J . Chem. SOC., Faraday Trans. I, 1982, 78, 2085-2094 Coupling between pH-induced Conformational Phenomena and Stereospecific Effects in Electron-transfer Reactions BY MARIO BARTERI AND BASILIO PISPISAT Istituto di Chimica Fisica, Universita di Roma, 00185 Roma, Italy Received 2 1 st September, 198 1 The oxidation by H,O, of L-( + )-ascorbate anion in the presence of 2,2’,2”,2”’-tetrapyridineiron(111) complex ions anchored to poly(L-glutamate) (FeL) or poly(D-glutamate) (FeD) has been studied at a complex-to-polymer-residue ratio of 0.10 and in the pH range 6-8. Evidence is produced that the reaction is a composite process reflecting contributions from parallel routes; one of these corresponds to a catalytic, [H,O,]-independent pathway and the other refers to an uncatalysed electron-transfer process between ascorbate anion and hydrogen peroxide. Stereospecific effects in the catalysis are observed with decreasing pH, accompanied by an increase in the amount of a-helix in the polypeptide supports &). Thus at pH 7.8 v;, z 0.13), k,,, = 1382.3 f 1 13.2 and kFeL = 1034.4 f 79.3 dm3 mol-l s-’ and the activation energy is 3.8f0.3 kcal mol-’ with both enantiomeric catalysts, whereas at pH 6.3 (fh x 0.84), kFeD = 70.9k5.5 and kFeL = 13.6f 1.1 dm3 mo1-ls-l and the activation energy is 18.0f 1.3 kcal mol-I in both cases.The results indicate that stereoselectivity is an entropy-controlled phenomenon. The effect is probably caused by conformational rigidity of the precursor complex, which arises from interactions between the optically active substrate molecules and the chiral residues of the ordered polymer surrounding the active centres.Effects of the stereochemical features of the substrate-catalyst adduct on the mechanism of electron transfer are discussed. The evidence suggests that the asymmetric [Fe(tetpy)(OH),]+-polyelectrolyte systems also behave as environmental controllers of the uncatalysed oxidation of ascorbate anion. In a previous paperf we showed that haemin-like 2,2’,2”,2”‘-tetrapyridineiron(111) complex ions anchored to sodium poly(L-glutamate) (PLG) or poly(D-glutamate) (PDG) are efficient catalysts for the oxidation of L-( +)-ascorbic acid, according to the reaction O=C-C-OH o=c-c=o I II catalyst I I 0 C-OH + HzOz 0 C=O + HZO \CL \CL I I CHZOH CHOH I I CHOH CH,OH It has been also reported that the conformational features of the polypeptide supports are primarily responsible for stereospecific effects in the catalysis.The specific rate constant of the reaction catalysed by [Fe(tetpy)(OH),]+-PDG (FeD) system was found to be higher than that obtained using the enantiomeric [Fe(tetpy)(OH),]+-PLG (FeL) material only when the amount of a-helix in the polymeric matrices was 1arge.l Since ordered structure in PLG or PDG can be achieved either by lowering the pH or increasing the amount of bound counter-ions,2 the effect of both these parameters on the kinetics of the reaction was extensively investigated. In the previous paper1 the t Permanent address: Istituto Chimico, Universita di Napoli, 80134 Napoli, Italy. 20852086 STEREOSPECIFIC EFFECTS IN ELECTRON TRANSFER results obtained at fixed pH (7.0) and varying complex-to-polymer-residue ratio [C]/[P] were illustrated, whilst the kinetic data obtained at fixed [C]/[P] (0.10) and varying pH are reported here.Evidence is produced that there is a close relationship between stereochemical features of the precursor complex and both mechanism and stereoselectivity of the electron-transfer reaction. The results are fully consistent with those previously reported1 and are discussed in the light of some general considerations concerning the structural characteristics of the catalytic systems. EXPERIMENTAL MATERIALS Sodium poly(L-glutamate) and poly(D-glutamate) were obtained as previously described.l Polymer concentration [PI, determined by U.V. absorption at 200 nm (E = 5500), is referred to the monomeric unit, i.e.monomol dmP3. [Fe(tetpy)(OH),]+ complex ions were prepared as previously reported.'* Under the experimental conditions used [pH 6.33 and 6.54 (phosphate buffer 4 x lo-, mol dm-3), 7.02, 7.48 and 7.76 (tris buffer 5 x lo-, mol dm-3)], the degree of association of complex counter-ions by polypeptides is ca. 84,89,94,98 and loo%, respectively, according to equilibrium dialysis measurements.2* Both ascorbic acid (Merck) and stabilizer-free H,O, (Erba) were analytical-grade reagents. All measurements were performed on freshly prepared solutions, using doubly distilled water with a conductivity < 2 x f2-l cm-l (20 "C). METHODS AND APPARATUS Kinetic experiments were carried out under pseudo-first-order conditions, measuring the disappearance of ascorbate anion (AH-) at 265 nm.5 (The pK, values of ascorbic acid are 4.04 and 1 1.34 at 25 0C.6) They were performed at two temperatures (25.9 & 0.1 and 16.0 & 0.1 "C) as already described.' At each pH the range of catalyst concentration explored was 0.5 < [C]/10-5 mol dm-3 < 5 ([C]/[P] = 0.10).In all cases, plots of log(A, -Am) as a function of time were linear over several half-lives. The observed pseudo-first-order rate constants k (s-l) were obtained from the slopes. Four kinetic measurements were performed for each run to obtain consistency in results. A slight oxidation of ascorbate anion by H,O, was detected in the presence of the sole polypeptide matrices,'?' its rate depending on both [PI and pH.All first-order rate constants were properly corrected for this effect. At each pH investigated, plots of k as a function of [C] always gave straight lines and the second-order rate constants kFeD and kFeL (dm3 mol-l s-l) were obtained from the slopes. Stereoselectivity of the catalysis is expressed as kFeD/kFeL. Absorption spectra were recorded on a Beckman DBGT or a Cary 219 spectrophotometer. Circular dichroism (c.d.) spectra were determined on a Cary 61 dichrograph, with appropriate quartz cells. The pH values were measured by a Radiometer 26 pH-meter using standard semimicroelectrodes. RESULTS The pseudo-first-order rate constants k of the oxidation reaction of L-( + )-ascorbate anion as a function of complex concentration [C], at fixed [C]/[P] of 0.10 and at pH 6.5 and 7.5, are reported in fig.1. As already observed for the same type of plot with the data obtained at fixed pH (7.0) and diverse [C]/[P] values,l the specific rate for electron transfer linearly increases with increasing [C]. This trend indicates true catalytic behaviour for the iron(m) chelate. Furthermore, within the whole range of pH explored (6.3-7.8) the straight lines exhibit intercept values which differ significantly from zero, i.e. k = k, + kcat [C]. This implies that the reaction is a composite process reflecting contribution from parallel pathways, one of which refers to the catalytic process (kcat/dm3 mol-l s-l) and the other corresponds to an uncatalysed route to dehydroascorbic acid (k0/s-l). The values of kcat and k, (25.9OC) at different pHM.BARTER1 A N D B. P I S P I S A 2087 100 - I m m 2 c 0 1 2 3 4 5 6 [C]/10-5 mol dm-3 FIG. 1 .-Catalytic effect of the oxidation of L-ascorbate anion in the presence of iron(w) complex ions bound to poly(L-glutamate) (empty symbols) or poly(D-glutamate) (filled symbols) at [C]/[P] = 0.10 and pH 6.5 (broken lines) and 7.5 (full lines), in 0.04 mol dmP3 phosphate buffer or 0.05 mol dm-3 tris buffer, respectively. T = 25.9 OC, [H,O,],/[AH-1, = 100. k is the difference between first-order rate constants in the presence and absence of bound complex ions. CATALYTIC (ko,D AND ko,L/s-l) OXIDATION OF L-ASCORBATE ANION AT DIFFERENT pH VALUES TABLE l.-RATE CONSTANTS FOR THE CATALYTIC (kFeD AND kFeL/dm3 mOl-' S-l) AND NON- T = 25.9 OC, [C]/[P] = 0.10, p = 0.04 dm3 mol-', [H,O,],/[AH-1, z 100.~~ PH fhU k F e D k F e L kFeD/kFeLb k O , D/ k O , L / k ~ , D / ~ o , L 7.76 0.13 1382.3k113.2 1034.4f79.3 1.3f0.2 110.3 106.1 1 .o 7.48 0.19 988.3f101.6 626.0k56.1 1.6f0.2 87.3 80.0 1.1 7.02 0.45 442.3f42.9 153.6k12.6 2.9f0.5 59.6 38.7 1.5 6.54 0.75 143.0f 13.1 40.2 k 3.7 3.7 k0.5 39.9 21.4 1.9 6.33 0.84 70.9f5.5 13.6 k 1.1 5.2 & 0.6 34.8 17.1 2.0 a-Helical fraction of polypeptide matrices at [C]/[PJ = 0.10 (see text); stereoselectivity factors of the catalytic reaction. values are reported in table 1, where subscripts FeD and FeL denote the enantiomeric catalysts, whereas D and L denote the asymmetric polymeric supports. In the same table the fraction of a-helix in the polypeptide matrices ( f h ) , at [C]/[P] = 0.10, is also shown.It was evaluated by the conventional expression A E o ~ s = f h AEh + ( -fh) AEc where AE = E~ - eR is the differential circular dichroism absorption (dm3 mol-l cm-l), using At+, = - 9.2 and A E ~ = 1.4, which represent the estimated values of ( E ~ - E ~ ) at2088 STEREOSPECIFIC EFFECTS IN ELECTRON TRANSFER 220 nm of helical and coil polypeptides at pH ca. 5 and ca. 9, respectively, in the presence of the complex ions.' The values of are those reported in fig. 2, where the change in ellipticity at 220 nm of poly(L-glumatate) solutions in the absence and presence of the iron(m) chelate molecules, at [C]/[P] = 0.10, is plotted as a function of pH. As already seen,' the binding of complex counter-ions determines a stabilization of the a-helical structure in the polypeptide in terms of an upward shift in the pH region of the conformational transition.Furthermore, the curve levels off at a value of ellipticity lower than that of the helical-polypeptide-free-complex solutions because of an induced c.d. band of opposite sign in the bound complex, within the same frequency 0 -10 I I I I 1 I 4 5 6 7 8 9 PH FIG. 2.-Variation of ellipticity at 220 nm of PLG solutions, in the absence (A) and presence (0) of chelate ions ([C]/[P] = 0.10) as a function of pH. Inspection of table 1 shows that the hydrogen-ion concentration markedly affects both parallel reactions in terms of specific rates as well as stereospecific effects. At around pH 8 a very small stereoselectivity is observed, despite the fact that the con- figurational dissymmetry2y8 of the active sites is relatively high, as indicated by the induced ellipticity of, say, the FeL system at 287 nm, where the chiral polymeric sup- port does not absorb ([el = - 32.0 x lo3 deg cm2 dmol-l, [C]/[P] = 0.10).With in- creasing [H+] the catalysis becomes stereoselective, kFeD being definitely higher than kFeL. In addition, the higher the hydrogen-ion concentration ( i e . the larger the amount of a-helix in the polypeptide matrices), the greater the stereoselectivity, which is seen to occur at the expense of catalytic efficiency because the rate constants decrease by ca. two orders of magnitude with respect to that of the non-stereospecific process. At the same time, the parallel uncatalysed reaction also shows some stereospecificity ; however it is much smaller than that of the catalytic process.When the stereoselectivity factors kFeD/kFeL are plotted as a function of pH, a trendM. BARTER1 A N D B. PISPISA 2089 similar to that followed by the a-helical fraction of the polypeptide supports (fh) is observed (fig. 3). This finding clearly indicates that the conformational dissymmetry2 of the active sites is primarily responsible for stereospecific effects in the catalysis. Similar conclusions were reached when studying the same reaction at pH 7.0 and varying [C]/[P].l In fact, an increase in the amount of bound counter-ions at fixed pH (7.0) has a similar (although less pronounced) effect on the coil-to-a-helix transition of the chiral polymeric supports to that of decreasing the pH at fixed [C]/[P] = 0.1 .o fh 0.5 0 6 5 i 3 * 2 1 PH FIG. 3.-Vanation of a-helical fraction in PLG, fh (a), and of stereoselectivity factor, kFeD/kFeL (vertical bars), as a function of pH. The parameterf, was evaiuated by c.d. measurements at 220 nm of complex-PLG solutions at [CJ/[P] = 0.10 (see text). TABLE 2.-RATE CONSTANTS (dm3 m0l-l S-l) FOR THE CATALYTIC OXIDATION OF L-ASCORBATE ANION AS A FUNCTION OF THE INITIAL CONCENTRATION OF HYDROGEN PEROXIDE AT TWO pH VALUES 7'= 25.9 O C , [C]/[P] = 0.10, [AH-] z 1 x mol dmP3. pH 7.0 pH 6.3 [H202],/mol dm-3 k F e o b e t . k F e D k F e L 1.ox 10-2 442.3 153.6 70.9 13.6 1.1 x 10-3 408.8 129.2 1.1 x 10-4 405.1 156.1 5 9 . 2 10.6 5.4x 10-5 47 1 .O 140.7 84.0 16.1 - - av . 431.8+30.6 144.9+ 10.8 71.4+ 10.1 13.4 2.0 The oxidation of L-ascorbate anion was also studied as a function of the initial concentration of hydrogen peroxide, in the range 0.5 < [H,O,],/[AH-], < 100.The results obtained at pH 6.3, which are entirely consistent with those observed at pH 7,' show that with increasing [H202] the slopes of the straight lines remain practically constant within experimental error (table 2), whilst the intercept values increase. This implies that the second-order rate constants of the catalytic oxidation of ascorbate anion is independent of [H202], at variance with the rate of the uncatalysed reaction. The dependence of k , on [H202],, at [H202],/[AH-], z 1 and pH 6.3, is illustrated in fig. 4. 68 FAR 12090 STEREOSPECIFIC EFFECTS I N ELECTRON TRANSFER On the basis of these results, the following empirical rate expression may be (1) where the second-order rate constant k,, app (dm3 mol-l s-l), which can be evaluated from the slope of the straight lines of plots of k , against [H2O2l0 (see fig.4), is a complicated function of [C]/[P]l as well as of [H+], as illustrated in table 3. formulated : d[AH-] dt --- - ko, app [AH-] [H202l+ kcat [AH-] [CI 30t ---- 10 0 5 10 [HzOzlo/ 10- mol dm-3 FIG. 4.-Dependence of pseudo-first-order rate constant k, of the uncatalysed oxidation of L-ascorbate anion on the initial concentration of hydrogen peroxide, in the presence of FeL (0) or FeD (A) systems ([C]/[P] = 0.10). [H,O,],/[AH-1, z 1, T = 25.9 O C , pH 6.3 (0.04 mol dmP3 phosphate buffer). TABLE 3 .-APPARENT SECOND-ORDER RATE CONSTANTS (dm3 m0l-l S-l) FOR THE UNCATALYSED OXIDATION OF L-ASCORBATE ANION.T = 25.9 O C , [H,O,],/[AH-1, z 1 . PH 6.3 0.10 178.3 93.3 7.0 0.10 230.6 157.0 7.0 0.20 287.8 156.0 Eqn (l), which is formally similar to that reported in our previous paper,l suggests that the rate-determining step of the uncatalysed reaction involves one molecule of substrate per molecule of hydrogen peroxide (which does not necessarily imply a two-electron step),g whereas that of the catalytic process occurs between one molecule of complex ion and one molecule of substrate. In the latter case, oxidation of both the lower-valence metal chelate and the ascorbate radical takes place in subsequent fast steps. A '- may also disproportionate very rapidly.1° These results are reminiscent of those obtained by other authors for the same reaction in acid solutions, catalysed by diverse iron(m) l1 Two further findings are worth mentioning.First, the activation energy of the catalysis is seen: (i) to vary as a function of pH following a sigmoid trend, like that shown by the stereoselectivity factor under the same experimental conditions, and (ii) to exhibit equal values with both enantiomeric catalysts, within experimental error (fig. 5). According to circular dichroism data, the helix content of poly(L-glutamate) in FelI1 chelate solutions ([C]/[P] = 0.10) at 16 O C does not practically differ from that evaluated at 26 OC, other experimental conditions being equa1.12 The variation of activation energies as a function offh may therefore be safely ascribed to someM.BARTER1 AND B. PISPISA 209 1 1250 1 0 5 6 7 0 PH FIG. 5 . 0 0.5 1.0 f h FIG. 6. FIG. 5.-Variation of the activation energies of the catalytic oxidation of L-ascorbate anion with pH. The different symbols refer to the enantiomeric catalysts ([C]/[P] = 0.10). FIG. 6.-Dependence of the difference in standard free energies of the diastereomeric transitions states on the amount of a-helix in the polypeptide matrices (see text); T = 25.9 O C . change in the electron-transfer mechanism with conformational features of the polypeptide matrices. In addition, stereoselectivi ty appears to be a process controlled by activation entropy, as already found in the study of the same reaction at fixed pH (7.0) and varying [C]/[P].l Secondly, the difference in the standard free energies of the diastereomeric transition states (G,Z, - GiL) is observed to increase with increasing a-helical fraction of the supports (fh), as shown in fig.6. The difference (GZ - GgL) was evaluated by the expression1 LL which relates the second-order rate constants of the electron-transfer reaction in FeD-L-ascorbate (DL) and FeL-L-ascorbate (LL) diastereomeric systems, on the assumption of ideal behaviour owing to the very low concentration of the reacting species. The data of fig. 6 indicate that stereoselectivity is closely connected with the ordered structure of the support in the sense that on increasing the amount of helical polypeptide the structural characteristics of the catalyst probably allow the chiral polymer residues to participate in the formation of the precursor complex.The local stereochemistry would be then such that the LL diastereoisomer experiences larger steric hindrances than does the DL diastereoisomer, which is responsible for a less efficient electron-transfer pro~ess.~’ 13* l4 Finally, according to the data reported in tables 1 and 3, the overall rate of the reaction varies as a function of pH in a rather complicated way. For sake of simplicity, we neglect here the uncatalysed process and focus attention on the variation of kcat with pH. 68-22092 STEREOSPECIFIC EFFECTS I N ELECTRON TRANSFER In principle, two concurrent factors may account for the observed dependence of k,,, on [H+], i.e. (i) the competition between non-stereospecific and stereospecific pathways in the overall catalytic cycle because two routes have different rate constants,l and (ii) the diverse reactivities of the ionic species of ascorbic acid toward the catalysts employed.6 By analogy, with the expression describing the dependence of kcat on [C]/[P],l the first effect is empirically describable as follows: kcst = k’f([H+I) + k”( 1 -f([H+I)) (3) where k’ is the specific rate constant at some low value of [H+] (say, ca.1 x 1 O-* mol dm-3), where the non-stereospecific catalysis predominates, k” is the rate constant at [H+] z 1 x lop6 mol dm-3, where kcst exhibits an asymptotic value and stereoselectivity approaches the maximum value, and f([H+]) is a function of hydrogen-ion concentration such that it tends to 1 or 0, respectively as [H+] approaches the former or latter value.On the other hand, since ascorbic acid (AH,) undergoes the following protolytic K , K2 equilibria : (4) and both the neutral and ionic forms can be oxidized, in principle, by the iron(rI1) derivative, the rate law of each catalytic pathway may be expressed as: AH, e AH- e A,- catalytic rate = [C] (k,[AH2] + k2[AH-] + k,[A2-]) ( 5 ) where k,, k, and k, are constants for the catalytic effect of the metal chelate on the un-ionised, monoionic and bi-ionic forms of ascorbic acid, respectively, on the assumption that the three substrate species react independently with the catalyst. Taking into account the equilibium constants in equilibrium (4), mass balance allows us to write eqn ( 5 ) as follows:1s [SI [Cl k, K, K, + k, K,[H+] + [H+I2 catalytic rate = K, K2 + K,[H+] + [€I+], where [S] denotes the total substrate concentration ([S] = [AH,] + [AH-] + [A2-]).Even simplifying eqn (6), by assuming that the concentration of AH, is negligibly small as compared with the others in the pH region l6 the concomitant occurrence of the effects described by eqn ( 3 ) and (6) emphasizes the difficulty in interpreting the change of kcat with pH. Additional complications arise not only from failing to have pK values of the substrate in the domains of the charged polymeric catalysts (owing to the fact that protolytic equilibria of ionic species in proximity to macroions are perturbed by the high electrostatic potential of the p o l y e l e ~ t r o l y t e ) ~ ~ - ~ ~ but also from having insufficient data within the range of pH explored.All these features make it possible, at present, to draw only qualitative conclusions on the dependence of the rate of oxidation of ascorbic acid on pH. The reaction is sensitive to [H+] in terms of rate constant as well as stereoselectivity, the former increasing and the latter decreasing as a function of pH. In addition, both effects were found to be more pronounced in the catalytic process than in the parallel uncatalysed reaction. Further study in this area is clearly required. DISCUSSION Evidence is produced that stereospecific effects in the catalytic oxidation of ascorbate anion are coupled with the amount of a-helix in the polypeptide matrices, which in turn depends on pH. At the same time, the activation energy is shown toM. BARTER1 A N D B.PISPISA 2093 increase with a sigmoid trend and to exhibit equal values with both enantiomeric catalysts. These results are suggestive of different routes to products, depending on the structural characteristics of the catalytic systems used. At or above pH 7, the negatively charged polymeric support repels the anionic substrate molecules, which may thus interact with the catalyst only through the bound, positively charged metal chelate. Under these conditions, the accessible axial position of the ligated complex ions would provide the electron-acceptor site,6- 16-21 and the transfer of one electron may occur directly between the two species through the bridge. A direct-attack mechanism by the substrate on the central metal ion has been proposed by us1 under conditions where similar structural features of the catalytic systems were matched, i.e.at pH 7 and very low [C]/[P]. Accordingly, this mechanism is expected to be coupled with a low stereoselectivity in that the degrees of rotational freedom of the diastereo- merically related transition states should be high, owing to the size of substituent in the substrate and the lack of assistance from the polypeptide in the formation of the intermediate. This is indeed the case, as experimentally observed (table 1 and In addition, a direct electron-transfer process between ascorbate and iron(1rr) ion within a mixed-ligand metal chelate should require a low activation 2 2 as observed in neutral or weakly alkaline solution (fig. 5). In contrast, the catalytic reaction in acid solution shows both high stereoselectivity and high activation energy.This suggests a different electron-transfer route to the products, probably owing to the diverse stereochemistry of the precursor complex. With increasing [H+] the negative charge density of the polypeptide matrix decreases, so that extensive interactions between substrate molecules and the ordered polymeric catalyst are expected to occur. For instance, hydrogen-bonding interactions between hydroxy groups of the substituent in ascorbate and y-carboxylate groups in the side-chains of the polymer may take place, leading to a much more rigid Michaelis adduct than that seen above. Such a loss of conformational mobility should enhance the difference in steric hindrances between the two diastereomeric transition states, which must be reflected in an increase of stereospecific effects.13 This is indeed the case, as shown in table 1 and fig.3 and 6. Furthermore, molecular models suggest that the stereochemistry of this type of adduct allows electron transfer from ascorbate to iron(m) ion to proceed only by a remote-attack mechanism, possibly through the peripheral quaterpyridine ligand of the active sites. This hypothesis, which is reminiscent of that proposed for the reduction of ferriporphyrins by ascorbic acid16 and for a number of redox reactions between metalloproteins and various reduc- tants,22 25 may account for the relatively high activation energy in acid solution. We may therefore conclude that there is a close relationship between stereochemical characteristics of the precursor complex and both mechanism and kinetic stereo- selectivity of the electron-transfer catalysis. All these features depend markedly on pH because it controls the structure of the chiral polymeric support, which appears to be primarily responsible for the observed phenomena.As far as the parallel uncatalysed oxidation of t-ascorbate anion is concerned, it was shown that with decreasing hydrogen-ion concentration the specific rate increases (tables 1 and 3 ) . By analogy with the catalytic reaction, this trend may be explained in terms of different rate constants for the oxidation of the ionic species of ascorbic acid (see above). Nevertheless, we are inclined to think that an additional, more subtle, factor connects the rate constant of the uncatalysed process with pH.It has been reportedly 2 * * that coordination of [Fe(tetpy)(OH),]+ ions by y-carboxylate groups of the side-chains of the polymer leads to the formation of positive ionic sites on the polypeptide because the ligated molecules keep the univalent positive charge of the free fig. 3 ) .2094 STEREOSPECIFIC EFFECTS I N ELECTRON TRANSFER chelate ions. Coulombic interactions between ascorbate molecules and the positively charged polymeric material are thus expected to take place, the effect being more pronounced the higher the pH because the degree of counter-ion association As a result, the specific rate of the uncatalysed oxidation of ascorbate anion is enhanced (table l), in agreement with the fact that the rate of ionic reactions is raised by polyions bearing fixed charges opposite in sign to those of the reacting 27 This hypothesis may also account for the fact that stereospecific effects in the uncatalysed reaction are definitely smaller than those in the true catalytic process.The formation of a Michaelis adduct as a preliminary step in the catalysis implies a more intimate contact between the asymmetric partners than that which one would predict on the basis of long-range Coulombic interactions alone. This should be reflected in a much larger difference between the standard free energies of the diastereomeric transition states, as experimentally observed (tables 1 and 3). In conclusion, although at present the pH dependence of the reaction can be described only qualitatively because of insufficient experimental data and quantitative knowledge of pK values of ascorbic acid in complex-polyelectrolyte solutions, the overall results are entirely consistent with a picture in which the complex-polypeptide system behaves not only as an efficient and stereospecific electron-transfer catalyst but also as an environmental controller of the uncatalysed oxidation of ascorbate anion.We thank the Italian National Research Council (C.N.R.) for partial financial support. M. Barteri and B. Pispisa, J. Chem. SOC., Faraday Trans. I , 1982, 78, in press. M. Branca and B. Pispisa, J. Chem. SOC., Faraday Trans. I , 1977, 73, 213. M. Branca, B. Pispisa and C. Aurisicchio, J. Chem. 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