J . Phys. Chem. 1985,89, 1767-1775
1767
Stereoseiective Electron Transfer between Chirai Catecholamines and Iron( III) Complex Ions Anchored to Asymmetric Polymers. A Kinetic and Conformational Investigation Basilio Pispisa,* Antonio Palleschi, Mario Barteri, and Stefanella Nardini Dipartimento di Chimica, Uniuersitd di Napoli, 801 34 Napoli, Italy, and Dipartimento di Chimica, Uniuersith di Roma, 00185 Roma, Italy (Received: September 18, 1984)
The oxidation of L-dopa (3,4-dihydroxyphenylalanine)and L-adrenaline (epinephrine) by [Fe(tetpy)(OH),]+ complex ions anchored to poly@-glutamate)(FeTL) or poly(D-glutamate) (FeTD) was studied at pH 7 (tetpy = 2,2’:6’,2”:6”,2”’-tetrapyridyl). Electron transfer from the chiral catecholamines to iron(II1) in the FeTL or FeTD system proceeds stereoselectively only when the polypeptide matrices are predominantly in the a-helical conformation and the accessibility of the active sites is, at least partially, hindered. Oxidant-reductant inferactions are thus mediated by the polymer, whase conformational asymmetry ensures a sterically constraining environment that affects differently the DL and LL reactions. Under these conditions, kDL = 31.4 f 2.1 and 31.2 f 2.1 M - I d and k L L = 7.9 f 0.4 and 7.5 f 0.5 M - ’ d for L-dopa and L-adrenaline, respectively, whereas the unimolecular rate constants for the electron-transfer step, which is rate determining, are kctDL= (1.9 f 0.2) X and (3.1 f 0.3) X lo-, s-l and k c l L ~= (0.7f 0.1) X and (0.9 f 0.1)X lo-, s-’, respectively (26 “C). Since the equilibrium constants for the formation of precursor complexes are KooL = (1.8 f 0.2) X lo3 and (1.0 f 0.1) X lo3 M-’ and K o = ~(1.2 ~ f 0.1) X lo3 and (0.8f 0.1) X lo3 M-’, respectively, stereoselectivity is largely controlled by kinetic effects. A hypothetical model of the diastereomeric noncovalent electron-transfer complexes is presented. The diastereomeric models are based on conformational energy calculations and supported by experimental results, as far as the limited data allow. They show significant differences in both the mutual orientation and separation distance of the redox centers and support the idea that stereoselectivityis coupled with a remote attack mechanism on the central metal ion, where the peripheral tetrapyridyl ligand of the active sites plays the role of electron-transfer agent.
Introduction Several attempts have been made in the last few years to observe rate difference in outer-sphere redox reactions between enantiomeric complex ions and chiral reductants,’ but most of them failed? This is probably because the reactants are not in contact in the transition state or the rate of racemization predominates.2 We have recently shown that specific steric requirements have to be met in order to observe stereoselective effects in electrontransfer reactions between asymmetric couple^.^,^ For instance, when one uses hemin-like [Fe(tetpy)(OH),]+ ions (hereafter called FeT, where tetpy = 2,2’:6’,2’’:6”,2’”-tetrapyridyl) bound to sodium poly&-glutamate) (FeTL) or poly(D-glutamate) (FeTD) as oxidant and L-ascorbic acid (I) or L-adrenaline (11) as reductant 0
CHOH
I
CH,OH (I)
(Ill)
(11)
(eq l), the reaction proceeds stereoselectively only when two @Fe”’T+
+
0A’ ‘OH
-
0. @F$’T
(11
A’
‘OH
conditions are matched. The polypepetide matrices must be predominantly in the a-helical conformation and the accessibility of the active sites to substrate molecules must be, at least partially, hindered. Under these conditions, oxidant-reductant interactions are mediated by the polypeptide, whose conformational asymmetry (1) B. Grossman and R. G. Wilkins, J . Am. Chem. Soc., 89,4230 (1967); J. H.Sutter and J. B. Hunt, ibid., 91, 3107 (1969); N. A. P. Kane-Maguire, R. M. Tollison, and D. E. Richardson, Inorg. Chem., 15, 499 (1976). (2) D. A. Geselowitz and H.Taube, J . Am. Chem. Sm., 102,4525 (1980). (3) (a) M. Barteri and B. Pispisa, J . Chem. Soc., Faraday Trans. I , 78, 2073 (1982); (b) ibid., 78, 2085 (1982). (4) (a) B. Pispisa, M. Barteri, and M. Farinella, Inorg. Chem., 22, 3166 (1983); (b) B. Pispisa and M. Farinella, Biopolymers, 23, 1465 (1984).
ensures a sterically constraining environment that affects differently the diastereomeric reaction^.^ The question arises as to whether such a difference is caused by kinetic or thermodynamic effects or both, the former depending mainly on a different mutual orientation and separation of the redox centers while the latter on a different stability of the diastereomeric adducts (which is related to the encounter distance of the reactants, too). The results so far obtained indicate that stereoselectivity is almost entirely kinetically ~ontrolled.~ The conformational order and rigidity of polypeptide chains in solution provide a good opportunity for investigating the foregoing topochemical effects by conformational energy calculations. It is the aim of this paper to present a hypothetical model of the diastereomeric (noncovalent) electron-transfer complexes, under conditions where stereoselectivity is observed. At the same time, the kinetic data on the oxidation of another catecholamine, such as ~-dopa(3,4-dihydroxyphenylalanine,111), by FeTL and FeTD systems are reported together with new results on L-adrenaline. Evidence is produced that in all cases stereoselectivity is largely controlled by kinetic effects and that the occurrence of suitable nonbonding interactions is a sufficient condition to account for significant differences in orientation and separation distance of the redox centers in the diastereomeric precursor complexes, while the total interaction energy in DL and LL models differs only by a few hundred calories.
Experimental Section Materials. The pseudooctahedral quaterpyridineiron(II1) complex was prepared as already described.5 It is an oxo-bridged dimeric compound in the solid state. In solution, at the pHs and concentrations with which we are primarily concerned, it is almost entirely in the mononuclear form.5b The polypeptides were purchased from Miles-Yeda and treated as already r e p ~ r t e d . ~ Concentrations of the complex, polymers, and catecholamines were determined by UV absorption at 300 (e 13 430), 200 ( E 5500), and 280 (e, 2720 for dopa and 2740 M-lcm-’ for adrenaline) nm, respectively (pH 7). Polymer concentration [PI is referred (5) (a) M. Branca, B. Pispisa, and C. Aurisicchio, J . Chem. Soc., Dalton Trans., 1543 (1976); (b) M. Cerdonio, F. Mogno, B. Pispisa, G. L. Romani, and S. Vitale, Inorg. Chem., 16, 400 (1977).
0022-3654/85/2089-1767$01.50/00 1985 American Chemical Society
1768 The Journal of Physical Chemistry, Vol. 89, No. 9, 1985
Pispisa et al.
TABLE I: Rate Constants and Activation Parameters of Electron Transfer from L - D o p a to Iron(II1) in FeTL or FeTD System at Different Complex to Polymer-Residue Ratios and Temperatures'
0.01
26.0 17.3 13.0 26.0 26.0 26.0 16.0 26.0 16.0 13.0 26.0 16.0 13.0
0.02 0.04 0.06 0.10 0.20
76.3 f 4.4 46.8 f 2.9
64.9 f 3.6 38.4 f 2.1 29.2 f 1.7 47.5 f 2.5 26.7 f 1.5 18.1 f 0.9 8.0 f 0.4 10.6 f 0.5 4.1 f 0.3 3.0 f 0.2 7.9 f 0.4 2.6 f 0.2 1.8 i 0.2
63.6 f 3.9 53.4 f 3.2 46.3 f 2.5 19.6 f 0.9 37.5 f 2.0 14.6 f 1.1 11.0 f 0.8 31.4 f 2.1 10.5 f 0.8 7.2 f 0.5
"pH 7, 0.05 M Tris buffer, [AH-], = 2
X lo4
1.2 f 0.1 1.2 f 0.1 1.3 f 0.1 2.0 f 0.2 2.6 f 0.2 2.5 f 0.2 3.5 f 0.3 3.6 f 0.4 3.7 f 0.4 4.0 f 0.3 4.0 f 0.4 4.0 f 0.5
HO
+
(111)
NH,
+
H,O,
FeTL or
FeTD
o ~ c ' ~ - c H,O ~ -+OH+ 0
+
9.8 f 0.5
-19
-17
14.2 f 1.0
13.4 f 1.0
-3
-8
15.4 f 1.0
15.7 f 1.1
0
1
* 1.0
18.7 f 1.3
10
8
18.4
M. bStereoselectivityratio of electron transfer (eq 1). CErrorswithin f5 eu.
to the monomeric unit (monomol/L). Tris buffer (Sigma) was employed in the chloride form, at a concentration of 0.05 M (pH 7.01 f 0.03). Under the experimental conditions used, the degree of association of complex ions by polyelectrolytes is higher than 93%, according to equilibrium dialysis experiments.6b L-Dopa (Merck), L-adrenaline (BDH), and stabilizer-free Hz02(Erba) were analytical-grade reagents and were used without further purification. All measurements were performed on solutions freshly prepared by using doubly distilled water. Methods. The oxidation of dopa to dopaquinone (IV; X, = 304 and 475 as that of adrenaline to adrenochr~me,~ was carried out in aqueous solution at pH 7 (0.05 M Tris buffer), according to the r e a ~ t i o n ' ~ -oDc'~H-COO-
9.2 f 0.6
NH,
I IV)
As already observed with the other substrate^,^,^*^ under the experimental conditions used the rate of electron transfer from dopa to Fe(II1) in the FeTL or FeTD system (eq 1) does not depend on hydrogen peroxide concentration. In fact, H202simply oxidizes the reduced iron ion and dopa radical in subsequent fast steps (see later), thus allowing one to follow the kinetics by measuring the formation of dopaquinone at 320 nm. A typical run consisted of adding hydrogen peroxide by a microsyringe into the 1-cm optical cell containing 2 mL of FeTL or FeTD and substrate, both systems being thermostated at 26.0 f 0.1 OC. The experimental conditions normally used were as follows: [AH-] = 1X M (unless otherwise stated) [HzOz]= 1 X M, M, and [C]/[P] = 0.01-0.20, AH-, [C] = 0.5 X 10-5-5 X C, and P denoting substrate, complex ions, and polypeptides, respectively. Measurements were also carried out at other temperatures (16.0 and 13.0 "C). It should be stressed that, according to circular dichroism data, the a-helical fraction of the polypeptide matrices at both these temperatures does not practically differ from that evaluated at 26 "C, other experimental conditions being equal.3b Plots of log (A, - A,) against time were linear over more than two half-lives and the observed rate constants ( k , s-l) were obtained from the slopes. After this time interval the deviation from linearity is ascribable to a rearrangement reaction of dopaquinone,'b which has a rate constant l order of magnitude lower s-l (27 "C). Four than that of its formation, Le., 0.16 X kinetic measurements were carried out for each run, to obtain consistency in the results. At each [C]/[P] ratio investigated, (6) (a) M. Branca, M. E. Marini, and B. Pispisa, Biopolymers, 15, 2219 (1976); (b) M. Branca and B. Pispisa, J. Chem. Soc., Faraday Trans. 1.73, 213 (1977). (7) (a) R. A. Heacock, Chem. Rev., 59, 181 (1959); (b) R. B. Martin, J . Phys. Chem., 75, 2657 (1971). ( 8 ) !a) B. Pispisa in 'The Coordination Chemistry of Metalloenzymes", I. Bertini, R.S. Drago, and C. Luchinat, Eds., NATO Advanced Institutes Series, Reidel, Dordrecht, 1983, p 279; (b) M. Barteri and B. Pispisa, Biopolymers, 21, 1093 (1982).
plots of k (SI) against complex concentration always gave straight lines and the second-order rate constants k- ( M - W ) of electron transfer (eq 1) were obtained from the slopes. It must be said that these straight lines have intercepts that differ from zero, i.e., k = ko + kow [C] or, when second-order conditions with respect to H202were adopted ([H202]0/[AH-]o z l), k = kOapp[HZOZ] kM [C]. This behavior, reminiscent of that observed with both ascorbic acid and adrer~aline,~,~ suggests the Occurrence of parallel pathways. One of these (kOapp, M - l d ) corresponds to an electron-transfer process from dopa to hydrogen perxide, while the other (/cow) refers to a [H20z]-independent route to products, whose rate-determining step involves one molecule of substrate per molecule of complex ion. For brevity, we neglect hereafter the former reaction, whose origin has been discussed Stereoselectivity of eq 1 is given by the ratio kDL/kLL, kDLand kLL(M-'-s-') being the observed specific rates of the diastereomeric electron-transfer processes. Initial reaction velocities, Vo= -d[AH-]/dt ( M d ) at different [C]/[P] ratios and fixed [C], were evaluated from the slope of dopaquinone or adrenochrome concentration against time curves at t 0, the extent of substrate oxidation being generally limited to about 20% of the original concentrations. Under pseudofirst-order conditions with respect to HzOz(as those normally used), the overall initial rate of reaction can be written as Vo = ko[AH-] Vet,4from which Vetmay be evaluated if one knows ko by independent measurements (see above). Absorption measurements were carried out on a Beckman DBGT or a Cary 219 spectrophotometer. Circular dichroism spectra were recorded on a Cary 61 instrument with appropriate quartz cells. Other apparatuses were already d e ~ c r i b e d . ~ ? ~
+
-
+
Results and Discussion Kinetics. For all complex to polymer residue ratios investigated for the FeT-poly@-glutamate) or FeT-poly@-glutamate) oxidant system, the rate of reaction 1 is given by -(d[Red.] /dt) = kOhd[Red.][Ox.] (2) where Red. refers to L-dopa or L-adrenaline. The values of kow at different temperatures and AH*owand AS*obsd(299 K) data are summarized in Tables I and 11. The reactions were studied at fixed pH (7; 0.05 M Tris buffer). From the results it appears that (i) the ratio kDL/kLL(to which we shall refer as the stereoselectivity ratio of electron transfer) is around 1 at [C]/[P] = 0.01 but it increases as the complex to polymer ratio increases, (ii) it reaches an almost asymptotic value of about 4 at [C]/[P] = 0.20, which corresponds to an enantiomeric excess of 60%, (iii) stereoselectivity occurs at the expense of the efficiency of reaction because the specific rates decrease with increasing [C]/[P], and (iv) at the same time, both activation parameters markedly increase, the values of AS*ow changing even sign. We next investigated the dependence of the initial rate of the reaction on the initial concentration of substrates, at fmed complex concentration and varying [C]/[P] ratio. Typical plots of Vet against [AH-], are shown in Figure 1. In all cases the reaction
The Journal of Physical Chemistry, Vol. 89, No. 9, 1985 1769
Stereoselective Electron Transfer
TABLE II: Rate Constants and Activation Parameters of Electron Transfer from L-Adrenaline to Iron(II1) in FeTL or FeTD System at Different Complex to Polymer-Residue Ratios and Temperatures"
~DL, [Cl/[PI
T, OC
0.01
25.9
86.9 f 4.9 63.1 f 2.9 33.7 f 2.3 30.1 f 1.9 14.2 f 0.8 29.7 f 1.8 11.1 f 0.8 9.0 f 0.7 31.2 f 2.1 10.3 f 0.7 7.3 f 0.5
15.8
0.02 0.04 0.06
25.9 25.9 25.9 15.8 25.9 15.8 12.8 25.9 15.8 12.8
0.10 0.20
~LL,
M-'.s-' 159.9 f 8.8 91.8 f 5.1 58.9 f 2.6 21.0 f 1.5 13.9 f 0.9 6.4 f 0.4 9.4f 0.8 3.5 f 0.3 3.0 f 0.3 7.5 f 0.5 2.4 f 0.2 1.7 i 0.2
M-I.s-1 158.0 f 7.6
"pH 7, 0.05 M Tris buffer, [AH-], = 1 X
lo4
kDL/kLLb 1.0 1.0 1.1 f 0.1 1.6 f 0.2 2.2 f 0.2 2.2 f 0.2 3.1 f 0.3 3.2 f 0.4 3.0 f 0.4 4.2 f 0.4 4.3 f 0.5 4.3 f 0.5
AH'DL, kcal/mol
AHILL, kcal/mol
9.5 f 0.6
8.8 f 0.8
LS*~,,' eu
AS*,,,' eu
-17
-1 9
12.2 f 1.0
12.6 f 1.1
-1 1
-8
14.6 f 1.1
14.1 f 1.3
-3
-7
17.5f 1.0
17.9 f 1.3
7
5
M. bStereoselectivity ratio. cErrors within f5 eu.
TABLE 111: Kinetic Parameters of the Lineweaver-Burk Plot for the Electron Transfer from dopa to Iron(II1) in FeTL or FeTD System"
io-)^,,
lO"1,b oxidant FeTD FeTD FeTL FeTD FeTL
[C]/[P]
i o - 3 ~ : s-l
0.01 0.10 0.10 0.20 0.20
1.63 f 0.12 1.03 f 0.14 2.96 f 0.30 1.48 f 0.12 5.72 f 0.44
s.L.mo1-l 3.95 f 0.24
2.59 f 0.20 6.00f 0.46 2.70 f 0.19 6.91 f 0.51
1O2ke1,s-I 2.53 f 0.27 1.93 f 0.21 0.83 f 0.11 1.85 f 0.21 0.72 f 0.08
mo1-l.L
kelKoC
2.42 f 0.23 2.51 f 0.39 2.03 f 0.26 1.82 f 0.20 1.21 f 0.13
61.2f 7.7 48.4 f 9.2 16.8 f 3.1 33.7 % 5.3 8.7 f 1.3
stereosd
2.9 f 0.8 3.9 f 0.8
"25.9"C, [C], = 2 X M but 1 X lop5M at [C]/[P] = 0.01,pH 7, 0.05 M Tris buffer. bObtained by the least-squares method. cM-l.~-', to be compared with koM. dStereoselectivity ratio: (kctKO)DL/(ketKO)LL, to be compared with kDL/kLL.
velocity follows first-order saturation kinetics at low [AH-],; Le., the straight lines in the double-log plots have unit slope, whereas the curves show the tendency toward saturation at high concentration of substrates. This suggests that a precursor complex forms in a preequilibrium step and that electron transfer from the catecholamine to the central metal ion takes place intramolecularly within the intermediate, i.e. FelIITL+ (or FeTD') FeII'TLAH
+ AH-
k k-I
FeII'TLAH
-% Fe"TL + AH.
10 -
Q
a-
(3)
(4)
Both the reduced iron ion and substrate radical are then oxidized by H202in subsequent fast steps (seeExperimental Section). AH. (or A-.) may also disproportionate very rapidly, as does the ascorbate radicaLg Assuming k-l >> ket (Le., that step 4 is rate determining), the initial rate of reaction can be written as vet
= ketK0[Clo[AH-1/(1
+ KoIAH-1)
I
_
(5)
where KO = kl/k-'. The Lineweaver-Burk plot of these data against [AH-],') gives, therefore, straight lines, whose intercepts I and tangents T (obtained by least-squares using weight factors) are reported in Tables I11 and IV. From the results one obtains the values of KOand k, shown in the same tables. They are of the same order of magnitude as those found in the oxidation of p-hydroquinone and homogentisic acid by the copper-poly(histidine) systern.'O When [AH-]Ko
