8414
Hydroxide Ion Catalysis of the Mono- and Bis ( triglycinato) cuprate (11) Interconversion and the Preference for Cis vs . Trans N-Peptide Bonding to Copper (11) Gary R. Dukes and Dale W . Margerum” Contribution from the Department of Chemistry, Purdue University, West Lafayette, Indiana 47907. Receioed April 20, 1972 Abstract: The Cu(H+GGG)- complex (where GGG- is the triglycinate ion and the protons are ionized from two : Cu(HP2GGG)peptide nitrogens coordinated to copper(I1)) reacts with excess GGG- to form CU(H-~GGG)~~GGGOHCU(H-IGGG)~~- OH- (ki, kr). The formation and dissociation rates are catalyzed by hydroxide ion, and the values of the rate constants (25.0”, 0.10 p ) are ki = (1.26 =t0.02) x IO7 M-2 sec-1 and k, = (8.8 i 0.3) x lo4 M-’ sec-l. In the proposed reaction mechanism the hydroxide ion deprotonates an incoming peptide group of one triglycine as the Cu-N(peptide) bond of another triglycine breaks. A combination of absorption and circular dichroism spectrophotometry, kinetic results, and model studies indicate that CU(H-~GGG)s2- exists in a planar form in which the two triglycine molecules are arranged around the copper in a cis bidentate configuration. Infrared and Raman results show that the doubly deprotonated bis complex of glycinamide with copper(I1) also adopts a cis geometry. It is postulated that strong u donation from the deprotonated peptide-nitrogen to copper(I1) causes a preference for the cis rather than the trans configuration.
+
*
+
+
T
riglycinate ion (GGG-) reacts with copper(I1) in neutral solutions to form a complex in which the ligand is tetradentate, Cu(H-,GGG)- (structure I). 1. With the addition of excess triglycinate ion in basic solution Dobbie and Kermack’ found potentiometric and spectrophotometric evidence for a bis complex. They proposed the formula CUH-,(GGG),~- and suggested that both “symmetric” (11) and asymmetric (111) structures were present in solution. Structure 111 was estimated to constitute 50% or more of the bis complex. Another bis structure which is more symmetric and has less steric hindrance also might be postulated (IV). Osterburg and Sjoberg3 carried out a potentiometric study in 3 M NaC104 and also found evidence for
?.
U
n
0
E? (1) H. Dobbie and W. 0. Kermack, Biochem. J., 59, 257 (1955). (2) H. C. Freeman and M. R. Taylor, Acta Crysrallogr., 18, 939 (1965). (3) R.
a bis complex (CUH-~(GGG),~-). Other binuclear complexes were detected at higher copper(I1) concentrations. Recent work in this laboratory4 has shown that excess triglycine catalyzes the exchange reaction between CU(H-~GGG)- and EDTA. Rapid formation of the bis complex is proposed in the mechanism for the catalysis. In the present work the equilibrium constant for reaction 1 is determined from spectrophotometric ki
CU(H-IGGG)-
+ GGG- + OH- Ikr_
+
CU(H-~GGG)~’- OH-
(1)
data and the kinetics of the forward and reverse reaction are determined by stopped-flow methods. Our studies indicate that only one bis species is present in solution and that this species must have both ligands in at least bidentate coordination. A comparison of the reactions of a number of tripeptides indicates that the cis isomer (structure 11) must be present. Experimental Section Triglycine (chromatographically homogeneous) was obtained from Mann Research Laboratories (New York, N. Y.). The Lalanine substituted tripeptides (glycylglycyl-L-alanine (GGA), glycyl-L-alanylglycine (GAG), and L-alanylglycylglycine (AGG)) were obtained from the Cyclo Chemical Division of Travenol Laboratories (Los Angeles, Calif.). A 9.85 X M Cu(I1) stock solution, prepared from twice recrystallized Cu(ClO& and standardized against EDTA, was used, The copper(I1)-triglycine solutions were freshly prepared before each series of kinetic runs. Ionic strength was maintained at 0.10 M with NaC104. The ionic strength of the solutions used to determine the stability constant of CU(H-,GGG)~~was 0.10 M NaCIOa plus that of the added triglycine (pLmaa= 0.17). Hydrogen ion concentrations were calculated from pH measurements by the relationship -log [H’] = pH -0.11,6 and hydroxide ion concentrations were calculated from pK, = 13.78. Glycinamide.HC1 was obtained from the Cyclo Chemical Co. The preparation of Cu(H--1Ga)2(where Ga is glycinamide) followed the method of ref 6 with the exception of using CuC12.2H20 instead (4) G. R. Dukes, G. K. Pagenkopf, and D. W. Margerum, Inorg. Chem., 10, 2419 (1971). ( 5 ) R. G. Bates, “Determination of pH,” Wiley, New York, N. Y., 1964. - .n c 92. - (6) T. Komorita, J. 42, 168 (1969). ~
Osterburg and B. Sjoberg, J . Biol. Chem., 243, 3038 (1968).
Journal of the American Chemical Society J 94:24 J November 29, 1972
Hidaka and Y. Shimura, Bull. Chem. SOC.JaP.,
8415 Table I A. Acid Dissociation Constants of the Tripeptides, 25” Ligand PKa 7.88 8.02 8.08 8.05
Glycylglycylglycine (GGG) Glycylglycyl-L-alanine (GGA) Glycyl-L-alanylglycine (GAG) L- Alanylglycylglycine (AGG) B. Species CU(H-1GGG) CU(H-zGGG)CU(H-zGGG)OH CU(H-iGGG)”CU(H-zGGA)Cu(H-zGAG)Cu(H-zAGG)-
’-
Log Log P
Ref
P Values of the Copper-Tripeptide Complexes
-0.01 -6.67 -7.03 -18.7 -4.43 -4.51 -6.91 -6.76 -7.01
P,
M
Ref
0 . 1 (KN03) 0 . 1 (KNO3) 0.16 (KCl) 0 . 1 (KN03) 0.10-.0.17 (NaC104) 0 . 1 (NaC104) 0.16 (KCl) 0.16 (KCl) 0.16 (KC1)
(7) B. G . Willis, J. A. Bittikoffer, H. L. Pardue, and D. W. Margerum, Anal. Chem.. 42. 1430 (1970). (8) W. M: Scbvell, G . C’Stocco, and R. S. Tobias, Inorg. Chem., 9, 2682 (1970). (9) D . D. Perrin and I. G. Sayce, Talanta, 14, 833 (1967).
a
b a
Spectrophotometricc Kinetics b b b
G. F. Bryce and F. R. N. Gurd, J. Bioi. Chem., 241,
H. Hauer, E. J. Billo, and D. W. Margerum, J. Amer. Chem. SOC.,93,4173 (1971). 1439 (1966). c This work.
of Cu(CzH302)2~Hz0. The fine purple crystals obtained were washed with ether and dried over PzO;. Kinetic runs were followed spectrophotometrically at 550 nm using a modified Durrum-Gibson stopped-flow spectrophotometer with a 2.0-cm cell path. The photomultiplier output was interfaced to a Hewlett-Packard 2115A general purpose digital computer as described elsewhere.7 All reactions in this study were run under first-order or pseudo-first-order conditions and each rate constant is the average of at least four kinetic runs. The standard deviations were calculated from the deviation of the individual rate constants from the mean. [The standard deviations of the rate constants calculated from the data points (100-250) for the individual runs are much smaller.] The stability constant of Cu(H-,GGG)zZ- was determined spectrophotometrically using a Cary Model 14 spectrophotometer thermostatted at 25.0” with cells of 10.0-cm light path. Circular dichroism spectra were obtained on a Cary Model 60 spectropolarimeter equipped with a circular dichroism accessory. Measurements were made at room temperature using a cell of 1.00 cm. Results are reported in terms of CL - CR, the difference in molar absorbance (based on [ C U ] ~ , ~between ,~) the left and right circularly polarized beams, Infrared spectra of Cu(H--lGa)Pwere measured with a Beckman IR-12 spectrophotometer as Nujol suspensions between polyethylene windows. The Raman spectra were obtained with an instrument built around a Spex Model 1400 double monochromator and a Coherent Radiation Model 52 argon laser (5145 and 4880 A). Signal detection and amplification are described elsewhere! The spectra were taken of powder samples in thin capillary tub:s by the transillumination technique. To verify that rhe Raman spectral peaks seen were due to the complex and not to artifacts caused by the nature of the sample, spectra were taken at both the 5145 and 4880 A lines of the argon laser and only the coincident bands considered. The band positions (Raman and infrared) reported in this work are the average of four different spectra. Attempts to obtain the Raman spectra o,f aqueous solutions of Cu(H-lGa)z, using both the 5145 and 4880 A lines, were unsuccessful due to the solution absorption of both the exciting and Ramanshifted light. The tripeptides used in this study, their acid dissociation constants, and the log values for their copper complexes (where is a cumulative stability constant with proton loss, Le., for Cu(H-lGGG), p = [Cu(H-,GGG)][H+]/[Cu*+][GGG-]) are listed in Table I. The constants are in terms of concentrations of all the species. The concentrations of the various species in solution were calculated by use of the species distribution program COMICS.~
U
40-
‘35
-
%
10
2.0
102[Triglycine]
3.0
4.0
,M
Figure 1. Dependence of kobsd on pH and [GGG] at 25.0”, /* = 0.10 (NaCIOa). Experimental points are given and the solid lines are the linear least-squares lines: 0 , pH 8.52; 0, pH 9.45; A , pH 10.00.
Results and Discussion Kinetics of Hydroxide Catalyzed Mono- and Bis(triglycinato)cuprate(II) Interconversion. 1. Reversible Conditions. The kinetics of the reaction shown in eq 1 have been determined under pseudo-first-order reversible conditions. The experimentally determined rate expression is - ~ [ C U ( H - ~ G G G ) - ] / ~ ~= k,b,d[Cu(H&GG)-] where kobsd = (kr[GGG-] -k k,)[OH-] as shown in Figure 1. At a given hydroxide ion concentration, a series of at least three reactions were run with varying triglycine concentrations. Values of km’ = kr[OH-] and k,’ = k,[OH-] were resolved from these data either graphically or by the method of simultaneous equations. The data used to obtain these values are given in Table 11. Figure 2 demonstrates Dukes, Margerum
N-Peptide Bonding to Copper(II)
8416
IO5 [OH-],
M
Figure 3. The effect of hydroxide ion concentration on the rate constant of the triglycine-catalyzed exchange reaction between Cu(H-&GG)- and EDTA at constant excess triglycine at 25.0" and p = 0.10 (NaC104). The points are experimental and the slope of the least-squares line equals kl (eq 2 and 4).
Figure 2. Demonstration of first-order dependence of kf' and k,' upon [OH-] at constant triglycine concentration at 25.0" and p = 0.10 (NaCIQi). Experimental points are given and the solid lines are drawii with a slope of 1.O: 0, k r ' ; 3 , ki'.
Table 11. Triglycine [GGG-] and pH Dependence of the Observed First-Order Rate Constant for the Reaction of Cu(H-*GGG)- with Excess G G G -
kr' and k,' US. [OH-] yields kf = (1.26 f 0.02) X lo7 M - 2 sec-' and k, = (8.8 j= 0.3) x I O 4 M-1 sec-1. 2. Forward Reaction Using EDTA as a Scavenger. The proposed4 rate-determining step in the triglycinecatalyzed exchange reaction between Cu(H,GGG)and EDTA below pH 9.7 is the formation of the bis(triglycine) complex with subsequent rapid attack by EDTA4- (eq 2 and 3). This reaction is not reversible Cu(H-%GGG)-
a
M
kobsd,sec-'
8.53 8.51 8.53 8.78 8.80 8.77 9.00 9.00 9.00 9.28 9.26 9.27 9.46 9.46 9.46 9.43 9.51 9.53 9.51 9.84 9.80 9.80 10.00 10.00 10.00 10.21 10.17 10.16 10.43b 10.41b 10.42b 10.76b 10.76b 10.77b
1.43 2.86 4.30 1.70 2.54 3.39 0.88 1.76 2.64 0.848 1.70 2.54 0,848 1.70 2.54 3.39 1.43 2.86 4.30 0.848 1.70 2.54 0.88 1.76 3.28 0.424 0.848 1.27 0.424 1.27 2.12 0.424 1.27 2.12
1.68 i 0.01 2.65 i 0.03 3.85 i 0.03 2.90 i 0.02 4.19 i 0.03 5.05 i 0.04 2.50 i 0.03 3.65 i 0.04 5.10 f 0.02 5.27 f 0.01 7.7 i 0 . 1 10.6 =k 0 . 1 8.75 i 0.07 13.0 i 0 . 1 19.0 i 0 . 1 21.6 i 0 . 1 10.0 i 0 . 1 17.1 i 0 . 2 23.4 i 0 . 4 20.8 i 0 . 2 30.4 i 0 . 3 41.8 f 0 . 3 17.4 f 0 . 2 28.0 i 0 . 8 43.8 f 0 . 8 31.5 f 0 . 5 39.6 i 0 . 8 51 i 2 43 f 2 75 f 1 112 i 3 104 f 8 182 i 5 310 i 20
25.0 i 0.1", p = 0.10 (NaClOJ. [Cu(H-*GGG)-] = 9.85 X M except where noted. b [CU(H-~GGG)-]= 4.92 X M.
the first-order dependence of kf ' and k,' upon hydroxide ion concentration. A linear least-squares treatment of Journal of the American Chemical Society
kl
CU(H-1GGG)z2-
+ OH-
(2)
rapid
+
CU(H-IGGG)~~- EDTA4- +products
102[triglycine], PH"
+ GGG- + OH- +
( 3)
because EDTA acts as a scavenger for Cu(H-1GGG)22-. The experimentally observed rate expression for the EDTA exchange reaction with excess triglycine (pH 8.2-9.6) is - ~ [ C U ( H - ~ G G G ) - ] / ~=~ kobsd[CU(H-2GGG)-] where kobsd = kd kl[GGG-][OH-]. The kd value is 0.12 sec-I. lo The rate constant expression may be rearranged to yield eq 4, which is plotted in
+
(kobsd - kd)/[GGG-]
kdOH-1
(4) sec-', Figure 3. This gives a kl value of 1.3 X lo7 which is in excellent agreement with the value determined for kf via reversible kinetics. The use of EDTA as a scavenger to prevent reversibility has been reported previously in the study of the proton transfer reactions of Cu(H-,GGG)- lo, and Ni(H-2GGG)-, l 2 and in the ligand exchange reactions of Ni(H-2GGG)-. EDTA acts as a scavenger in these reactions because a tertiary nitrogen (as in EDTA) is sterically hindered from directly attacking the planar site vacated by the carboxylate groupl4 but will rapidly attack a species which does not present these steric difficulties (i.e., Cu(H-1GGG), Ni(H-lGGG), and Cu(H-lGGG)GGG-). 3. Reverse Reaction Using trien as a Scavenger. Triethylenetetramine (trien), on the other hand, is a very effective nucleophile in its reaction with Cu(H-2GGG)- (k = 1.1 X IO7 M-I ~ e c - ' ) . ' ~ In contrast t o its reaction with CU(H-~GGG)-, the reaction of trien with Cu(H-1GGG)22- to form Cu(trien)2+ is much =
(10) G. I
