Kinetics of acid-catalysed dissociation of lead(II) and copper(II) complexes of ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA)

The acid-catalysed dissociation rate constants for PbEGTA²⁻ and CuEGTA²⁻ complexes (where EGTA is ethylenebis(oxyethylenenitrilo) tetraacetic acid) were measured in acetic acid-acetate buffer medium (pH: 3.0–4.8) and perchloric acid solutions ([H⁺] = 0.05–0.15 M), respectively, at a constant ionic strength of 0.15 (NaClO₄). The rate laws shown by the lead(II) and copper(II) complexes are of the form, Rate = {k_d + k_H[H⁺]}[complex] and Rate = {k_d + k_H²[H⁺]²}[complex], respectively. Enthalpy and entropy of activation for acid-independent and acid-catalysed pathways for both the complexes were obtained by the temperature-dependence studies of resolved rate constants in the 16–45°C range. The rate of dissociation of PbEGTA²⁻ is not enhanced by increasing the concentration of acetate ion in the buffer, and the amount of total electrolyte in the reaction mixture has no pronounced effect on the dissociation rates of their the lead(II) or copper(II) complex. Attempts to study the kinetics of stepwise ligand unwrapping in the binuclear Cu₂EGTA complex were unsuccessful due to the extremely rapid dissociation of this complex to yield mononuclear CuEGTA²⁻.     

Kinetics of the membrane-bound inorganic pyrophosphatase from Rhodospirillum rubrum chromatophores: Effect of the transmembrane electrical potential on the rate constants

The behaviour of the membrane-bound proton-translocating pyrophosphatase (H⁺-PPase) in Rhodospirillum rubrum chromatophores upon application of an electrochemical potential is studied. The rate constants are shown to be affected in an asymmetric fashion. The forward rate constant (PP_i synthesis) is shown to be at least 45-times larger during illumination than when there is no proton-motive force. The hydrolysis rate is increased maximally 8-times when the potential is dissipated. The effect of the electrical field gradient is thus mainly to increase the forward rate of the reaction. The H⁺-PPase also seems to be a functionally simpler enzyme than the H⁺-ATPase, lacking the hydrolysis activation step during energization found in the latter.     

(Na+,K+)-ATPase of mammalian brain: Effects of temperature on cation and ATP interactions regulating phosphatase activity

The effects of temperature on interactions between univalent cations or ATP and the p-nitrophenylphosphatase activity associated with brain (Na⁺,K⁺)-ATPase were examined. The apparent affinity for K⁺ activation under conditions favoring the moderate affinity site was temperature dependent, increasing with decreasing temperature. A comparison of univalent cations showed that the negative apparent ΔH and ΔS for cation binding increased with increasing apparent cation affinity. In contrast to the case with the moderate affinity sites, apparent affinity for the high affinity K⁺ site was independent of temperature. As temperature decreased, properties of moderate affinity site binding approached those of the high affinity site. The temperature dependence of ATP inhibition was opposite to that for K⁺ activation, with positive apparent ΔH and ΔS. The apparent ΔH and ΔS for cation binding approached those for the overall conformational change to K⁺-sensitive enzyme as cation affinity increased. These data suggest that E2, the K⁺-sensitive form of (Na⁺,K⁺)-ATPase, is stabilized by forces that require a decrease in entropy, explaining the predominant existence of E1 at physiologic temperatures. A conformational change leading to stabilization of E2 at higher temperatures can be produced by binding of univalent cations to a moderate affinity, presumably intracellular, site. This effect is counteracted by ATP. ATP also appears to alter the selectivity of this site to favor Na⁺ over K⁺ binding.     

Stabilization of glucoso-6-phosphate dehydrogenase by its substrate and cofactor in an ultrasonic field

The inactivation kinetics of glucoso-6-phosphate dehydrogenase (GPDH) and its complexes with glucoso-6-phosphate and NADP⁺ was characterized in aqueous solutions at 36–47°C under treatment with low frequency (27 kHz, 60 W/cm²) and high frequency ultrasound (880 kHz, 1 W/cm²). To this end, we measured three effective first-order inactivation rate constants: thermal k _in ^* , total (thermal and ultrasonic) k _in, and ultrasonic k _in(US). The values of the constants were found to be higher for the free enzyme than for its complexes GPDH-GP and GPDH-NADP⁺ at all temperatures, which confirms the enzyme stabilization by its substrate and cofactor under both thermal and ultrasonic inactivation. Effective values of the activation energies (E _a) were determined and the preexponential factors of the rate constants and thermodynamic activation parameters of inactivation processes (ΔH*, ΔS*, and ΔG*) were calculated from the temperature dependences of the inactivation rate constants of GPDH and its complexes. The sonication of aqueous solutions of free GPDH and its complexes was accompanied by a reduction of E _a and ΔH* values in comparison with the corresponding values for thermal inactivation. The E _a, ΔH*, and ΔS* inactivation values for GPDH are lower than the corresponding values for its complexes. A linear dependence between the growth of the ΔH* and ΔS* values was observed for all the inactivation processes for free GPDH and its complexes.     

Dissociation of pig liver carboxylesterase measured by quantitative micro-complement fixation

Rate and apparent equilibrium constants for the dissociation of pig liver carboxylesterase into three subunit molecules have been determined by complement fixation. The dependence of the dissociation equilibria on pH are consistent with dissociation reactions involving the addition of two protons per subunit, a pH-independent dissociation, and a dissociation upon the loss of one proton per subunit. The rate constants for dissociation are consistent with terms first order in hydrogen and hydroxide ions and a pH-independent path. The equilibrium constants in the range 3–35 °C at pH 7.2 exhibit no dependence on temperature; the association reaction is entropy driven with $\text{ΔS = 68}\text{cal mol}{}^{\mathrm{?1}}{}^{\mathrm{°}}\text{K}{}^{\mathrm{?1}}$. The rate constants for the pH-independent dissociation follow ΔH^≠ ? 6 kcal mol^?1. The order of effectiveness of concentrated salts in promoting denaturation is correlated with their effect on the activity coefficient of acetyltetraglycine ethyl ester and suggests that peptide groups become more exposed upon dissociation. The increased dissociation in the presence of urea derivatives containing alkyl substituents suggests exposure of hydrophobic regions upon dissociation; this is also consistent with ΔH = 0 for dissociation. It is likely that hydrophobic interactions contribute to the stability of the trimeric whole molecule.     

The temperature dependences of some types of gaseous ionic reactions of astrochemical interest

The rate constants of ion-molecule reactions which are of potential significance in astrochemical systems are found to exhibit significant, and in many cases, negative temperature dependences. The rate constants of fast ion-polar molecule reactions (e.g., XH⁺+B»BH⁺+X) may increase by a factor of 5–10 between 1000 and 10K. Slow reactions that proceed via reaction complexes (e.g., H-transfer and association reactions) often exhibit temperature dependences of the formk=AT ⁻ⁿ ,n=1–5. Both transition state theory considerations and the coupled-oscillator RRK-type model are seen to be able to account qualitatively for the behavior of slow ion-molecule, reactions.     

Kinetics and mechanism of oxidation of d-glucopyranose 6-phosphate and d-ribofuranose 5-phosphate by vanadium(V) in perchloric acid media

The second-order rate constants for the oxidations of d-glucopyranose 6-phosphate and d-ribofuranose 5-phosphate by vanadium(V) in perchloric acid media have been measured spectrophotometrically in the visible region. The order with respect to [H⁺] is less than unity for each reaction. The enthalpy and entropy of activations of the reactions are higher than those for the corresponding two-electron oxidations by chromium(VI). The oxidation involves free-radical intermediates.     

Transport Properties of the Tomato Fruit Tonoplast : III. Temperature Dependence of Calcium Transport

Calcium transport into tomato (Lycopersicon esculentum Mill, cv Castlemart) fruit tonoplast vesicles was studied. Calcium uptake was stimulated approximately 10-fold by MgATP. Two ATP-dependent Ca²⁺ transport activities could be resolved on the basis of sensitivity to nitrate and affinity for Ca²⁺. A low affinity Ca²⁺ uptake system (K_m > 200 micromolar) was inhibited by nitrate and ionophores and is thought to represent a tonoplast localized H⁺/Ca²⁺ antiport. A high affinity Ca²⁺ uptake system (K_m = 6 micromolar) was not inhibited by nitrate, had reduced sensitivity to ionophores, and appeared to be associated with a population of low density endoplasmic reticulum vesicles that contaminated the tonoplast-enriched membrane fraction. Arrhenius plots of the temperature dependence of Ca²⁺ transport in tomato membrane vesicles showed a sharp increase in activation energy at temperatures below 10 to 12°C that was not observed in red beet membrane vesicles. This low temperature effect on tonoplast Ca²⁺/H⁺ antiport activity could only by partially ascribed to an effect of low temperature on H⁺-ATPase activity, ATP-dependent H⁺ transport, passive H⁺ fluxes, or passive Ca²⁺ fluxes. These results suggest that low temperature directly affects Ca²⁺/H⁺ exchange across the tomato fruit tonoplast, resulting in an apparent change in activation energy for the transport reaction. This could result from a direct effect of temperature on the Ca²⁺/H⁺ exchange protein or by an indirect effect of temperature on lipid interactions with the Ca²⁺/H⁺ exchange protein.     

Distinct unfolding and refolding pathways of ribonuclease a revealed by heating and cooling temperature jumps

Heating and cooling temperature jumps (T-jumps) were performed using a newly developed technique to trigger unfolding and refolding of wild-type ribonuclease A and a tryptophan-containing variant (Y115W). From the linear Arrhenius plots of the microscopic folding and unfolding rate constants, activation enthalpy (ΔH^#), and activation entropy (ΔS^#) were determined to characterize the kinetic transition states (TS) for the unfolding and refolding reactions. The single TS of the wild-type protein was split into three for the Y115W variant. Two of these transition states, TS1 and TS2, characterize a slow kinetic phase, and one, TS3, a fast phase. Heating T-jumps induced protein unfolding via TS2 and TS3; cooling T-jumps induced refolding via TS1 and TS3. The observed speed of the fast phase increased at lower temperature, due to a strongly negative ΔH^# of the folding-rate constant. The results are consistent with a path-dependent protein folding/unfolding mechanism. TS1 and TS2 are likely to reflect X-Pro¹¹⁴ isomerization in the folded and unfolded protein, respectively, and TS3 the local conformational change of the β-hairpin comprising Trp¹¹⁵. A very fast protein folding/unfolding phase appears to precede both processes. The path dependence of the observed kinetics is suggestive of a rugged energy protein folding funnel.     

The effect of temperature on the activity of (Naplus plus K plus)-ATPase

The marked temperature dependence of product formation by microsomal preparations of ouabain-sensitive (Na⁺ + K⁺)-ATPase prepared from rabbit renal cortex has been confirmed by extremely sensitive pH measurement. It was possible to confine measurements of H⁺ release to a change of less than 0.02 pH units. With periods of observation not exceeding 20 sec, the linearity of H⁺ release could be maintained. Below 14 ° there is no detectable H⁺ release which is sensitive to inhibition by 0.2 mm ouabain. Above this critical temperature, however, the reaction increases markedly throughout the range examined. The ouabain-insensitive or “residual” ATPase of these preparations also shows a dependence upon temperature, but only above a critical temperature of 24 °.     

Reaction of hydrogen peroxide with hexaaquacobalt(III) perchlorate

The reaction of with H₂O₂ in 1.0 M HClO₄/LiClO₄ was found to be first-order in both reactants and the [H⁺] dependence of the second-order rate constant is given by k_2obs = b/[H⁺], b at 25 °C is 26.4 ± 0.5 s⁻¹. The rate law shows a simple inverse dependence on [H⁺] that is consistent with a rapidly maintained equilibrium between and its hydrolyzed form Co(H₂O)₅(OH)²⁺, followed by the rate controlling step, i.e. oxidation of H₂O₂ by Co(H₂O)₅(OH)²⁺.     

Differential Effects of Mutations on the Transport Properties of the Na+/H+ Antiporter NhaA from Escherichia coli

Na⁺/H⁺ antiporters show a marked pH dependence, which is important for their physiological function in eukaryotic and prokaryotic cells. In NhaA, the Escherichia coli Na⁺/H⁺ antiporter, specific single site mutations modulating the pH profile of the transporter have been described in the past. To clarify the mechanism by which these mutations influence the pH dependence of NhaA, the substrate dependence of the kinetics of selected NhaA variants was electrophysiologically investigated and analyzed with a kinetic model. It is shown that the mutations affect NhaA activity in quite different ways by changing the properties of the binding site or the dynamics of the transporter. In the first case, pK and/or K_D^Na are altered, and in the second case, the rate constants of the conformational transition between the inside and the outside open conformation are modified. It is shown that residues as far apart as 15–20 Å from the binding site can have a significant impact on the dynamics of the conformational transitions or on the binding properties of NhaA. The implications of these results for the pH regulation mechanism of NhaA are discussed.     

Equilibrium constants under physiological conditions for the reactions of the nonphosphorylated pathway of L-serine biosynthesis

The observed equilibrium constants (K_obs) for the reactions of d-2-phosphoglycerate phosphatase, d-2-Phosphoglycerate^3? + H₂O → d-glycerate^? + HPO₄^2?; d-glycerate dehydrogenase (EC 1.1.1.29), d-Glycerate^? + NAD⁺ → NADH + hydroxypyruvate^? + H⁺; and l-serine:pyruvate aminotransferase (EC 2.6.1.51), Hydroxypyruvate^? + l-H · alanine^± → pyruvate^? + l-H · serine^±; have been determined, directly and indirectly, at 38 °C and under conditions of physiological ionic strength (0.25 m) and physiological ranges of pH and magnesium concentrations. From these observed constants and the acid dissociation and metal-binding constants of the substrates, an ionic equilibrium constant (K) also has been calculated for each reaction. The value of K for the d-2-phosphoglycerate phosphatase reaction is 4.00 × 10³m [ΔG⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)]([H₂0] = 1). Values of K_obs for this reaction at 38 °C, [K⁺] = 0.2 m, I = 0.25 M, and pH 7.0 include 3.39 × 10³m (free [Mg²⁺] = 0), 3.23 × 10³m (free [Mg²⁺] = 10^?3m), and 2.32 × 10³m (free [Mg²⁺] = 10^?2m). The value of K for the d-glycerate dehydrogenase reaction has been determined to be 4.36 ± 0.13 × 10^?13m (38 °C, I = 0.25 M) [ΔG⁰ = 73.6 kJ/mol (17.6 kcal/mol)]. This constant is relatively insensitive to free magnesium concentrations but is affected by changes in temperature [ΔH⁰ = 46.9 kJ/mol (11.2 kcal/mol)]. The value of K for the serine:pyruvate aminotransferase reaction is 5.41 ± 0.11 [ΔG⁰ = ?4.37 kJ/mol (?1.04 kcal/mol)] at 38 °C (I = 0.25 M) and shows a small temperature effect [ΔH⁰ = 16.3 kJ/ mol (3.9 kcal/mol)]. The constant showed no significant effect of ionic strength (0.06–1.0 m) and a response to the hydrogen ion concentration only above pH 8.5. The value of K_obs is 5.50 ± 0.11 at pH 7.0 (38 °C, [K⁺] = 0.2 m, [Mg²⁺] = 0, I = 0.25 M). The results have also allowed the value of K for the d-glycerate kinase reaction (EC 2.7.1.31), d-Glycerate^? + ATP^4? → d-2-phosphoglycerate^3? + ADP^3? + H⁺, to be calculated to be 32.5 m (38 °C, I = 0.25 M). Values for K_obs for this reaction under these conditions and at pH 7.0 include 236 (free [Mg²⁺] = 0) and 50.8 (free [Mg²⁺] = 10^?3m).     

Proton transport by gastric membrane vesicles

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H⁺ uptake into an osmotically sensitive space. The apparent K_m for ATP was 1.7 · 10^?4 M in the absence of a K⁺ gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺ and is inhibited by ATPase inhibitors such as N,N′-dicylclohexylcarbodiimide. Maximal H⁺ uptake occurs with an outward K⁺ gradient but the minimal apparent K_A is found in the absence of a K⁺ gradient. The pH optimum for H⁺ uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphorylation of the enzyme, but is considerably less than the pH maximum of the K⁺ dependent dephosphorylation. In the presence of an inward K^? gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H⁺ gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K⁺ conductance but a measurable H⁺ conductance, hence a K⁺ gradient can produce an H⁺ gradient in the presence of valinomycin. The uptake and spontaneous leak of H⁺ are temperature sensitive skin with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p-nitrophenylphosphatase inactivation. It is concluded that H⁺ uptake by these vesicles is probably due to a dimeric (H⁺ + K⁺)-ATPase and is probably non-electrogenic.     

Electron acceptors of bacterial photosynthetic reaction centers II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex

The photoreduction of ubiquinone in the electron acceptor complex (Q₁Q₁₁) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H⁺ in the reduction was revealed by the pH dependence of the electron transfer events and by net H⁺ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash Q₁₁ receives an electron via Q₁ to form a stable ubisemiquinone anion (Q?^?₁₁); the second flash generates Q?^?₁. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H⁺, to produce Q₁₁H₂. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H⁺ uptake. Above pH 6 there is a progressive increase in H⁺ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H⁺ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to Q?^?₁₁. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H⁺ binding electron transfer following the second flash.     

Essential Arginyl Residues in the Plasma Membrane H-ATPase from Vigna radiata L. (Mung Bean) Roots

Proton-translocating ATPase (H⁺-ATPase) was purified from mung bean (Vigna radiata L.) roots. Treatment of this enzyme with the arginine-specific reagent 2,3-butanedione in the presence of borate at 37°C (pH 7.0), caused a marked decrease in its activity. Under this condition, half-maximal inhibition was brought about by 20 millimolar 2,3-butanedione at 12 minutes. MgATP and MgADP, the physiological substrate and competitive inhibitor of the ATPase, respectively, provided partial protection against inactivation. Loss of activity followed pseudo-first order kinetics with respect to 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration gave a curve with a slope of 0.984. Thus, inactivation may possibly result from reaction of one arginine residue at each active site of the enzyme. The results obtained from the present study indicate that at least one arginyl residue performs an essential function in the plasma membrane H⁺-ATPase, probably at the catalytic site.     

Thermodynamics and coordination characteristics of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 extraction complex

The extraction equilibrium of the hydronium-uranium(VI)-dicyclohexano-24-crown-8 complex was carried out in the crown ether1,2-dichloroethaneHCl aqueous solution system at different temperatures. The extraction complex has the overall composition (L)₂·(H₃O⁺·χH₂O)₂·UO₂Cl₄²⁻ (L = dicyclohexano-24-crown-8). The values of the extraction equilibrium constants (K_ex) increase steadily with a decrease in temperature: 13.5 (298 K), 7.96 (301 K), 4.20 (303 K) and 2.07 (305 K). A plot of log K_ex against 1/T shows a straight line. The value of the enthalpy change, ΔH°, was calculated from the slope and equals −212 kJ mol⁻¹. The value of the entropy change, ΔS°, was calculated from ΔH° and K_ex and equals −690 J K⁻¹ mol⁻¹, whereas ΔG° = −6.45 kJ mol⁻¹. Comparing these thermodynamic parameters with those of the dicyclohexano-18-crown-6 isomer A [1] (ΔS° = −314 J K⁻¹ mol⁻¹, ΔH° = −101 kJ mol⁻¹ and ΔG° = −8.37 kJ mol⁻¹), it can be seen that ΔH° and ΔS° are more negative for the former than for the latter, and both are enthalpy-stabilized complexes. The molecular structure of the complex has the feature that there are two H₅O₂⁺ ions in it, in contrast to the H₃O⁺ ions in the dicyclohexano-18-crown-6 isomer A complex [1]. Each of the H₅O₂⁺ ions is held in the crown ether cavity by four hydrogen bonds. The H₅O₂⁺ ion has a central bond. The uranium atom forms UO₂Cl₄²⁻ as a counterion away from the crown ether. The formation of this complex is in good agreement with more negative entropy change and less negative free energy change, as mentioned above.     

Spectroscopic study of the complexation of benzo-15-crown-5 and dibenzo-30-crown-10 with sodium and potassium ions in binary acetonitrile-water mixture

The interaction between Na⁺ and K⁺ ions and benzo-15-crown-5 and dibenzo-30-crown-10 in CH_3⁻ CNH₂O mixtures has been studied spectrophotometrically, at 25 °C and at ionic strength of 0.015. The formation constants for 1:1 complexes at different weight percentages of acetonitrile in water were determined. In all solvent mixtures used, the stabilities of the complexes vary in the order DB30C10-K⁺ > B15C5-K⁺ > B15C5-Na⁺. A linear relationship is observed between log K_f of the three complexes and the mole fraction of acetonitrile. The solvent dependence of the stability constants is most notable with B15C5-K⁺ and the least with DB30C10-K⁺ complex.     

Theoretical fluorescence induction curves derived from coupled differential equations describing the primary photochemistry of photosystem II by an exciton-radical pair equilibrium

Fluorescence induction curves were calculated from a molecular model for the primary photophysical and photochemical processes of photosystem II that includes reversible exciton trapping by open (PHQ_A) and closed (PHQ^-_A) reaction centers (RCs), charge stabilization as well as quenching by oxidized (P⁺HQ^(-)_A) RCs. For the limiting case of perfectly connected photosynthetic units (“lake model”) and thermal equilibrium between the primary radical pair (P⁺H^-) and the excited singlet state, the primary reactions can be mathematically formulated by a set of coupled ordinary differential equations (ODE). These were numerically solved for weak flashes in a recursive way to simulate experiments with continuous illumination. Using recently published values for the molecular rate constants, this procedure yielded the time dependence of closed RCs as well as of the fluorescence yield (= fluorescence induction curves). The theoretical curves displayed the same sigmoidal shapes as experimental fluorescence induction curves. From the time development of closed RCs and the fluorescence yield, it was possible to check currently assumed proportionalities between the fraction of closed RCs and either (a) the variable fluorescence, (b) the complementary area above the fluorescence induction curve, or (c) the complementary area normalized to the variable fluorescence. By changing selected molecular rate constants, it is shown that, in contrast to current beliefs, none of these correlations obeys simple laws. The time dependence of these quantities is strongly nonexponential. In the presence of substances that quench the excited state, the model predicts straight lines in Stern-Volmer plots. We further conclude that it is impossible to estimate the degree of physical interunit energy transfer from the sigmoidicity of the fluorescence induction curve or from the curvature of the variable fluorescence plotted versus the fraction of closed RCs.     

The enthalpy of oxidation of flavin mononucleotide

The oxidation enthalpy of reduced flavin mononucleotide at pH 7.0 in 0.2 m phosphate buffer has been studied by determining the heat associated with the reaction: FMNH₂ + 2 Fe(CN)^?3₆ ? FMN + 2 Fe(CN)^?4₆ + 2 H⁺. (a) (The quinone, semiquinone, and hydroquinone forms of FMN are represented as FMN, FMNH, and FMNH₂, respectively.) Calorimetric experiments were performed in a flow microcalorimeter which was modified to prevent sample contamination by oxygen. The enthalpy observed for reaction (a), after correction for dilution and buffer effects, was ?39.2 ± 0.4 kcal (mole FMNH₂)^?1 at 25 °C. The potential difference, ΔE′, developed by reaction (a) was determined potentiometrically and corresponded to a free energy change, ΔG′, of ?30.3 kcal (mole FMNH₂)^?1. The resulting entropy change, ΔS′, was thus calculated to be ?29.8 e.u. Reaction (a) was also studied at temperatures of 7 °C and 35.5 °C. ΔC_p′ for the reaction was calculated as ?155 ± 18 cal deg^?1 (mole FMNH₂)^?1 at 20 °C. ΔH′ for the reaction (b), FMNH₂ ? FMN + H₂, (b) was calculated as +14.2 ± 0.7 kcal mole^?1 at 25 °C, relative to the enthalpy of the hydrogen electrode being identically equal to zero at all values of pH and temperature. The free energy at pH 7.0 for reaction (b), calculated from the potential was found to be ?9.7 kcal mole^?1, which resulted in an entropy for reaction (b) of 80.2 e.u. A thermal titration of reaction (a) was used to calculate the thermodynamic parameters for the formation of semiquinone dimer according to the reaction FMNH₂ + FMN ? (·FMNH)₂. (c) The free energy, enthalpy, and entropy changes for reaction (c) were estimated to be ?6.1 kcal mole^?1, ?7 kcal mole^?1, and ?3 e.u., respectively.