion out of the plane of the heme without substantial changes in the Fe-N distance. It was shown that the X-ray absorption fine structure above the Fe K-edge is sensitive to both local geometry changes near the Fe ion in the active site of the protein and the spin state of the Fe ion itself. The change in the symmetry of Fe coordination lead to modifications of the spectrum shape in the entire interval up to 40 eV above the main edge. It was found that the spin state effects mostly the rising edge, and at energies above 15 eV becomes negligibly small.     

气道上皮细胞的抗氧化损伤保护及微环境调控

本工作建立了臭氧对原代培养的兔支气管上皮细胞（ＢＥＣ）损伤模型,观察到血管活性肠肽（ＶＩＰ）、表皮生长因子（ＥＧＦ）、热应激等微环境理化因子可减轻细胞损伤,具有细胞保护作用,其保护机制与提高还原谷胱甘肽（ＧＳＨ）含量有关,并依赖于蛋白激酶的磷酸化调节及基因转录。ＢＥＣ细胞在基础情况下有ｂｃｌ－２基因的低水平表达。ＶＩＰ和ＥＧＦ可促进ｂｃｌ－２基因转录,增强ＢＥＣ的抗氧化损伤能力。ＥＧＦ或热应激促进     

Influence of inositol hexaphosphate binding on subunit dissociation in methemoglobin.

The tetramer-dimer equilibria of various forms of methemoglobin have been measured by sedimentation equilibrium to test the hypothesis of Perutz that high spin derivatives can be switched by inositol hexaphosphate (Inos-P6) from the R state to the T state more readily than low spin derivatives. Since transitions from the R state to the T state are accompanied by a decrease in the tetramer-dimer dissociation constant (K4,2), this parameter is a quantitative indicator of the conformational state. Measurements of K4,2 were performed using an analytical ultracentrifuge with absorption optics and a scanner-computer system. Statistical analysis of the sedimentation data indicated that the stoichiometry if Inos-P6 binding is 1 molecule/hemoglobin tetramer and 2 molecules/hemoglobin dimer. The apparent affinity of the dimer sites for Inos-P6 is much lower than the corresponding value for the tetramer site. As a result of the stoichiometries, at low concentrations Inos-P6 shifts the tetramer-dimer equilibrium in favor of the tetramer, but at high concentrations Inos-P6 shifts the equilibrium in favor of the dimer. Te tetramer binding site for Inos-P6 of various liganded forms of hemoglobin appears to be the same as has been established for deoxyhemoglobin, since the effect of Inos-P6 on subunit dissociation is reduced in pyridoxylated derivatives. Values of K4,2 for aquo-, azido- and cyanomethemoglobin in 0.01 M 2,2-bis(hydroxymethyl)-2,2',2'-nitroethanol buffer, pH 6.0/0.1 M NaCl, are all near 2 X 10(-5) M. Upon addition of 50 muM Inos-P6 the values of K4,2 for all three forms are shifted to near 10(-9) M. Since the aquo derivative is high spin, while the azido and cyano derivatives are low spin, the similarity of values for the derivatives in the presence and absence of Inos-P6 indicate that the changes in K4,2 are not spin-spin state dependent. For another high spin derivative, fluoromethemoglobin, such high concentrations of NaF are required that ionic strength effects are encountered. When data at several NaF concentrations are extrapolated to 0.1 M NaF to correct for the ionic strength effects, values of K4,2 of 7 X 10(-6) M and 10(-8) M are obtained for solutions in the absence and in the presence of 50 muM Inos-P6, respectively. Therefore the results with the fluoro derivative, in conjunction with the other forms of methemoglobin, support the view that high spin derivatives do not exhibit a greater response to Inos-P6 than low spin derivatives.     

Electronic state of heme in cytochrome oxidase. I. Magnetic circular dichroism of the isolated enzyme and its derivatives.

Magnetic circular dichroism (MCD) spectra have been recorded for beef heart cytochrome oxidase and a number of its inhibitor complexes. The resting enzyme exhibits a derivate shape Faraday C term in the Soret region, characteristic of low spin ferric heme, which accounts for 50% of the total oxidase heme a. The remaining heme a (50%) is assigned to the high spin state. MCD temperature studies, comparison with the MCD spectra of heme a-imidazole model compounds, and ligand binding (cyanide, formate) studies are consistent with these spin state assignments in the oxidized enzyme. Furthermore, the ligand binding properties and correlations between optical and MCD parameters indicate that in the resting enzyme the low spin heme a is due solely to cytochrome a3+ and the high spin heme a to cytochrome a33+. The Soret MCD of the reduced protein is interpreted as th sum of two MCD curves: an intense, asymmetric MCD band very similar to that exhibited by deoxymyoglobin which we assign to paramagnetic high spin cytochrome a3(2+) and a weaker, more symmetric MCD contribution, which is attributed to diamagnetic low spin cytochrome a2+. Temperature studies of the Soret MCD intensity support this proposed spin state heterogeneity. Ligand binding (CO, CN-) to the reduced protein eliminates the intense MCD associated with high spin cytochrome a3(2+); however, the band associated with cytochrome a2+ is observed under these conditions as well as in a number of inhibitor complexes (cyanide, formate, sulfide, azide) of the partially reduced protein. The MCD spectra of oxidized, reduced, and inhibitor-complexed cytochrome oxidase show no evidence for heme-heme interaction via spectral parameters. This conclusion is used in conjunction with the fact that ferric, high spin heme exhibits weak MCD intensity to calculate the MCD spectra for the individual cytochromes of the oxidase as well as the spectra for some inhibitor complexes of cytochrome a3. The results are most simply interpreted using the model we have recently proposed to account for the electronic and magnetic properties of cytochrome (Palmer, G., Babcock, F.T., and Vcikery, L.E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210).     

Spin state transitions of liver microsomal cytochrome P-450.

Spin state transitions of membrane-bound cytochrome P-450 were investigated by difference spectrophotometry using the 'D'-charge transfer absorbance band at 645 nm as a measure of the amount of hemin iron present in the 5-coordinated state. The magnitude of the 'D'-absorbance band in the absence of exogenous substrates, e.g., the concentration of native high spin cytochrome P-450, was evaluated from the difference in absorbance at 645 nm between ferric cytochrome P-450 and the carbon monoxide derivative of the pigment in its ferrous state. The contribution of the native high spin species to the total cytochrome P-450 content of microsomes was calculated to be between 40% and 65% after induction with phenobarbital and polycyclic hydrocarbons, respectively. Up to 80% of the cytochrome P-450 was found to be present in the high spin state after the addition of exogenous substrates. Further, the steady state concentrations of high spin cytochrome P-450, observed in the presence of reduced pyridine nucleotides, suggest that the rate limiting step for microsomal mixed function oxidation reactions is variable and dependent on the substrate under investigation.     

Relationship between vegetation structure and microenvironment in Fagus grandifolia subsp. mexicana forest relicts in Mexico

Aims Changes in the structure and composition of forests, whether caused by natural or anthropic events, alter the microenvironment, sometimes irreversibly. Since the local environment has a direct impact on basic ecological processes, this has become a key component of research. Mexican beech forests (Fagus grandifolia subsp. mexicana) in the Sierra Madre Oriental are restricted to sites with specific climate, soils and topography, making them an ideal natural system for ecological research. The objectives of this study were to identify the relationship between the microenvironment and the tree and shrub structure and composition of Mexican beech forests in the state of Hidalgo, and to compare the floristic similarity of these forests on the country scale using data from seven localities.Methods Specimens were collected for a period of one year at all localities in the state of Hidalgo where beech forests are located. At each locality, five 400 m 2 plots were established, and structural attributes (basal area, coverage, density and species richness) and six environmental variables were measured in the plots. The relationship between structure and microenvironment was estimated by simple correlation and canonic correspondence analysis (CCA). In addition, floristic similarity between different beech forest localities in the Sierra Madre Oriental was estimated by correspondence analysis (CA).Important findings Twenty tree species and eight shrub species were identified; at all localities studied F. grandifolia subsp. mexicana dominated the canopy. The multivariate analysis indicated that (i) in the four localities in the state of Hidalgo, all microenvironmental variables except pH are related to the variation observed in species composition and structure; (ii) the El Gosco locality had both tree and shrub species and microenvironmental factors different from those observed in the Fagus forests at the other localities in the study and (iii) the localities studied in order to draw country-scale comparisons could be divided into three groups by floristic similarity. The first group consisted of the Hidalgo localities, the second of the Veracruz localities, and the third, more different from the others, of the Tamaulipas locality. The results of this study provide the first reference for the relationship between the range of microenvironments and species structure in Mexican beech forests. Microenvironmental conditions in the larger beech forests could be used as a model for designing management and conservation programs for this plant association. Because of its particular ecological and historical characteristics, this association could serve as an example of biodiversity conservation in Mexico.