Oxidation of L-thiols in the presence of iron (III) complex ions anchored

J . Phys. Chem. 1988, 92, 3422-3429

3422

Oxidation of L-Thiols in the Presence of Iron(II1) Complex Ions Anchored to Asymmetric Polymers. A Kinetic and Conformational Investigation B. Pispisa,*,t G. Paradossi,t A. Palleschi,t and A. Desiderig

Downloaded by GEORGETOWN UNIV on August 22, 2015 | http://pubs.acs.org Publication Date: June 1, 1988 | doi: 10.1021/j100323a023

Dipartimento di Chimica, Universitci di Napoli, 801 34 Napoli, Italy, Dipartimento di Chimica, I Universitri di Roma. 00185 Roma, Italy, and Dipartimento di Biologia, II Universitci di Roma, 001 73 Roma, Italy (Received: August 4, 1987: In Final Form: December 30, 1987)

The H202oxidation of L-cysteine in the presence of enantiomeric systems formed by [Fe(tetpy)(OH),]+ (FeT) ions anchored to ordered poly(o-glutamate) (FeTD) or poly(1-glutamate) (FeTL) matrices has been studied at pH 7 (tetpy = 2,2’,2’’,2’’’-tetrapyridyl). The reaction follows a total third-order kinetics (kox= 6.1 X lo4 M-2 s-l, 26 “C) and does not exhibit any stereoselectivity. These results markedly differ, in terms of both rate law and chiral discrimination, from those obtained under similar conditions by using optically active o-dihydroxy substrates, such as L-ascorbic acid, L-adrenaline, and L-dopa. They also differ from those obtained with reduced L-glutathione, which follows a total second-order kinetics ( k = 6.4 M-’ s-’, 26 “ C ) . In the absence of hydrogen peroxide, evidence is produced for the formation of a stable, polymer-supported, Fe”’T-cysteinate complex, to which no stereoselectivity is associated because of the high conformational mobility of the axially bound substrate. This complex is characterized by electron paramagnetic resonance (EPR) signals (10 K) at g = 2.20, 2.15, and 1.92 and by a rather slow, [H+]-dependent kinetics of formation ( k = 12.7 M-’ SKI, 26 “C) that are suggestive of a ligand-exchange process at the polymer-shielded active sites, within an outer-sphere compound involving the entering substrate molecule. Conformational energy calculations on the noncovalent diastereomeric adducts show the absence of a sterically preferred pathway for the closest approach of cysteine to the central metal ion and support the hypothesis of the ligand-interchangereaction on steric and geometric grounds. Implicationsof these effects on the absence of stereoselectivity in the reaction investigated are briefly discussed.

Introduction The oxidation of thiols to disulfides is an important biological process, and the role of metal ions in the reactions of sulfhydryl-containing substrates is of significance in view of the nature of electron-transport enzymes, which frequently incorporate such a group. In this regard iron(II1)- and iron(I1)-thiolate complexes are of particular current interest because they represent possible models for heme iron enzymes, such as cytochrome P-450,’-4or for nonheme iron proteins containing iron-sulfur linkage^.^.^ Available evidence indicates that five- or six-coordinate Fe(II1) porphyrin (P) derivatives, containing a thiolate species (RS), may be inherently unstable by virtue of the redox reaction 1 or 2, in 2Fe”’P(SR) 2Fe”’P(SR)L

+ 2L

+ RSSR 2Fet1PL2+ RSSR

2Fe”P

-

-+

(2)

which the initial process is presumably intramolecular reduction of iron(II1) by bound t h i ~ l a t e .These ~ ~ reactions may be preventable by hindering radical coupling,3b e.g., through immobilization of the iron(II1)-thiolate complex on a polymeric matrix and storage as solvent-free solid,2aor by lowering the temperature below -60 oC.4b A similar redox process seems to take place by using Fe3+ions under acid condition^.'.^ The final products are thought to originate from a dimerization step yielding a binuclear Fe(I1I)-thiol intermediate,* where the iron center acts as a concerted two-electron oxidant in the proximity of two sulfiydryl groups, or from a binuclear Fe(I1)-thiyl radical adduct9 that forms after intramolecular electron transfer within the mononuclear complex has occurred. In the past few years we were interested in stereoselective oxidation of chiral ortho-dihydroxy compounds, such as L(+)ascorbic acid, L-dopa, and L-adrenaline, catalyzed by Fe(II1) complex ions anchored to Using [Fe(tetpy)(OH),]” ions (FeT; tetpy = 2,2’,2”,2”’-tetrapyridyl) bound to sodium poly(D-glutamate) (FeTD) or poly(L-glutamate) (FeTL) as enantiomeric catalysts, we have found that the reaction proceeds stereoselectively only when structurally ordered and partially *To whom correspondence should be addressed at Dipartimento di Chimica, I1 Universiti di Roma, Torvergata, 00173 Roma, Italy. ‘UniversitB di Napoli. Dipartimento di Chimica, I Universiti di Roma. Dipartimento di Biologia, I1 UniversitB di Roma.

*

0022-3654/88/2092-3422$01.50/0

shielded active sites prevent easy approach for substrate molecules. The hindered accessibility of reactive centers makes the chiral residues of the helical polypeptide behave as primary sites of binding for substrate molecules, thus ensuring different steric constraints that affect the binding properties and structural features of diastereomeric adducts differently. The ultimate result is a chiral discrimination in the catalysis, corresponding to an enantiomeric excess as high as 65%. Extension of this investigation to optically active thiols, such as L-cysteine (eq 3, where RS- denotes S C H 2 C H N H 2 C 0 Fspecies 2RS-

+ H202

FeTD

RSSR

+ 20H-

(3)

and RSSR denotes cystine), seemed to us interesting to examine whether the structural and electronic factors’, that produce stereochemical control in the oxidation of the dihydroxy compounds are still operative when a quite different substrate is used. In addition, the role of hydrogen peroxide in the oxidation of thiols ~~~~

~~~

~~

(1) Cytochromes P-450;Sato, R., Omura, T., Eds.; Academic: New York,

1978. (2) (a) Collman, J. P.; Sorell, T. N.; Hoffman, B. M. J . Am. Chem. SOC. 1975, 97, 913. (b) Collman, J. P.; Groh, S. E. Ibid. 1982, 104, 1391. (3) (a) Koch, S.; Tang, S. C.; Holm, R. H.; Frankel, R. B.; Ibers, J. A. J . Am. Chem. Soc. 1975, 97,916. (b) Tang, S. C.; Koch, S.; Papaefthymiou, G. C.; Foner, S.; Frankel, R. B.; Ibers, J. A,; Holm, R. H. Ibid. 1976, 98, 2414. (4) (a) Chang, C. H.; Dolphin, D. J . Am. Chem. SOC.1975, 97, 5948. (b) Ruf, H. H.; Wende, P. Ibid. 1977, 99, 5499. (c) Kau L. S.; Svastits, E. W.; Dawson, J. H.; Hodgson, K. 0. Inorg. Chem. 1986, 25, 4307. (5) Averill, B. A,; Orme-Johnson, W. H. In Metal Ions in Biological Systems; Sigel, H., Ed.; Dekker: New York, 1978; Vol. 7, Chapter 4. (6) Holm, R. H. In Biological Aspects of Inorganic Chemistry; Addison, A . W . , Cullen, W. R., Dolphin, D., James, B. R., Eds.; Wiley: New York, 1977; p 71. (7) (a) Leussing, D. L.; Mislan, J. P.; Goll, R. J. J . Phys. Chem. 1960, 64, 1070. (b) Stadtherr, L. G.; Martin, R. B. Inorg. Chem. 1972, l J , 92. (8) Lappin, A. G.; McAuley, A. J . Chem. SOC.,Dalton Trans. 1975, 1560. Ellis, K. J.; Lappin, A. G.; McAuley, A. Ibid. 1975, 1930. (9) (a) Hamed, M. Y.; Silver, J.; Wilson, M. T. Inorg. Chim. Acta 1983, 78, 1. (b) Jensen, P.; Hamed, M. Y . ;Wilson, M. T.; Silver, J. Ibid. 1986, 125, 17. ( I O ) (a) Barteri, M.; Pispisa, B. J . Chem. Soc., Faraday Trans. 1 1982, 78, 2073. (b) Ibid. 1982, 78, 2085. Makromol. Chem., Rapid Commun. 1982, 3, 715. ( 1 1) Pispisa, B.; Palleschi, A. Macromolecules 1986, 19, 904. Pispisa, B.; Palleschi, A,; Paradossi, G. J . Mol. Catal. 1987, 42, 269. (12) (a) Pispisa, B.; Palleschi, A,; Barteri, M.; Nardini, S. J . Phys. Chem. 1985, 89, 1767. (b) Pispisa, B.; Palleschi, A,; Paradossi, G . Ibid. 1987. 91, 1546.

0 1988 American Chemical Society

Oxidation of L-Thiols

[H2O2I0was also linear, and the third-order specific rate (k,,, M-2 s-') was obtained from the slope. The rate constant of the "uncatalyzed" oxidation of cysteine (k,, M-I s-]) was obtained from a plot of kfo (s-I) as a function of [H20,], (see later). Independent measurements on the H202oxidation of cysteine, under second-order conditions ([H202]/[RSH] 1; 26 "C, pH 7, 0.05 M Tris buffer), gave a specific rate constant of 1.5 f 0.1 in excellent agreement with ko = 1.5 f 0.2 M-' SKI(Table M-I SKI, 11). Addition of sodium poly@-glutamate) or pOly(D-glUtamate) did not practically affect the rate of this reaction. Furthermore, blank experiments showed that autoxidation of cysteine in doubly distilled (trace-metal free) water and in the absence of H202is an extremely slow process (pH 7).lSa It seems likely therefore that intervention of adventitious metal ions and coordinating buffer effects have sometimes led to improper mechanistic interpretations of cysteine autoxidative reaction. A set of runs were also carried out in the absence of H202,but Experimental Section in aerobic conditions, to examine the nature of the intermediate (see later). These experiments were performed by mixing FeTL Materials. (Quaterpyridine)iron(III) complex ions, sodium or FeTD and substrate buffered solutions at 26 "C into the optical poly@-glutamate), and sodium poly@-glutamate) were obtained cell. The range of pH investigated was 7-8. The kinetics of as already described.I6 L-Cysteine (Sigma) and stabilizer-free formation of the intermediate complex, under pseudo-first-order H202(Erba) were analytical-grade reagents and used without conditions with respect to cysteine, were followed at 327 or 550 further purification. Concentrations of complex ions and polymers nm, the results being quite comparable at both wavelengths. For were determined by UV absorption,I0 that of polymer [PI being instance, at [C] = 2.8 X M ([C]/[P] = 0.20), [RSH], = referred to monomeric unit (monomole per liter). Concentrations 4X M, pH 7 (0.05 M Tris buffer), T = 26.0 f 0.1 "C, kfobsd of cysteine and cystine (the disulfide product of oxidation) were = 0.97 X and 1.08 X s-' at 327 and 550 nm, respectively, = 246 nm, emax = 76 and 335 M-l determined at 250 nm (A,, the observed rate constants ( k L b d ) being obtained by plots of log cm-I, respectively). Formation of cystine was also checked by ( A , - A , ) versus t , which were linear over more than 2 half-lives. HPLC measurements (cystine blank from Sigma). Tris buffer Within the range of substrate concentration explored plots of kLw (Sigma) was employed in the chloride form and in concentration against [RSH], show a good linearity at each pH investigated of 0.05 M. All measurements were carried out on freshly prepared and second-order rate constants k' (M-I SKI)for the formation solutions, with use of doubly distilled water. of the intermediate complex were obtained from the slopes (Table Methods and Apparatuses. The stoichiometry of eq 3 was 111). M catalyst checked by titration of 6 X 10-3 M cysteine in 1 X A number of experiments were also carried out on the H 2 0 2 solution with H 2 0 2 ,by monitoring the absorbance at 250 nm due to cystine. As a result, the mean of [cysteine]reancd/[H202]mnsumcd oxidation of L-glutathione (Sigma). The formation of oxidized L-glutathione (the 'disulfide product) was monitored at 250 nm was 1.9 f 0.2, suggesting a 2:l stoichiometry for the reaction. ( E = 334 M-' cm-I) and was further checked by HPLC meaKinetic experiments measuring the formation of cystine were surements. The experimental conditions were quite similar to those carried out under a variety of conditions and at fixed pH (pH 7, reported above for cysteine, but the second-order rate constants 0.05 M Tris buffer). A typical run consisted of adding hydrogen (k,,,, M-I s-,), as obtained by plots of k,, (s-') against complex peroxide by a microsyringe into the 1-cm optical cell containing concentration at fixed [C]/[P] = 0.20 and varying [H2O2Io,were 2 mL of catalyst and substrate, both systems being thermostated found to be [H202]independent within experimental errors. at 26.0 f 0.1 "C. The experimental conditions were as follows: Absorption measurements were carried out on a Beckman [ H 2 0 2 J 0= 1 X 10-3-1 X M, [RSH], = 4 X M (unless DU-50spectrophotometer, and circular dichroism spectra were otherwise stated), [C] = 0.5 X 10-5-4 X M, [C]/[P] = 0.20, recorded on a Cary 61 or a Jasco 5-500 A instrument with apthis latter being the complex to polymer ratio of the enantiomeric propriate quartz cells. HPLC measurements were performed by catalysts corresponding to the highest stereoselective activity in a Varian SO00 apparatus, with a 50-cm Micropak TSK-SW the oxidation of dihydroxy derivatives.lOJ1(RSH, C, and P denote column and a refractive index detector. cysteine, complex ions, and polypeptides, respectively.) MeaEPR spectra were recorded at X-band and 10 K on a Varian surements were also carried out at 12 "C without practically E 9 spectrometer, with an Air Products and Chemicals LT-3-110 affecting the a-helical content in the polymeric matrices.Iob Plots liquid-transfer Cryo-Tip refrigerator with automatic temperature of log ( A , - A,) versus t were normally linear over more than 85% controller. of the reaction progress, and the observed rate constants (kobsd, Other apparatuses were already described.lo-l2 s-I) were obtained from the slopes. At least 3 (and sometimes as many as 10) kinetic measurements were performed for each Results and Discussion run to obtain consistency in the results. At each [ H 2 0 z l oinvestigated, plots of k&, against complex concentration always Structural Features of Catalysts. The structural features of gave straight lines and pseudo-second-order rate constants ( k , M-l the enantiomeric FeT-poly(L-glutamate) and FeT-poly(Ds-l) were obtained from the slopes. Finally, a plot of k against glutamate) systems have already been reportedI2 and may be summarized as follows. (1) (Quaterpyridine)iron(III) ions form an inner-sphere complex with the polyelectrolytes (pH 7), in which (13) Cavallini, D.; De Marco, C.; Dupre, S.;Rotilio, G. Arch. Biochem. the coordination of an apical site of FeT by a y-carboxylate group Biophys. 1969,130, 354. Zwant, J.; Van Wolput, J. H. M. C.; Koningsberger, of side chains of the polymer occurs. ( 2 ) At high complex to D. C. J . Mol. Catal. 1981, 12, 85. (14) (a) Saez, G.; Thornalley, P. J.; Hill, H. A. 0.;Hems, R.; Bannister, = 0.20, the polymeric matrix polymer-residue ratio, e.g., [C]/[P] J. V. Biochim. Biophys. Acta 1982, 719, 24. (b) Motohashi, N.; Mori, I. J . is predominantly in a a-helical conformation even in neutral Inorg. Biochem. 1986, 26, 205. Florence, T. M. Ibid. 1984, 22, 221. solution'6band forms a three-dimensional network where most (15) (a) Winterbum, C. C . Biochem. J . 1981,198, 125. Biochem. J . Lett. of the iron molecules act as bridging groups between helical 1982, 205, 463. (b) Gutteridge, M. C.; Richmond, R.; Halliwell, B. Biochem. segments of different chains or of the same chain after partial J . 1979, 184, 469. Tien, M.; Bucher, J. R.; Aust, S.D. Biochem. Biophys. Res. Commun. 1982, 107, 279. folding. (3) The stereochemistry of these active sites is such that (16) (a) Branca, M.; Pispisa, B.; Aurisicchio, C. J . Chem. Soc., Dalton the oxygen atoms of the side chains of the polymer closest to Trans. 1976, 1543. Cerdonio, M.; Mogno, F.; Pispisa, B.; Vitale, S.Inorg. Fe(1II) lie on the axis normal to the equatorial plane of the Chem. 1977, 16, 400. (b) Branca, M.; Pispisa, B. J . Chem. Soc., Faraday Trans. 1 1977, 73, 213. complex, at a separation distance of 2.34 (0,)and 2.50 8, (0,) has been investigated, and the effect of added metal ions has been ~ t u d i e d ' ~especially *'~ in view of the metabolic activity of the reactive intermediates in a number of biological processes,1s but little attention has been so far payed to the environmental control of the reaction by macromolecular ensembles. Contrary to expectation, L-cysteine does not undergo a stereoselective oxidation by the foregoing enantiomeric catalysts. Our main goal was therefore that of understanding why in this case the polymeric matrix fails to control chiral discrimination and also of examining the interacting systems by conformational energy calculations. We also produce evidence for the formation of a polymer-bound Fe"'T-SR intermediate with unusual stability (in the absence of H202) at room temperature. Finally, we shall present a few results on the oxidation of L-glutathione to disulfide, showing how the bulkiness of this three-peptide-like substrate leads to a different pathway to the reaction products.


The Journal of Physical Chemistry, Vol. 92, No. 12, 1988 3423

-

Pispisa et al.

3424 The Journal of Physical Chemistry. Vol. 92. No. I2, I988

10'ElIMI

Figure 2. Dependence of the observed first-order specific rates for the oxidation of L-cysteine(3 X 10.' M), in the presence of an FeTL (open symbols) or FeTD (solid symbls) enantiomeric system at [Cl/[P] = 0.20, on the concentration of bund complex ions at varying initial concentrations of hydrogen peroxide: [H2021a= 1.0 X lWzM (triangles), 2.8 X I F ' M (squares), and 0.9 X IO-' M (circles); T = 26 'C, pH 7, 0.05 M Tris buffer.

TABLE I: Dependence of Slopes and Intercepe of the Straight Lines = k[C] + kb (Figure 2) on Hydrogen Peroxide Concentration" IO'[H,OiIo, M k, M-' s-' 103k6,8-1


,k

no

47

..

17

1.9 2.8 3.7 7.0 10.0

120 173 223 430 665

2.5 3.7 5.2 10.8 16.2

...

'FeTL or FeTD system ([C]/[P] = 0.20), 26 OC, pH 7,0.05 M Tris buffer. Flare 1. Molecular model of structurally ordered FeT-poly@glutamate) system (see text). The oxygen atom of the side chains of the polymer closest to Fe(II1) are indicated as 0, and 02. Two types of active sites are shown, in agreement with Mhsbauer data,'lb those exposed to bulk solvent bcing presumably around 30%. from the central metal ion (Figure (4) Under the same conditions, a minor portion of bound FeT ions are, in contrast, exposed to the bulk solvent.lZb It is worth recalling"" that where the oxidation of dihydroxy compounds (L-ascorbic acid and L-catecholamines) is performed with the enantiomeric catalysts a t high [C]/[P] ratio, the active sites capable of stereoselectivereaction are those FeT ions "buried" into the ordered polypeptide matrix. This is because their hindered accessibility requires a polymer-assisted pathway for the formation of catalystsubstrate complexes that ensures an efficient sterically discriminating environment. It would be possible, however, that the "distal" oxygen atom (0,)of these sites undergoes a ligandexchange reaction under suitable conditions. While this process should basically retain the helical conformation of the polymeric matrix because it is stabilized by the still bound complex ions through the "proximal" oxygen atom 0,'" (Figure I), the breaking of the steric arrangement around the active centers should lead to a loss of stereoselectivity. This is very likely the case with cysteine, because of the small dimensions of the molecule as compared to those of the aforementioned dihydroxy compounds and the tendency of the mercaptosulfur donor atom to bind fairly strongly to polymer-supported FeT ions, as shown below. Kinetics of Oxidation. Typical data of H,O, oxidation of L-cysteine (eq 3) at pH 7 are presented in Figure 2, where the observed pseudo-first-order specific rates (kOw,SKI)are plotted as a function of complex concentration [C], at fixed complex to polymer-residue ratio [C]/[P] = 0.20 and different initial concentrations of H202(26 "C). Under the experimental conditions used, both the intercepts and slopes of the straight lines in the figure (kaM = kb + k[C]) depend on hydrogen peroxide concentration, as shown in Table I. From the results, the following empirical rate expression may be formulated1' -d[RS-]/Z df = ko,[C][RS-][H,O,] + ko[RS-][H202] (4) (17) The ionized form of cysteine (RS-)is used in cq 4 in anticipation of pH, shown in the next section.

the findings obtained as a function of

which indicates the Occurrence of parallel pathways. One of these (ko, M-' S-I)refers to the oxidation of cysteine by H,O, in bulk solution and the other (kOx,M-, sC1) to a "catalytic", [H202]dependent route to products, whose ratedetermining step involves one molecule of both substrate and complex ion per molecule of hydrogen peroxide. In addition, no stereoselectivity is observed, since k,. = 6.1 X IO' M-,sC1 for both DL and LL diastereomeric reactions (Table 11). These results differ markedly from those obtained under similar conditions in the H202oxidation of L-ascorbic acid, L-dopa, and L-adrenaline (AH-) in terms of both rate law (en 5 ) and stereoselectivitylOJt (Table 11).

-d[AH-]/dt

= k,,,[C][AH-]

+ ko[AH-][H202]

(5)

A termolecular process is rare, so that oxidation of cysteine should be a multistep reaction where a stable FeT-thiolate adduct very likely forms in a preliminary step. W e then investigated the formation of such a n intermediate, which would also shed light on the absence of stereoselectivity in eq 3. Intermediate Complex: Kinetics and Spectroscopy. When neutral solutions of excess L-cysteine and FeTL or FeTD system ([C]/[P] = 0.20) are mixed together, a pink-red color rapidly appears. A typical spectrum of the mixture, after equilibrium has been attained, is shown in Figure 3 together with the spectrum of the catalyst. The insert in the figure illustrates the kinetics of the process. Two new bands a t 327 and 550 nm, together with a shoulder at around 500 nm, form, and several clear isosbestic points are evident. The observations that (1) the final spectrum is stable under aerobic conditions for more than 10 h (pH 7) and (2) the observed pseudo-first-order rate constants evaluated a t 327 and 550 nm are quite comparable (seeExperimental Section) lead us to conclude that spectrum h in Figure 3 pertains to a dead-end complex, possibly a polymer-bound Fe(II1)-thiolate species that forms according to the following equilibria:

c + RSH A cD+ H+ k+

(6)

k

C+RS-&CD k-r

(7)

where the active sites are abbreviated as C and the dead-end complex is abbreviated as CD,while RSH denotes HSCH2CH-



TABLE 11: Specific Rate Constants for the Catalytic and Complex Ion-Uncatalyzed Oxidation of Chiral Substrates by the Enantiomeric FeTD and FeTL Systems at ICl/IPI = 0.20" substrates

rate law eq eq eq eq

L(+)ascorbic acid L-adrenaline L-dopa L-cysteine

5 5 5 4

kLL, M-l s-'

kDL/kLLb

kODL,M-I s-I

414.0 f 33.4 106.4 f 9.2 31.2 f 2.1 7.5 f 0.5 31.4 & 2.1 7.9 f 0.4 (6.1 f 0.5) X

3.9 f 0.5 4.2 f 0.4 4.0 f 0.3 1

287.8 f 26.2 4.9 f 0.5

~ D L M-' , s-l

kOLL, M-I

s-l

156.0 f 14.8 3.2 f 0.3 10.3 f 0.8 6.5 f 0.6 1.5 f 0.2

kODL/koL; 1.8 f 0.2 1.5 f 0.2

ref 10

1.6 f 0.2 1

11 e

11

"26 OC, p H 7, 0.05 M Tris buffer. bStereoselectivity ratio of the catalysis. CStereoselectivity ratio of the complex ion-uncatalyzed oxidation of substrates. dThird-oider specific rate; at 12 OC: k,, = (6.7 f 1.0) X lo3 M-2 s-] (AH,w* = 26.0 f 1.0 kcal/mol). 'This work.

TABLE 111: Kinetic Data for the Formation of the Dead-End Complex between FeTL or FeTD System ([C]/[P] = 0.20) and L-Cysteine" pH 1O3[RSH],, M 103k6bsd,bs-I kt,C M-1 s-l 6.97

3.0 4.0 5.0 6.0 7.0 2.0 3.0 4.0


7.20

0.85 1.04 1.29 1.52 1.65 1.54 1.95 2.88 3.90 0.97 1.87 2.69 3.11 2.34 4.03 6.12 8.92 2.54 4.61 8.05

5.0 7.48

300

350

400

450

500

550

600

650

Figure 3. Absorption spectra of FeT-poly(g1utamate) system at [C]/[P] = 0.20 (curve a) and of cysteine-FeT-poly(g1utamate) mixture (b), after M, [cysteine] = 2 X equilibrium has been attained. [C] = 3.5 X M, p H 7, 0.05 M Tris buffer. Optical path length of 2 cm within the range of 300-400 nm and of 5 cm above 400 nm. Insert: Variation of the spectrum of the mixture as a function of time of mixing, under the same experimental conditions but with [C] = 2.8 X loT5M ( I = 2 cm). Curve, time (minutes): 1, 1; 2, 6; 3, 9; 4, 16; 5, 20; 6, 29; 7, 180.

- (k+[H+]

+ k-7)[C~]

(8)

where k ' = k6 + k7Ka/[H+]

(9)

and [RS-] = [RSH]K,/[H+], K, being the dissociation constant of the thiol group. At equilibrium d[CD]/dt = 0 and, under the conditions used, [RSH] = [RSH], and [C], = [C], + [cD],q, so that eq 8 may be written as d[CDl/dt = k'[RSHl[CIO(l - ([cDl/[cDleq))

(lo)

According to eq 9, a plot of k'against [H+]-' should give a straight line with a slope T = k7K, and intercept I = k6. This is indeed the case, with T = (6.0 f 1 . l ) X s-] and I = 0. The results indicate, therefore, that the un-ionized thiol group is totally inactive for the reaction, Le., that the observed specific rates apply entirely to the species SCH2CHNH2C02-(eq 7). Assuming pK, = 8.33,18 the rate constant for the formation of the dead-end complex is k7 = 12.7 f 2.4 M-l s-I (26 "C). Reaction 7 thus appears to be (18) Borsaok, H.; Ellis, E. L.; Huffman, H. M. J . Biol. Chem. 1937, 117, 281. Edsall, J. T.; Wyman, J. Biophysical Chemistry; Academic: New York, 1958; Vol. 1, pp 496-504. Benesch, R. E.; Benesch, R. J . Am. Chem. SOC. 1955, 77, 5877. Elson, E.; Edsall, E. Biochemistry 1962, I, 1. Wilson, E. W., Jr.; Martin, R. B. Arch. Biochem. Biophys. 1971, 142, 445.

1.o 1.5

2.0 0.5 1.o 1.5 2.0 0.5 1.o 1.5

7.82

7.95

0.73 f 0.06

1.79 f 0.17

4.31 f 0.31 5.02 f 0.38

M. bEach entry is the "26 "C, 0.05 M Tris buffer, [C], = 3 X average of at least three determinations. CEquation 9 (see text).

NH2C02-and RS- denotes -SCH,CHNH2C0; species of substrate. The results of kinetic experiments followed at 327 nm, within the pH range of 7-8, appear in Table 111. Since the spectral patterns and the observed rate constants are quite similar when using FeTL or FeTD system ([C]/[P] = 0.20), no stereoselectivity is associated with formation of the dead-end complex. Furthermore, a first-order dependence on [C] and an inverse dependence of initial reaction rates on hydrogen ion concentration are observed, so that the empirical rate law may be expressed in the form d [ C ~ ] / d t= k'[C][RSH]

0.5

0.26 f 0.02

-1 0 2

-0

0, '5,

--lo -20

200

220

240

260

280

300

320

340

Aim

Figure 4. Typical differential circular dichroism spectrum (curve a, left-hand ordinate) of L-cysteine-FeT-poly(L-glutamate) mixture, after equilibrium has been attained, against L-cysteine, and ellipticity (curve b, right-hand ordinate) of the original FeTL solution under the same experimental conditions: [C] = 2.5 X M, [C]/[P] = 0.20, [cysteine] = 1 X lo-) M, pH 7, 0.05 M Tris buffer. Optical path length normalized to 1 cm.

a rather slow process as compared to that in which thiolate species8 or other monodentate ligandsI9 bind to Fe(II1) ions (lo3-lo4 M-' s-'). This finding together with both the absence of stereoselectivity and the observation that the helical content of polypeptide in the equilibrium mixtures is close to that of the initial solutions of FeT-poly(g1utamate) systems at [C]/[P] = 0.2011*20 (Figure (19) Seewald, D.;Sutin, N. Inorg. Chem. 1963, 2, 643. Moorhead, E. G.; Sutin, N. Ibid. 1966, 5 , 1866. (20) The dichroic bands of the ordered polypeptide at 222 and 207 nm21 are somewhat lowered upon addition of L-cysteine (Figure 4a). Nevertheless, a conservative estimate of the a-helical fraction is heref, 0.5 as compared toyh 0.7 of the starting FeTL material at [C]/[P] = 0.20 and pH 7.16b Furthermore, since the extrinsic dichroic bands above 260 nm originate solely from the electronic transitions of the bound achiral FeT ions and are chiefly due to conformationally induced Cotton effects,16b,22 the ellipticity above this wavelength is also lowered but still indicates that the a-helical structure in the polymeric matrix is largely stabilized by the bound complex ions.

-

-

3426 The Journal of Physical Chemistry, Vol. 92, No. 12, 1988

4 28

I

2 fO


n

-

Figure 5. EPR spectra of FeT-poly(g1utamate) system a t [C]/[P] = 0.20 ([C] 2X M; curve a) and of cysteine-FeT-poly(g1utamate) mixtures ([cysteine] 8 X lo-) M, pH 7, 0.05 M Tris buffer). The

-

delay times between mixing a t room temperature and quench freezing were (b) 90 s and (c) 30 min. The spectra were taken at 10 K, microwave power 20 mW, and microwave frequency 9.21 GHz.

4) strongly suggests that the rate-determining (spectrophotometrically measurable) step of reaction 7 is a ligand-exchange process involving displacement of the "distal" oxygen atom (0,) in the buried active sites by the ionized thiol group of cysteine. Therefore, it is the rate of ligand exchange in the buried centers that at least partially determines the global kinetics, and the foregoing value of k7 only applies to eq 7 when this kind of sites is involved.23 W e next evaluated the equilibrium constant of eq 7 (K7) by absorption measurements at 550 nm as a function of total cysteine concentration [RSHIo, after equilibrium has been attained (pH 7, 0.05 M Tris buffer). At this wavelength the absorption of the catalyst is extremely small and, as a first approximation, may be From the results,24 esse = (3.9 f 0.3) X lo3 M-' cm-' and K7 = (1.8 f 0.3) X lo4 M-I, assuming pKa = 8.33.Is Then K6 = K7Ka = 8 X and k-7 = k7/K7 = (7 f 2) X low4 SI. Interestingly, the value of k-7 is close to that found for dissociation of monodentate ligands (including thiolate species) s - ' ) . ~ , ~This ~ gives further support to the from iron(II1) ( foregoing hypothesis and suggests that the pathway for the formation of the dead-end complex is similar to but much slower than the associative mechanism of monodentate ligands to Fe3+ N

(21) Myer, Y. P. Macromolecules 1969, 2, 624. (22) Branca, M.; Marini, M. E.; Pispisa, B. Biopolymers 1976, 15, 2219. Barteri, M.; Pispisa, B.; Primiceri, M. V. Ibid. 1979, 18, 3115. (23) The structural features of the FeT-poly(g1utamate) system at high [ C ] / [ P ]ratio, e.g., 0.20 (Figure l ) , make predictable the occurrence of a biphasic process for the formation of the dead-end complex. However, that involving the "exposed" active sites is too rapid to study by conventional spectrophotometric methods, requiring the use of stopped-flow technique^.^^'^ (24) From mass balance ([RSH], = [RSH] + [RS-] + [C,]) one can easily obtain the following expression:

where A is the absorbance at 550 nm,c the molar extinction coefficient of the dead-end complex, [C], the total complex ion concentration, and I the optical path length ( A = cl[CD]). A plot of ([RSH],[C],)A-' against ([RSH], [C],), at fixed [C], and [H*], gives therefore a straight line that allows us to determine K7 and c.

+

Pispisa et al. ions, in which the rate-determining step is the loss of the "distal" carboxylic group instead of a water molecule.19 This idea is consistent with the finding that steric hindrances dramatically slow down ligand-interchange reaction^,,^ and it even better applies to the buried centers if they are viewed as basically formed by a five-coordinate [N40Fe"'] unit, owing to the rather long Fe-0, bond distance (2.50 A, Figure 1). On the other hand, the expected lability of the cysteinate-Fe(II1) complex and its reactivity in side processes involving intermediate species of the very slow spontaneous o ~ i d a t i o n l of ~ ~excess J ~ ~ cysteine in bulk solution (pH 7 ; see Experimental Section) are very likely depressed by virtue of the environmental effects of the polypeptide.2a Finally, we examined the formation of the dead-end complex by EPR spectra at 10 K. Typical spectral patterns are presented in Figure 5 . The initial solution of substrate-free FeT-poly(glutamate) system ([C]/[P] = 0.20, pH 7) shows only a signal at g = 4.28, which is characteristic of high-spin (S = 5 / 2 ) Fe(II1) species with rhombic distortion.26 (No other resonance was detected in the region 1000-2000 G or elsewhere where a signal from a S = 1 ' , system might be e ~ p e c t e d . , ~ ,Upon ~ ) addition of cysteine this signal is replaced with new signals at g = 2.20, 2.15, and 1.92, whose intensity increases with an increase of the time of mixing at room t e m p e r a t ~ r e ,and ~ reaches a maximum (constant) value after about 30 min (Figure 5c). A variety of low-spin ( S = I/,) [N,LFe"'S] centers with axial sulfur coordination exhibit rhombic spectra with similar g including oxidized cytochrome P-45030 as well as myoglobin and hemoglobin in the presence of thiols.31 Furthermore, ample evidence exists to show a spin state-structure correlation in synthetic iron(II1) porphyrin complexes such that five-coordinate high-spin derivatives change to six-coordinate low-spin structures upon substrate b i n d i ~ ~ g . ~ ~ , ~ ~ From the results it appears that the polymer-supported, high-spin FeT ions change to low-spin [N,OFe"'S] units upon axial coordination of ionized thiol group of cysteine, in full agreement with the foregoing findings. In summary, the rather rigid assembly of polymer-supported FeT ions does not permit the occurrence of bimolecular phenomena, like those of eq 1 and 2, once cysteine tightly binds to Fe(II1). This leads to a stable Fe"'T-S-Cys complex. An important environmental effect of polypeptide matrix is hence that of thwarting coupling of Fe(II1)-thiolate molecules rather than that of hindering radical coupling,3b once an intramolecular (25) Margerum, D. W. ACS Symp. Ser. 1982, No. 198, 3. (26) (a) Peisach, J.; Blumberg, W. E.; Lode, E. T.; Coon, M. J. J . Bioi. Chem. 1971, 246, 5877. Osterhuis, W. T. Struct. Bonding (Berlin) 1974, 20, 59. (b) Palmer, G.In Methods for Determining Metal Ion Environments in Proteins; Darnall, D. W., Wilkins, R. G., Eds.; Elsevier: New York, 1980; Chapter 6. (27) EPR monitoring of reaction mixtures has also shown that, if further thaw-quench cycles were performed after the initial low-temperature quench, they were accompanied by a decrease in the intensity of the low-spin Fe(II1) set of rhombic signals (see above). This phenomenon is reminiscent of that observed by Holm et al.3bin thaw-quench cycles of low-spin Fe"'P(SR)L complexes (P = porphyrins) and is ascribable to reduction of the central metal ion. Since it has been demonstrated**that on freezing aqueous Tris-buffered solutionsdown to 77 K the apparent pH increases approximately 2-3 pH units, it is likely that repeated freezings of catalyst-cysteine buffered mixtures determine irreversible changes in the ordered polypeptide matrix, possibly accompanied by a release of bound complex ions. This would make quite unstable the low-spin Fe"'T-S-Cys intermediate.*,' In fact, both the color and electronic spectra of such solutions differ from those of the dead-end complex, the color being light blue and the spectra exhibiting an intense band at 342 nm together with two shoulders at around 330 and 365 nm and a small band at 544 nm. (28) Williams-Smith, D. L.; Bray, R. C.; Barber, M. J.; Tsopanakis, A. D.; Vincent, S. P. Biochem. J . 1977, 167, 593. Orii, Y.; Morita, M. J . Biochem. 1977, 81, 163. (29) Peisach, J.; Blumberg, W. E.; Adler, A. Ann. N. Y . Acad. Sei. 1973, 206, 310. (30) Miyake, Y.; Mori, K.; Yamano, T. Arch. Biochem. Biophys. 1969, 133, 318. Whysner, J. A.; Ramseyer, S.; Harding, B. W. J. Bioi. Chem. 1970, 245, 5441. Cheng, S . C.; Harding, B. W. Ibid. 1973, 248, 7263. (31) Bayer, E.; Hill, H. A. 0.;Roder, A.; Williams, R. J. P. Chem. Commun. 1969, 109. Blumberg, W . E.; Peisach, J. Adu. Chem. Ser. 1971, 100, 271. (32) Schaeffer, C.; Momenteau, M.; Mispelter, J.; Loock, B.; Huel, C.; Lhoste, J. M. Inorg. Chem. 1986, 25, 4571.



TABLE IV: Molecular Parameters of the Diastereomeric Noncovalent Models” substrate L-cysteine L-dopa L-adrenaline

diastereomer DL LL DL LL DL

LL

R,b

A

5.2 f 0.1 5.2 f 0.1 7.1 7.3 7.0 7.7

R‘,c A

Ro,d A

U,ot(Ri,),ekcal/mol

4.2 f 0.2 4.2 f 0.2 1.6 7.6 7.1 8.1

3.3 f 0.2 3.3 f 0.1 3.2 3.2 3.3 3.3

-3.84 f 0.53 -3.81 f 0.62

f

-7.52

12b

-7.28 -7.96 -7.71

12b

ref


“Ail distances and energies with cysteine are mean values over 40 different encounter complexes for each diastereomeric pair, while the same parameters with catecholamines are those corresponding to the deepest minimum in the total interaction energy (see text). *Closest mercaptide S--Fe or catecholic O--Fe separation distance. CCIosest approach of mercaptide S- or catecholic 0- to the median distance between Fe and the “distal” oxygen atom O2in the buried centers (see Chart I). dClosest approach of mercaptide S- or catecholic 0- to the edge of the peripheral tetrapyridyl ring of the active site. eCalculated from eq 11. The torsional potential function was only used with cysteine. /This work. CHART I: Schematic Drawing of the “Buried” Active Site (See electron transfer has occurred in a preliminary step, otherwise a Fe(I1)-thiyl radical species would have been ~ b t a i n e d . ~ ~ , ~ ~Figure 1)” On the other hand, t h e foregoing process can be hardly stereoselective because t h e inner-sphere P-FeII’T-S-Cys complex forms a t the expense of the structural features of the buried sites, eliminating any stereochemical control of the ordered polymeric matrix. Instead, L-catecholamines were shown t o form an outer-sphere precursor complex in the close environment of the same sites,’, and discrimination in the binding was found to result from a complex interplay of both steric effects and ionic interactions in which t h e polypeptides play a basic role.’2b CYS Conformational Energy Calculations. The steric feasibility “The closest approach of mercaptide S- to the median distance beof the aforementioned mechanism in which ionized cysteine (RS-) tween the central metal ion and the ”distal” oxygen atom O2 is indireplaces t h e “distal” oxygen atom O2in the buried active centers cated as R’. For clarity, only one direction of approach of the substrate was examined by t h e methods of theoretical conformational is shown. analysis. T h e complex-forming properties of cysteine have not yet been satisfactorily clarified,35abut t h e main purpose of these CHART II: Partial Atomic Charges Used for Ionized Cysteine“ calculations was t h e investigation of t h e following specific -1.104 questions. (1) Assuming t h a t the given coordinates provide a 0.043 precise definition of the buried active site,12what pathway is there H\ for the closest approach of the entering RS- molecule toward the ‘ c 0.029 - 0.687 central metal ion to achieve axial coordination? (2) W h a t difH’ o 087 ference is there in both t h e total interaction energy and closest

’I 1

‘\c-

O’ ; ; ,o

(33) This seems to be the case with reduced L-glutathione (R’S-). EPR spectra a t 10 K of catalyst-glutathione mixtures show only a resonance at g = 2.0, which slowly and partially replaces that at g = 4.3. Since this signal was not saturated at power levels up to 100 mW ( I O K), it seems likely that it arises from a radical species in the close proximity of a metal center, possibly a thiyl radical weakly coupled to a high-spin ( S = 2) Fe(I1) center, giving a total S = 3 / 2 system.9b*26b Furthermore, we have found that this substrate behaves differently from cysteine toward oxidation, despite the similarity of A different their reduction potentials, which differ only by about 0.01 V.3433s behavior in the oxidation reaction of cysteine and glutathione by Mood2-has alreadv been r e ~ o r t e d .In~ ~fact.. H,O, .,.oxidation of L-elutathione follows an empirLcal rate jaw like that observed with the dihydr&y compounds (eq 5), i.e., d[R’SSR’]/dt = k,,,,,[R’S-][C] k0[R’S-][H2O2],with k,,,,, = 6.4 f 0.6 M‘I s-I and k, = 0.4 M-’ SKI(26 “C, pH 7, 0.05 M Tris buffer, [C]/[P] . ~ . = 0.20). While the process does not exhibit any stereoselectivity, as one could expect owing to the high degree of mobility of this three-peptide-like substrate, the kinetics suggest that a stable P-Fe”’T-SR’ complex does not form in this case. This is probably ascribable to steric hindrances owing to the bulkiness of the molecule. Further experiments are clearly needed, but the results so far collected suggest that besides a slow autoxidative rea~tion,~’” electron transfer from glutathione to iron(II1) at the “exposed”active sites does take place intramolecularly within a loosely bound precursor complex, where the interaction of mercaptide ligand cannot closely approach the strength of normal axis bond or else conversion to low-spin iron would have occurred. The reduced central metal ion is then oxidized by H202in subsequent fast steps,ll while R‘S. radicals yield oxidized glutathione (the disulfide product) very rapidly.38aApparently, the more strongly bound Fe(II1) does not undergo reduction as the more weakly bound metal ion, which in turn suggests that other subtler (possibly electronic) factors than the environmental protection of the polymeric matrix also play a role in the redox process of iron(II1)thiolate systems. (34) Rost, J.; Rapoport, S. Nature (London) 1964, 201, 185. (35) (a) Gergely, A,; Sovago, I. In Metal Iom in Eiologiral Systems; Sigel, H., Ed.; Dekker: New York, 1979; Vol. 9, Chapter 3. (b) Rabenstein, D. L.; Guevremont, R.; Evans, C. A. Ibid.; Chapter 4. (36) Martin, J. F.; Spence, J. T. J . Phys. Chem. 1970, 74, 2863. (37) (a) Albro, P. W.; Corbett, J. T.; Schroeder, J. L. J . Inorg. Eiochem. 1986, 27, 191. (b) Searle, A. J. F.; Tomasi, A. Ibid. 1982, 17, 161. (38) (a) Hoffman, M. Z . ; Hayon, E. J . Am. Chem. SOC.1972, 94, 7950. (b) Adams, G. E.; Armstrong, R. C.; Charlesby, A,; Michael, B. D.; Willson, R. L. Trans. Faraday SOC.1969, 65, 732.

C -H

361

0.053

I

+N 0.086

/I\ H ; ( H 0 259

“Those of FeT-poly(g1utamate) system are reported in ref 12b

+

.

separation distance of the reacting centers for the diastereomeric encounter complexes with L-catecholamines and t h e equivalent hypothetical complexes with L-cysteine? To answer these questions, we adopted t h e strategy of approaching cysteine to the unperturbed buried active site along the line of energy gradient, allowing the system to minimize the total interaction energy in terms of three sets of parameters, Le., t h e six (translational and rotational) degrees of freedom of the entering substrate molecule, the torsional angle about the NC‘-C@SS-bond of ionized cysteine, and the closest separation distance between t h e sulfhydrilic S- and iron(II1) ( R ) . A t the s a m e time, we also searched for t h e closest approach of S- to t h e median distance between Fe and the “distal” oxygen atom 0, (R’, Chart I) because, with the fixed-catalyst coordinates used in connection with question 1 , this position appears to be just where the attack of t h e ionized group of cysteine is likely t o occur for axial binding. Several minimizations were then carried o u t by starting from different mutual orientations of the reactants as well as different directions of approach of cysteine, t h e total conformational energy being calculated as the sum of all pairwise nonbonded (NB), electrostatic ( C O U L ) , and hydrogen-bond ( H B ) interactions (eq 11, where

Ri, (angstroms) is t h e separation distance of the pertinent atoms i a n d j and t h e energy terms a r e similar to those recently rep~rted’~,~~,~~).

3428 The Journal of Physical Chemistry, Vol. 92, No. 12, 1988

Pispisa et al.

The set of parameters employed in the present analysis includes been extensively investigated also in the presence of both H202 (1) partial atomic charges for each atom of the reactants, as shown and transition-metal ions, and several mechanistic interpretations in Chart I1 for cysteine, those of the FeT-poly(g1utamate) system have been g i ~ e n . ' ~ - ' ~There % ~ ' is, however, a surprising paucity having already been (2) bond lengths and bond angles of thorough kinetic studies to support such mechanisms. Furfor all species considered,40a (3) parameters for nonbonded, thermore, the involvement of reactive intermediates has been ~ u g g e s t e d , ' ~but J ~only ~ recently some of these intermediates, such electrostatic, and hydrogen-bond interactions,ab and (4) a torsional as hydroxyl and cysteinyl free radicals, have been identified.'4a,37b energy contribution (TOR) to rotation about the NC"-CPS- bond of ionized cysteine, Le., TOR(x2) = (2.7/2)(1 + cos 3 ~ ~x ) , We ~ have ~ ~also observed HO. intermediates in reaction 3 by spinbeing the dihedral angle. trapping experiment^,^^ while cysteinyl radicals could not be Briefly, the small dimensions of cysteine as compared to those detected, probably because the ratio [cysteine]/ [H202]was not of catecholamines enable a close approach of this substrate to the high e n 0 ~ g h . lEPR ~ ~ signals due to DMPO-OH (AN = AH = buried sites of both enantiomeric systems from several directions, 14.9 G) and DMPO-a-hydroxyethyl (AN = 15.8 G, A H = 22.6 G) adducts were obtained by using DMPO (5,Sdimethyl- 1with rather comparable energies. In some cases, LL pairs are pyrroline N-oxide) as a spin trap and ethanol as a competitive favored, while in others DL pairs are preferred, so that, on the average, a negligible difference in the stability of diastereomers inhibitor of DMPO-OH p r o d ~ c t i o n . ~ ~ , ~ ~ is attained. This is illustrated in Table IV, where the most relevant The results shown in the previous sections indicate that eq 3 is not a true catalytic process, at variance with the oxidation of data of the computational studies are presented. The answer to both ascorbic acid and catechol derivatives performed under similar question 2 is therefore that even if the structural features of the buried sites could remain unperturbed by the entering cysteine experimental The results also allow us to put forward Scheme I for the H 2 0 2 oxidation of cysteine in the molecule, very poor discriminating effects would have been observed. In contrast, the closest approach of L-dopa and L-adreSCHEME I naline to the buried centers was already shown to be coupled with RS- H202 RS. + HO. + HO(a) the formation of a sterically preferred adduct for each pair, characterized by an energy minimum 2-3 kcal/mol deeper than RS- e (Fe"'TSR) (Fe"'T') (b) the other relative minima of total interaction energy that substrate (FeTSR) H 2 0 2 (FeT') + RS. + HO. + HOmolecules encounter during their approach to the active site.12 (c) This leads to steric discrimination between the diastereomers (FeTSR) + HO. (FeT') RS. HO(d) (Table IV), in agreement with the idea that stereoselectivity is enhanced by conformational rigidity of the adducts.42 Therefore, RS- + HO. -.+ RS. + HO(e) L-catecholamines are "good" substrates to constitute "productive" RS. + RSRSSR'(f complexes for stereoselective reaction,I2 while L-cysteine is not because of the lack of specific interactions with the polymeric RSSR'RS. RSSR RS(g) matrices of the enantiomeric systems used. 2RSRSSR (h) Inspection of Table IV shows some further interesting features of the noncovalent models with cysteine. Not only the mean presence of the FeT-poly(g1utamate) system, where, for clarity, interaction energy43and the closest mercaptide S--Fe separation the active sites are abbreviated as (FeT'). The scheme is condistance ( R ) but also the closest approach of S- to the median sistent with stoichiometry and includes oxidation of substrate in distance between Fe and the distal oxygen atom O2 (R?are, on bulk solution (steps a and e). average, quite similar for both diastereomers. In addition, the Conventional steady-state approximation for HO., RS., values of RtDLand R I L L are around 4.2 %, or even smaller (-3.8 RSSR'-,38 and (FeTSR) leads to eq 12, where RSSR denotes A) at a still negative interaction energy (-2 kcal/mol). These findings are relevant to question 1 (see above) because they strongly suggest that the proposed mechanism for the occurrence of a ligand-interchange reaction is feasible on steric and geometric cystine, k ' = k-b/k, and k " = kakd/(k,k,). Equation 12 corregrounds and that this process cannot be stereoselective, as exsponds to eq 4, provided that certain assumptions are made reperimentally observed. garding the relative magnitude of k'and k". The close analogy Mechanism of Oxidation. We now return briefly to the H202 of reactions a and e with c and d makes it reasonable to assume oxidation of cysteine (eq 3). Autoxidation of this substrate has that k,/k, = k,/k,. Then k " = 1, and eq 12 reduces to eq 4 if k'>> 2[H202]. This implies that under the experimental con(39) The nonbonded (12-6 Lennard-Jones type function) and hydrogenditions used, k, must be smaller than 0.1 M-' s-' since k-b = bond energy terms used here are those recently reported for computational s-'.~,'~This conclusion appears reasonable in view of the fact that studies of naturally occurring compounds, including sulfur derivatives.mb Furthermore, as in earlier calculations with L-catecholamines,lZbthe dielectric the specific rate (k,) of the H202oxidation of RS- in bulk solution constant in the Coulombic term was expressed as c = c'(1 + /cR,~), with c' = is 1.5 M-'s-' (Table 11). However, k,, = 6.1 X lo4 M-2 s-I, so R$.4' The dielectric constant was thus interpreted as the numerical, dimenthat k, = k,,k-,,/kb = 3 M-' s-' if kb = 12.7 M-' s-I, this latter sionless value of the distance (R{7)between nonbonded atoms i and j . corrected for the inverse of Debye-Hkkel screening length k (0.07 at 25 OC and = being the value of the rate constant for the formation of a cys0.04 M).Izb teinate-Fe(II1) complex involving the buried centers (see above). (40) (a) Momany, F. A.; McGuire, R. F.; Burgess, A. W.; Scheraga, H. In this case the assumption k'>> 2[H20,] is therefore untenable. A. J . Phys. Chem. 1975, 79, 2361. (b) Nemethy, G.; Pottle, M. S.; Scheraga, Instead, the same assumption fully applies to the foregoing H. A. Ibid. 1983, 87, 1883. mechanism when oxidation of a (FeII'TRS) complex involving the (41) McCammon, J. A,; Wolynes, P.G.; Karplus, M. Biochemistry 1979, 18,927. Blaney, J. M.; Weiner, P. K.; Daring, A,; Kollman, P. A,; Jorgensen, "exposed" active sites is considered. In this latter case kb is E. C.; Oatley, S. J.; Burridge, J. M.; Blake, C. C. F. J . Am. Chem. SOC.1982, presumably of the order of 103-104 M-' s-l, like that found for 104, 6424. a number of monodentate ligands upon binding to iron(III).*J9 (42) Hwang, F. J.; De Bolt, L. C.; Morawetz, H. J . Am. Chem. SOC.1976, Then k, becomes less than 0.1 M-' SKI,in agreement with pre98, 5890. (43) The mean van der Waals intermolecular energymbover 40 different diction.46 It is worth mentioning that numerous other mechanisms


+

-+

+

+

-

+

encounter complexes for each diastereomeric pair with L-cysteine (Table IV) is -3.26 f 0.54 and -3.29 f 0.58 kcal/mol for DL and LL pairs, as compared to a van der Waals energPbof -4.79 and -5.23 kcal/mol and -5.78 and -5.37 kcal/mol for DL and LL complexes with L-dopa and L-adrenaline, respectively, in the deepest minimum of total energy.Izb The mean torsional energy in both diastereomeric adducts with cysteine amounts to about 0.15 f 0.08 kcal/mol, and the mean electrostatic energy39is -0.73 f 0.33 and -0.67 f 0.35 kcal/mol for DL and LL pairs with cysteine, as compared to -2.73 and -2.05 kcal/mol and -2.18 and -2.34 kcal/mol for the same complexes with dopa and adrenaline, respectively,Izbin the deepest minimum.

-

+

-

+

+

+

(44) Paradossi, G.; Desideri, A,; Palleschi, A,; Pispisa, B., to be submitted for publication. (45) Finkelstein, E.; Rosen, G. M.; Rauckman, E. J. Arch. Biochem. Biophys. 1980, 200, 1. J . Am. Chem. SOC.1980, 102, 4994. (46) Independent kinetics data on the H 2 0 2oxidation of Feii'TRS (C,) complex, which will be shown elsewhere,44confirm the second-order behavior of step c. Furthermore, the presence of SOD (superoxide dismutase) did not apparently affect the rate of the reaction.

J. Phys. Chem. 1988,92, 3429-3437 were considered but were found to be inadequate. This does not imply that the foregoing proposal must be unique but rather that a good alternative was not found. For instance, if reactive intermediates other than OH., such as H 0 2 . radical (OH- + H202 H02. H2047),are taken into account the rate law no longer agrees with the empirical one.

-

+


Concluding Remarks From the data set considered here it is evident that the H202 oxidation of L-cysteine in the presence of structurally ordered FeTD and FeTL enantiomeric systems involves decomposition of a Cysteinate-FeII’T complex that rapidly forms at the “exposed” active sites (Figure 1). A negligible sterical discrimination between FeTL-L-cysteine and FeTD-L-cysteine diastereomers results from the conformational mobility of the axially bound substrate, and this is reflected in the absence of stereoselective effects in the reaction. The same complex forms too slowly in the “buried” centers to participate in the oxidation process, because the polymeric matrix partially hinders the accessibility of the central metal ion to the entering substrate molecule. In the absence of (47) Walling, C. Acc. Chem. Res. 1975,8, 125. Weinstein, J.; Bielski, B. H. J. J. A m . Chem. SOC.1979, 101, 58.

3429

hydrogen peroxide, the polypeptide matrix stabilizes the Fe(1II)T-SXys structure but does not control chiral discrimination between the diastereomeric complexes, even when the buried centers are involved. Conformational energy calculations support these conclusions. At variance with the results obtained with L-catecholamines,12 they show both the absence of a sterically preferred pathway for the closest approach of L-cysteine to the central metal ion and the steric feasibility for the formation of a number of almost equally populated diastereomeric adducts. Very poor discriminating effects would have been hence observed even if the structural features of the polymer-shielded active sites could remain unperturbed by the entering cysteine molecule. These findings highlight the difficulty so far associated with modeling a general pattern for stereoselective reactions between chiral species.

Acknowledgment. We thank P. De Santis, G. A. Lappin, and G. Nemethy for helpful discussions. Collaboration of E. Cataldo and G. Musci in some experiments is also acknowledged. This work was supported by MPI (Rome) and the Italian Research Council (CNR). Registry No. [Fe(tetpy)(OH),]*, 6 14 12-01-9; H202,7722-84- 1; sodium poly(L-glutamate), 26247-79-0; sodium poly(D-glutamate), 3081 1-79-1; L-cysteine, 52-90-4; cystine, 56-89-3.

Vibrational Relaxation and Intersystem Crossing in N2(a‘ng) William J. Marinel&,* Byron D. Green, Margrethe A. DeFaccio, Physical Sciences Inc., Research Park, PO Box 3100, Andover, Massachusetts 01810

and William A. M. Blumberg Air Force Geophysics Laboratory, Hanscom AFB, Massachusetts 01 731 (Received: August 10, 1987; In Final Form: December 17, 1987)

We have observed both electronic quenching and very fast level-specific vibrational deactivation of N,(allI,) by N2. Rate coefficients for these processes have been determined, and the role of the collisional coupling of the N2(a’II,) state to the N2(a”Z[) state in the relaxation of the a’II, state has been assessed.

Introduction Lyman-BirgeHopfield (LBH) band emission due to transitions from N,(alII,) to N2(X1Zg+)ground state is a prominent feature in the ultraviolet spectrum of the quiescent as well as the aurorally disturbed atmosphere. Observations of LBH emission’” from high altitude auroras are consistent with the predictions of models that include Franck-Condon type excitation of ground-state molecular N 2 to the N2(a) state by energetic electrons followed by radiative r e l a ~ a t i o n . ~However, the band shape of LBH emissions from penetrating lower altitude auroras and from the dayglow and nightglow where N,(a)-state vibrational distributions are affected (1) Gentieu, E. P.; Feldman, P. D.; Meier, R. R. Geophys. Res. Lett. 1979, 6, 325. (2) Park, H.; Feldman, P. D.; Fastie, W. G . Geophys. Res. Left. 1977, 4 , 41. (3) Huffman, R. E.; LeBlanc, F. J.; Larrabee, J. C.; Paulsen, D. E. J. Geophys. Res. 1980, 85, 2201. (4) Takacs, P. Z.; Feldman, P. D. J. Geophys. Res. 1977, 82, 5011. (5) Rottman, G. J; Feldman, P. D.; Moos, H. W. J . Geophys. Res. 1973, 78, 258. (6) Paresce, F.; Lumpton, M.; Holberg, J. J. Geophys. Res. 1972, 77,4773. (7) Ajello, J. M.; Shemansky, D. E. J. Geophys. Res. 1985,90,9845-9861.

0022-3654/88/2092-3429$01 S O / O

by electronic quenching, vibrational relaxation, and intersystem collision-induced cascade are not u n d e r s t o ~ d . ~ ~ ~ ~ ~ Collisional processes involving N2(a) are complicated by the existence of two other states with comparable term energies: the a’*& and the wIA,. This trio of electronic states gives rise to a system of nested vibronic levels that are radiatively as well as collisionally coupled. The relative energies of these states is displayed by using a ladder diagram in Figure 1. The allowed radiative transitions w1AU a l n , and a ’ n , a’l& comprise the McFarlane infrared systemg of emissions in the 3-8.5-pm wavelength range. Transitions from the wlAu state to the a’n, state dominate the McFarlane bands9 The transition probabilities for these bands have not been experimentally determined. No emission resulting from a’ a state transitions has been observed,

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(8) Torr, M. R.; Torr, D. G.; Eun, J. W. J. Geophys. Res. 1985, 90, 4427-4433. (9) McFarlane, R. A. IEEE J. Quantum Electron. 1966, 2, 229-232. (10) Herzberg, G.; Herzberg, L. Nature (London) 1948, 161, 283. (11) Douglas, A. E.; Herzberg, G. Can. J. Phys. 1951, 29, 294-300. (12) Lofthus, A. Can. J. Phys. 1956, 34, 780-789. (1 3) Vanderslice, J. T.; Tilford, S.G.; Wilkinson, P. G. Astrophys. J . 1965, 141, 395-426.

0 1988 American Chemical Society

Oxidation of L-thiols in the presence of iron (III) complex ions anchored

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