Conformational transitions and vibronic couplings in acid ferricytochrome c: a resonance Raman study.

Resonance Raman spectral changes in ferricytochrome c as a function of pH between 6.7 and 1.0 are reported and the structural implication is discussed in terms of the "core-expansion" model advanced by L. D. Spaulding et al. [(1975) J. Am. Chem. Soc. 97, 2517]. The data are interpreted as indicating the iron in high-spin ferricytochrome c (at pH 2.0) with two water molecules as axial ligands lies in the plane of the porphyrin ring. At pH 1.0 there is a different high-spin form of cytochrome c which has an estimated iron out-of-plane distance of approximately 0.46 A. The effect of a monovalent anion at pH 2.0 is to produce a thermal spin mixture with predominant low-spin species. Excitation at approximately 620 nm in acid cytochrome c (pH 2.0) enhances only three depolarized ring vibrations at 1623, 1555, and 764 cm-1. Marked enhancement of depolarized modes relative to polarized and anomalously polarized modes is attributed to the vibronic coupling between porphyrin pi leads to pi and porphyrin pi leads to iron (dpi) charge-transfer states.     

Tuna cytochrome c at 2.0 A resolution. I.Ferricytochrome structure analysis.

The crystal structure of oxidized cytochrome c from tuna hearts has been solved by x-ray diffraction to a resolution of 2.0 A, using four isomorphous heavy atom derivatives. The crystals, space group P43, have 2 independent cytochrome molecules in the asymmetric repeating unit. No significant difference is seen between these 2 molecules, aside from conformations of a few surface side chains. The molecular folding observed is essentially that reported for tuna ferrocytochrome c. In particular, the ring of phenylalanine 83 lies against the heme group and closes the heme crevice, and is not swung out into the surroundings as had been believed from the 2.8 A horse ferricytochrome c structure.     

Kinetic mechanism of beef heart ubiquinol:cytochrome c oxidoreductase.

The electron transfer from ubiquinol-2 to ferricytochrome c mediated by ubiquinol:cytochrome c oxidoreductase [E.C. 1.10.2.2] purified from beef heart mitochondria, which contained one equivalent of ubiquinone-10 (Q10), was investigated under initial steady-state conditions. The Q10-depleted enzyme was as active as the Q10-containing one. Double reciprocal plots for the initial steady-state rate versus one of the two substrates at various fixed levels of the other substrate gave parallel straight lines in the absence of any product. Intersecting straight lines were obtained in the presence of a constant level of one of the products, ferrocytochrome c. The other product, ubiquinone-2, did not show any significant effect on the enzymic reaction. Ferrocytochrome c non-competitively inhibited the enzymic reaction against either ubiquinol-2 or ferricytochrome c. These results indicate a Hexa-Uni ping-pong mechanism with one ubiquinol-2 and two ferricytochrome c molecules as the substrates, which involves the irreversible release of ubiquinone-2 as the first product and the irreversible isomerization between the release of the first ferrocytochrome c and the binding of the second ferricytochrome c. Considering the cyclic electron transfer reaction mechanism, this scheme suggests that the binding of quinone or quinol to the enzyme and electron transfer between the iron-sulfur center and cytochrome c1 are rigorously controlled by the electron distribution within the enzyme.     

Structure of bonito heart ferricytochrome c and some remarks on molecular interaction in its crystalline state.

The structure analysis of bonito heart ferricytochrome c was carried out at 2.8 A resolution by X-ray diffraction. The overall features of the molecule are virtually identical with those of bonito ferrocytochromes c and other cytochromes c. In the present work, the modes of molecular packing among cytochromes c were also compared by means of intermolecular distance maps. Some differences in the structures of ferro- and ferricytochrome c may exist on the surface of the molecules.     

Tuna cytochrome c at 2.0 A resolution. II. Ferrocytochrome structure analysis.

The x-ray crystal structure analysis of tuna ferrocytochrome c has been extended from 2.45 to 2.0 A resolution. The overall folding is unchanged and is the same as has been reported for tuna ferricytochrome c (Swanson R., Trus, B.L., Mandel, N., Mandel, G., Kallai, O.B., and Dickerson, R.E. (1977) J. Biol. Chem. 252, 759-755). No significant structural differences are observed between oxidation states. Difference map studies using reoxidized crystals of ferrocytochrome c confirm the absence of a conformation change. A detailed analysis of hydrogen bonding shows the presence of six beta or 310 bends of type II with obligatory glycines in the 3rd residue position. This explains 6 of the 10 nearly invariant glycines in the molecule. Close packing contacts account for three more, and only the invariant glycine 1 remains a mystery.     

Study of the biological significance of cytochrome methylation. I. Thermal, acid and guanidinium hydrochloride denaturations of baker's yeast ferricytochromes c.

The iso-cytochromes c from baker's yeast: iso-1 methylated and unmethylated forms and iso-2 have been purified and their stabilities towards denaturants compared to that of horse heart cytochrome c. Thermal, acid and guanidinium hydrochloride denaturations were followed using fluorescence emission of their tryptophan 59 and/or the absorbance in the Soret region as the physical parameters. Very few differences could be evidenced among the ferricytochromes investigated in this study insofar as the acid denaturations are concerned. This is to be contrasted with the conclusions of the thermal and guanidinium hydrochloride denaturations studies which clearly showed the ferricytochrome from horse heart to be much more stable than those from baker's yeast. No appreciable differences could be measured among the methylated and unmethylated forms of iso-1 cytochrome c nor among iso-1 and iso-2 cytochromes from baker's yeast. Our results suggest that a stabilizing effect of methylation on the tridimensional structure of ferricytochrome c must probably be discarded. Other possible physiological roles of methylation are suggested taking into account the relative instability of ascomycetes's cytochromes as compared to mammalian ones.     

Raman spectroscopic studies of dimyristoylphosphatidic acid and its interactions with ferricytochrome c in cationic binary and ternary lipid-protein complexes.

The vibrational Raman spectra of both pure 1-alpha-dimyristoylphosphatidic acid (DMPA) liposomes and DMPA multilayers reconstituted with ferricytochrome c at pH 7 and pH 4, with either sodium or calcium as the cation, are reported as a function of temperature. Multilayers composed of a 1:1 mol ratio DMPA and dimyristoylphosphatidylcholine with perdeuterated acyl chains (DMPC-d54) have also been reconstituted with approximately 10(-4) M ferricytochrome c for Raman spectroscopic observation. Total integrated band intensities and relative peak height intensity ratios, two spectral Raman scattering parameters used to characterize bilayer properties, are sensitive to the presence of both ferricytochrome c and the cation in the reconstituted liposomes. Temperature profiles, derived from the various Raman intensity parameters for the 3,100-2,800 cm-1 lipid acyl chain C-H stretching mode region specifically reflect bilayer perturbations due to the interactions of ferricytochrome c. At pH 4 the calcium DMPA multilamellar gel to liquid crystalline phase transition temperatures Tm, defined by either the C-H stretching mode I2850/I2880 and I2935/I2880 peak height intensity ratios, are 58.5 +/- 0.5 degrees C and 60.0 +/- 0.3 degrees C, respectively. This difference in Tm's resolves the phase transition process into first an expansion of the lipid lattice and then a melting of the lipid acyl chains. At pH 7 the calcium DMPA liposomes show no distinct phase transition characteristics below 75 degrees C. For sodium DMPA liposomes reconstituted with ferricytochrome c at either pH 4.0 or pH 7.0, spontaneous Raman spectra show altered lipid structures at temperatures above 40 degrees C. Resonance Raman spectra indicate that ferricytochrome c reconstituted in either calcium or sodium DMPA liposomes changes irreversibly above Tm. For either the binary lipid or ternary lipid-protein systems reconstituted with DMPC-d54, linewidth parameters of the DMPC-d54 acyl chain CD2 symmetric stretching modes at 2,103 cm-1 provide a sensitive measure of the conformational and dynamic properties of the perdeuterated lipid component, while the 3,000 cm-1 C-H spectral region reflects the bilayer characteristics of the DMPA species in the complex. Although calcium clearly induces a lateral phase separation in the DMPA/DMPC-d54 system at pH 7.5 (Kouaouci, R., J.R. Silvius, I. Grah, and M. Pezolet. 1985. Biochemistry. 24:7132-7140), no distinct lateral segregation of the lipid components is observed in the mixed DMPA/DMPC-d54 lipid system in the presence of either ferricytochrome c or the sodium and calcium cations at pH 4.0.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformation change of cytochrome c. I. Ferrocytochrome c structure refined at 1.5 A resolution

Tuna ferrocytochrome c has been crystallographically refined at a resolution of 1.5 Å using the Diamond real-space method followed by Jack-Levitt restrained energy and reciprocal space refinement, monitoring progress continuously with superimposed Fourier and difference Fourier maps: The final R factor for cytochrome plus 53 solvent molecules, using 13,840 reflections with intensities greater than 2 σ, is 17·3%. The overall structure remains as described earlier (Takano et al., 1977), but structural details have been clarified to the point where meaningful comparison can be made with the oxidized molecule (following paper). Main and side-chain flexibility as judged by isotropic temperature parameters correlate with position in the molecule, with greatest flexibility at external chain loops. The haem group is held tightly in place by its attachments and neighbours, and is deformed slightly into a saddle shape. The iron does not deviate significantly from the best mean plane of the haem, and bond lengths to ligands are as expected from model compounds.A water molecule buried in the haem crevice is bonded to Asn52, Tyr67 and Thr78, the latter two being bonded also to Met80 and the outer haem propionate. It is proposed that this buried water molecule is involved in the reduction of ferricytochrome c by chromous ion, and the reactions of Tyr67 with KI₃ and tetranitromethane. Two other buried water molecules occur beneath the 20's loop at the right, and within the 40's loop at the bottom. Reasonable if tentative functional assignments can be made for all 24 of the evolutionarily invariant residues in the cytochrome molecule.     

The kinetics of the reduction of cytochrome c by the superoxide anion radical.

1. At neutral pH ferricytochrome c is reduced by the superoxide anion radical (O2-), without loss of enzymatic activity, by a second order process in which no intermediates are observed. The yield of ferrocytochrome c (82-104%), as related to the amount of O2- produced, is slightly dependent on the concentration of sodium formate in the matrix solution. 2. The reaction (k1 equals (1.1+/-0.1) - 10(6) M-1 - s-1 at pH 7.2, I equals 4 mM and 21 degrees C) can be inhibited by superoxide dismutase and trace amounts of copper ions. The inhibition by copper ions is removed by EDTA without interference in the O2- reduction reaction. 3. The second-order rate constant for the reaction of O2- with ferricytochrome c depends on the pH of the matrix solution, decreasing rapidly at pH greater than 8. The dependence of the rate constant on the pH can be explained by assuming that only the neutral form of ferricytochrome c reacts with O2- and that the alkaline form of the hemoprotein is unreactive. From studies at pH 8.9, the rate for the transition from the alkaline to the neutral form of ferricytochrome c can be estimated to be 0.3 s-1 (at 21 degrees C and I equals 4 mM). 4. The second-order rate constant for the reaction of O2- with ferricytochrome c is also dependent on the ionic strength of the medium. From a plot of log k1 versus I1/2-(I + alphaI1/2)-1 we determined the effective charge on the ferricytochrome c molecule as +6.3 and the rate constant at I equals 0 as (3.1+/-0.1) - 10(6) M-1 - s-1 (pH 7.1, 21 degrees C). 5. The possibility that singlet oxygen is formed as a product of the reaction of O2- with ferricytochrome c can be ruled out on thermodynamic grounds.     

The role of the complex formation between cytochrome c and NAD in the manifestation of the photoprotective effect of the dinucleotide in relation to hemeprotein

UV irradiation of free ferricytochrome solutions, pH 8, induces photorecovery of protein molecules. Hemoproteide photorecovery does not occur after irradiation of the ferricytochrome c/NAD mixture, pH 6 and 8: dinucleotide exerts a photoprotective effect with respect to ferricytochrome. This NAD effect is not observed after exposure of the ferricytochrome c/NAD system, pH 4. With this pH value, each component of the above mixture is eluted from a gel chromatographic column by its peak, whereas with pH 6 and 8, NAD and ferricytochrome c leave the column as one fraction. This indicates that the photoprotective effect of the coenzyme manifests itself upon formation of complex with hemoprotein.     

Crystallization and preliminary X-ray crystallographic studies of benzamidine-inhibited trypsin from the North Atlantic salmon (Salmo salar)

Crystals of benzamidine-inhibited trypsin from the North Atlantic salmon (Salmo salar) have been grown from ammonium sulphate solution at pH 5.0. Two crystal forms suitable for X-ray structure analysis, obtained from a hanging-drop experiment, have been characterized. Both belong to space-group P22(1)2(1) with cell dimensions a = 39.2 A, b = 62.4 A, c = 84.6 A and a = 31.4 A, b = 74.8 A, c = 83.5 A, for forms I and II, respectively. Intensity data to 1.82 A have been collected for crystal form I on a CAD4 diffractometer, and initial phases have been obtained by molecular replacement methods. The conventional R-factor after two rounds of model building and subsequent refinement is 0.25 for data between 6.0 and 2.0 A. So far no water molecules have been included in the model.     

The role of superoxide radical in the autoxidation of cytochrome c.

The net rate of autoxidation of ferrocytochrome c was decreased by ferricytochrome c. Superoxide dismutase accelerated this autoxidation to a limit and overcame the inhibitory effect of ferricytochrome c. This was the case whether the autoxidationwas observed in the presence or in the absence of denaturants, such as alcohols orurea, and whether the superoxide dismutase used was the Cu-2+-Zn-2+ enzyme from bovine erythrocytes or the Mn-3+-enzyme from Escherichia coli. It can be deduced that the autoxidation of ferrocytochrome c, under a variety of conditions, geenerates O2 minus which can then dismute to H202 + O2 or can reduce ferricytochrome c back to ferrocytochrome c. Superoxide dismutase, by accelerating the dismutation of O2 minus, prevents the back reaction and thus exposes the true rate of reaction of ferrocytochrome c with molecular oxygen.     

Different types of interaction of oxidized and reduced cytochrome c with phospholipid vesicles]

The binding constants of ferri- and ferrocytochrome c interactions with phosphatidylcholine or cardiolipin-containing vesicules were determined. It was found that affinity of ferricytochrome c to phospholipids is one order of magnitude higher that of ferrocytochrome c. A comparative investigation of circular dichroism spectra of free and phospholipid-bound ferri- and ferrocytochrome c was undertaken and it was shown that alpha-helix content of free ferrocytochrome c is higher than that of ferricytochrome c. The formation of ferricytochrome c containing lipoprotein complex led to decrease of alpha-helix content of the protein. In the case of ferrocytochrome c on the other hand interaction with phospholipids did not cause any changes in alpha-helix content. Distribution of ferri- and ferrocytochrome c in different two-phase systems consisting of dextran and polyethylenglycol or dextran and polyethylenglvcol-palmitate was also studied. A comparison of distribution constants shows that higher alpha-helix content of ferrocytochrome c results in the formation of hydrophobic clusters in the protein molecules. In previous communications it was reported that binding of ferrocytochrome c to phospholipids is determined by hydrophobic interactions while in the case of ferricytochrome c the interactions with phospholipids are mainly electrostatic. On the basis of the results obtained in this work it is supposed that it is hydrophobic clusters which determine the binding of ferrocytochrome c to phospholipid membranes.     

Superoxide radical production during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium perchlorate.

1-Methyl-4-phenyl-2,3-dihydropyridinium perchlorate (MPDP+), an intermediate in the metabolism of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, was found to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected by their ability to reduce ferricytochrome c. Superoxide dismutase inhibited this reduction in a dose-dependent manner. The rate of reduction of ferricytochrome c was dependent not only on the concentration of MPDP+ but also on the pH of the system. Thus, the rate of autoxidation of MPDP+ and the sensitivity of this autoxidation to superoxide dismutase-inhibitable ferricytochrome c reduction were both augmented, as the pH was raised from 7.0 to 10.5. The rate constant (Kc) for the reaction of superoxide radical with ferricytochrome c to form ferricytochrome c was found to be 3.48 x 10(5) M-1 s-1. The rate constant (KMPDP+) for the reaction of MPDP+ with ferricytochrome3+ c was found to be only 4.86 M-1 s-1. These results, in conjunction with complexities in the kinetics, lead to the proposal that autoxidation of MPDP+ proceeds by at least two distinct pathways, one of which involves the production of superoxide radicals and hence is inhibitable by superoxide dismutase. It is possible that the free radicals so generated could induce oxidative injury which may be central to the MPTP/MPDP(+)-induced neuropathy.     

X-ray crystal structure of the serine proteinase inhibitor eglin c at 1.95 A resolution.

The crystal structure of eglin c, naturally occurring in the leech Hirudo medicinalis, is known from its complexes with various serine proteinases, but the crystallization of free eglin c has not yet been reported. A method is described for growing well-diffracting crystals of free eglin c from highly concentrated protein solutions (approximately 200 mg/ml). The space group of the orthorhombic crystals was determined to be P2(1)2(1)2(1) with unit cell parameters a = 32.6, b = 42.0, c = 44.1 A. The structure of free eglin c was resolved at 1.95 A resolution by Patterson search methods. The final model contains all 70 amino acids of eglin c and 125 water molecules. In comparison to the eglin structure known from its complexes with proteinases, only small differences have been observed in free eglin c. However, the reactive site-binding loop and a few residues on the surface of eglin have been found in different conformations due to crystal contacts. In contrast to the complex structures, the first seven amino acids of the highly flexible amino terminus can be located. Crystallographic refinement comprised molecular dynamics refinement, classical restrained least-squares refinement and individual isotropic atomic temperature refinement. The final R-factor is 15.8%.     

Proton NMR comparison of noncovalent and covalently cross-linked complexes of cytochrome c peroxidase with horse, tuna, and yeast ferricytochromes c.

Proton NMR spectroscopy at 500 and 361 MHz has been used to characterize the noncovalent or electrostatic complexes of yeast cytochrome c peroxidase (CcP) with horse, tuna, yeast isozyme-1, and yeast isozyme-2 ferricytochromes c and the covalently cross-linked complexes of cytochrome c peroxidase with horse and yeast isozyme-1 ferricytochromes c. Under the conditions employed in this work, the stoichiometry of the predominant complex formed in solution (which totaled greater than 90% of complex formed) was found to be 1:1 in all cases. These studies have elucidated significant differences in the proton NMR absorption spectra and the one-dimensional nuclear Overhauser effect difference spectra of the complexes, depending on the specific species of ferricytochrome c incorporated. In particular, the results indicate that the noncovalent complexes formed between CcP and physiological redox partners (yeast isozyme-1 or yeast isozyme-2 ferricytochromes c) are distinctly different from the noncovalent complexes formed between CcP and ferricytochromes c from horse and tuna. Parallel chemical cross-linking studies carried out using mixtures of cytochrome c peroxidase with horse ferricytochrome c, and cytochrome c peroxidase with yeast isozyme-1 ferricytochrome c further emphasize such cytochrome c-dependent differences, with only the covalently cross-linked complex of physiological redox partners (cytochrome c peroxidase/yeast isozyme-1) displaying NMR spectra characteristic of a heterogeneous mixture of different 1:1 complexes. Finally, one-dimensional nuclear Overhauser effect experiments have proven valuable in selectively and efficiently probing the protein-protein interface in these complexes, including the environment around the cytochrome c heme 3-methyl group and Phe-82.     

The recognition of a special ubiquinone functionally central in the ubiquinone-cytochrome b-c2 oxidoreductase.

Although the energy conserving membranes of the photosynthetic bacterium Rhodopseudomonas sphaeroides contain a 25 (+/- 3)-fold molar excess of ubiquinone over the photochemical reaction center, the activity of the ubiquinone-cytochrome b-c2 oxidoreductase is unaffected by quinone extraction until only 3, or at most 4, ubiquinones remain; only then does further extraction prevent the function of the oxidoreductase. Since 2 of these last ubiquinones are integral parts of the photochemical reaction center, we conclude that the ubiquinone-cytochrome b-c2 oxidoreductase requires only 1, or at most 2, molecules of ubiquinone-10 for its function. Earlier kinetic data identified a major electron donor to ferricytochrome c2 as a single molecule (known as Z) which requires 2 electrons and 2 protons for its equilibrium reduction. Hence, we identify a single molecule of quinone, probably ubiquinone-10 in a special environment, as a major electron donor to ferricytochrome c2 in the ubiquinone cytochrome b-c2 oxidoreductase.     

Reaction of cytochrome c with nitrite and nitric oxide. A model of dissimilatory nitrite reductase.

The reaction of bovine heart ferrocytochrome c with nitrite was studied under various conditions. The reaction product was ferricytochrome c at around pH 5, whereas at around pH 3 it was Compound I, characterized by twin peaks at 529 and 563 nm of equal intensity. However, ferrocytochrome c decreased obeying first-order kinetics over the pH range examined, irrespective of the presence or absence of molecular oxygen. The apparent first-order rate constant was proportional to the square of the nitrite concentration at pH 4.4 and it increased as the pH was lowered. At pH 3 the reaction was so rapid that it had to be followed by stopped-flow and rapid-scanning techniques. The apparent rate constant at this pH was found to increase linearly with the nitrite concentration. Based on these results the active species of nitrite was concluded to be dinitrogen trioxide at pH 4.4 and nitrosonium ion, no+, at pH 3. Compound II was formed by reaction of ferrocytochrome c and NO gas at acidic and alkaline pH values. The absorption peaks were at 533 and 563 nm at pH 3, and at 538 and 567 nm at pH 12.9. This compound was also formed by reducing Compound I with reductants. Compound I prepared from ferricytochrome c and NO was stable below pH 6. However, appreciable absorption peaks for ferrocytochrome c appeared between pH 8 and 10, because Compound I was dissociated into ferrocytochrome c and NO+, and because ferrocytochrome c thus formed reacted with NO very slowly in this pH region. Saccharomyces ferricytochrome c under NO gas behaved differently from mammalian cytochrome, indicating the significance of the nature of the heme environment in determing the reactivity. Only at extreme pH values was Compound II formed exclusively and persisted. A model system for dissimilatory nitrite reductase was constructed by using bovine heart cytochrome c, nitrite and NADH plus PMS at pH 3.3, and a scheme involving cyclic turnover of ferrocytochrome c, Compound I and Compound II is presented, with kinetic parameters.     

Ion binding to lysine-modified derivatives of cytochrome c.

The effect of Cl- and K+ ions on the apparent equilibrium constant of the reaction between horse ferricytochrome c and potassium ferrocyanide was studied. Unmodified cytochrome was compared with two lysine-modified derivatives. One, guanidinated, had all lysyl groups converted to homoarginine (but retained the same positive charge); the other was trinitrophenylated at one lysine (measured spectrophotometrically). Both modified derivatives had a somewhat larger equilibrium constant in the reaction of the reduced protein with ferricyanide, but, unlike trifluoroacetylated cytochrome c (which has a negative charge), the redox properties were not dramatically different. The native protein and the lysine-modified cytochromes showed differential K+ binding in Tris-cacodylate buffer at constant ionic strength (0.003-0.005 M). More K+ was bound to ferrocytochrome c. This redox-linked binding, however, was unaffected by modification of lysine. All three derivatives also showed redox-linked differential Cl- ion binding (more Cl- ion was bount to ferricytochrome); however, in this case, the binding was reduced in the lysine-modified molecules. This was interpreted as loss of a single anion site. This anion site critically depends on one or a few lysines which are more reactive with trinitrobenzene sulfonate.     

Electrogenic events in the ubiquinone-cytochrome b/c2 oxidoreductase of Rhodopseudomonas sphaeroides.

The reductant of ferricytochrome c2 in Rhodopseudomonas sphaeroides is a component, Z, which has an equilibrium oxidation-reduction reaction involving two electrons and two protons with a midpoint potential of 155 mV at pH 7. Under energy coupled conditions, the reduction of ferricytochrome c2 by ZH2 is obligatorily coupled to an apparently electrogenic reaction which is monitored by a red shift of the endogeneous carotenoids. Both ferricytochrome c2 reduction and the associated carotenoid bandshift are similarly affected by the concentrations of ZH2 and ferricytochrome c2, pH, temperature the inhibitors diphenylamine and antimycin, and the presence of ubiquinone. The second-order rate constant for ferricytochrome c2 reduction at pH 7.0 and at 24 degrees C was 2 - 10(9) M-1 - s-1, but this varied with pH, being 5.1 - 10(8) M-1 = s-1 at pH 5.2 and 4.3 - 10(9) M-1 - s-1 at pH 9.3. At pH 7 the reaction had an activation energy of 10.3 kcal/mol.