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Cupric complexes of poly(L-Lysine, L-Tyrosine): Spectroscopic determination of structure in aqueous solution
Cupric Complexes of Poly(GLysine, LTyrosine): SpectroscopicDeterminationof Structurein Aqueous Solution Arlette Gamier and Lucia Tosi D&amnent de Recherches Physiques. Laboratoire associk au CNRS W 71. Universite’ Pierre et Marie Curie, Paris (L. Tosi and A. Gamier), UER de Medecine et BioiQie Hurnaine Universit6 Paris Nerd, Bobigqv, France (A. Gamier)
ARCRACT The formation and structure of two CucII)-(L-Lys, L-Tyr), complexes have been investigated using potentiometric. absorption, circular dichroism (CD), and resonance Raman measurements. Two complexes
have been detected. The first, which is fully defined at
pH 7.8, contains four nitrogens-two from amino groups of lateral chains and two from peptide groups-and a phenolate oxygen, presumably in apical position, bound to the metal. The second complex that forms at pH 11.6-12.2 contains a cupric ion coordinated to four peptide nitrogens.
INTRODUCTION The invo1vemer.t of the phenolate oxygen of tyrosine residues at the metal binding sites of Fe(W), Cu(II), and several trivalent metal transferrins have been inferred from physicochemical studies [ 1, 2]_ Several spectrometric techniques, such as difference spectra [3-5 ] , nmr [6] , and fluorescence spectra [7], have supported this assumption. ‘Ihe visible absorption spectra of various metal transferrins in water solution present a moie--l - cm-l - 1) centered at 410470 ~TI strong absorption (E - 2000-8000 18, 9]_ Resonance-enhanced Raman spectra (RR) on excitation in the metal-transferrin absorption band has been applied to further elucidate the nature of ligands intervening in complexation. Tomimatsu et al. [lo] obtained the fir& RR spectrum of Fe(W) transferrin and by comparison with the Rarnan spectra of qrosine and histidine, Carey et al_ [ 1 l] assigned the RR-enhanced bands to tyrosine and histidine vibrational modes. Subsequently, in a RR study on Fe(II1) and Cu(I1) transferrin and on the Fe(II1) complex of ethylendiamine bis(o-hydroxyphenylacetate), Gaber et al. 1123 were able to demonstrate that all the enhanced Raman bands originate from phenolate vibrational modes only. More recently, the compound bis(2,4,6-tri-ehlorophenolato)diimidazole Cu(I1) was prepared as a possible model for Cu(I1) binding to transfer&s [ 131, and its crystal and molecular structure has been determined [ 141. The absorpJournalof InorganicBiochemistry10, 147-158 0 Elsevier North Holland, Inc., 1979
(1979) 0162-0134/79/020147+12$01.75
147
A_ Gzunier and L. Tosi
148
tion spectrum of this complex in acetone solution is very similar to that of Cu(II)transferrins presenting the strong b3nd referred to above_ By studying the RR spec-
trum of this complex 3nd the Raman spectra of the ligands. the specific vibrational modes of the pftenol3te moiety were characterized and 3 norm31 coordinate analysis enabled their unambiguous rtssignment. These data confirmed Gaber et al. [ 121 results clearly identifying tyrosine as 3 ligand in transferrins, and the strong band in their visible absorption spectrrt 3s arisin, 0 form 3 ligand-metal charge-transfer transition [I;] _ The Fe(II1) and Cu(I1) model compounds mentioned ertrlier do not contain the tyrosine moiety_ In f3ct. from the three isomers of tyrosine. only the phenolate group of orrlro-tyrosine psrticipes in bondin g to copper [ 15. I6]_ In mera- and para-tyrosine. the phenolate osygen is unable to bind to the metal. presumably due to steric constraints [ IS]_ In the case of transferrins. however. it tlppears tflat tfie engagement of 05 amino and carbonyl groups in the peptide bond enrlbles the free access of the tyrosine phenol3te oxygen at the met31 binding site. In this article we report the identification of 3 comples contrtining 3 para-tyrosine phenolate osygen bound to copper. The results were obtained by studying the interaction of cupric ions with two poly (arunino acid;): !L-Tyr. L-L_vs), and (L-Tyr),. using potentiometric, absorption. CD, and resommce Rrtmsn me3surements.
EXPERIXlEi\;TAL Poly( L-Tyr, L-Lys) I IBr 1: 1 and poly( L-Tyr) were purchased from Miles-Yeda Ltd. atid were used without further purification The average molecular weight of the L(Tyr. L-Lys) copolymers obtained by the Archibald method using 0.1-N KC1 3s supporting electrolvte was 25,000 at pf1 6. Sedimentation velocity was measured in a Beckmrtn Spin& Model E ultracentrifuge equipped with Schlieren optics. The average molecular weight of (L-Tyr), was 2 i 200, as indicated by the manufacturers. Potentiometric titrations were performed on a Tacussel ~11 meter. type ISIS AdAgCl-glass Methrom electrode, Model El47. The 20.000, using 3 combined electrode calibration ~3s made following Irving et al. procedure [17] : NaOI-1 WCIS rzsgent gmde (hlerck Co). Concentmtions of solutions were detemlined by 3 micro Kjeldahl method_ Absorption specrrrt of 185-900 nm were recorded on 3 Can; 13 spectrometer_ Circular dichroism specrirt of 185-800 nm were obtained by means of3 Jobin Yvon Dichrograph Model Mark III; it ~3s crtlibrated with 3 solution of epiandrosterone in dio.xane 3s indic3ted by tfte manufacturers [IS] . For CD measurements. the solutions were prepared to give values of optic31 density not exceeding 1.5. For messurements under 200 nm. 311 spectrometers were purged from oxygen prior to recording. Results are reported in terms of E (molar ttbsorption coefficient) and .k = ei - E, (molar circular dic!troism coefficient ). Tile values of E 3nd & are expressed either in temls of [Cu] or in terms of [LyTy] or of [Tyr] _ whiclr represent the molar residue concentration of tyrosine in eiich polymer. Kesommce Rrrm3n spectm were recorded with 3 Coderg PI10 spectrometer equipped with holographic gratings. using the escitation lines of 3 Spectra Physics 3rgon ion frtser. Samples vtere conmined in 3 specially designed Hellma R3m3n cuvette using 90” viewing optics. Rrtmrtn intensities were mettsured using tire vr mode of
Cupric Complexes
of Poly(L-Lysine,
149
L-Tyrosine)
cm-l) as internal standard. Potentiometric and spectroscopic meas(936 urements were made at 25°C under argon atmosphere_ All solutions were prepared with doubly distilled water. Gaussian analysis was performed by the use of a tenchannel DuPont de Nemours curve analyzer_ ClO*-
RESULTS Cu(II)-(L-Tyr,
L-Lys), System
Porerzriomerric Tirrafions. The copolymers (L-Tyr, L-Lys), HBr 1: 1 is soluble in water of the order of 10e3 M [19] I it redisand precipitates at pH 9.2 at concentrations solves at pH 11.6 giving clear solutions. In the presence of Cu(II) ions a complex is formed from pH 6 and is fully formed at pH 7.8 before precipitation occurs (at pH 8.5). The results of potentiotnetric titrations are shown in Fig_ 1. where curve a gives the number of protons neutralized per LyTy residue as a function of pH and curve b, the number of protons neutralized per copper ion at molar ratio [LyTy]/ [Cu] = 4. As can be noticed, when (L-Tyr. L-Lys), is titrated in the absence of cupric ions, the releasing of protons starts at about pH 7 and approsimately at pH 6 in the presence of copper: up to pH 7-S, rive protons per cupric ion are removed. yielding complex A.
FIGURE 1. Potentiometric titration curves of (L-Tyr. L-Lys), in absrncc (curve a) and presence (curve b) of cupric ions. Curve ;i gives number of protons neutralized per LyTp residue, [ LyTy] = 2 X lo-3 31; curve b gives number of protons neutralized per cupric ion at [!_yTy]/[c~] = 4, [ LyTy] = 4 X IO-3 M. __._region of polymer precipitation.
5
6
7
8 P"
9
10
150
A. Gamier and L. Tosi
Absorption
and Circular DichrotXm
Complex A. TIlis complex is formed at molar ratios higher than 3. At wavelengths higher than 300 nm, absorption spectra show one band and two shoulders at 390 nm, 320 nm_ and 520 nm. respectively_ The molar absorption coefficient per cupric ion (e!Cu) and the moIar CD coefticient per cupric ion (&/Cu) of these bands increase 3s the pH is raised from 6 to 7.8; at pH 7.8-8.5 (where precipitation occurs), no further variation is noticeabIe. The plots of e/Cu at 390 nm and &z/Cu at 520 run and 320 nm as a function of pH at [LyTy] /[Cu] = 4 are reported in Fig. 2. The amount of compies A that is formed depends on [LyTy] /[Cu] molar ratio. At pH higher than 7.8 the amplitudes of the absorption and CD bands increase as the molar ratio is raised from 2 to -I and then become independent of molar ratio. Figure 3 gives the plots of &/Cu at 520 and 320 nm as a function of [LyTy]/[Cu] at pH 8. From these data we can conclude that at pH higher than 7.8 (and lower than S-5) and at [LyTy]/[Cu] higher than 4. ali the cupric ions are bound to the copolytner forming complex A. Figure 4 shows the absorption and CD spectra of this complex. Spectral data are reported in Table
1 _ A preliminary
FIGURE
report on this complex
has been published
[20] -
II). Variations of mohr absorption and moIar CD coeffiat [ LpTy]/(Cu] = 4, [Cuj = IO-3 M. Figureson curves indicrtte wavelengths of the different bands; . . . . region of polymer precipitation. cients
2. Systsm ( L-Tyr, L-Lys),-Cu(
of different
+I
bands
3s pH function
I I
to.5 [ 01 <
----_-_------AE 320
/
1
.
E390 --------
600
;ii+ 500
_-_--------AE 520
-1 ;
400
! I
300
-1.5t I
200
-2c
'I S
6
100
I 7
8
?W
9
lo
11
12
< w
Cupric Complexes
of Poly(L-Lysine,
L-Tyrosine)
151
+l PH 8
GO ZJ
y\--??_-_
pH8
-1
-2
pH12.2
12
7
3
a
~W&J]~ FIGURE 3. System (L-Tyr, L-Lys),-Cu(II). Variations of molar CD coefficients of different bands as function of molar ratio at pH 8 and 12.2. [Cu] = 10-a M. Figures on curves indicate wavelengths of the different bands.
Complex
B. Whatever
the
value
of
the
molar
ratio
[LyTy]
/
[Cu], a
precipitate
occurs at pH 8.5 and redissolves at pH higher than 11.6. At wavelengths higher than 300 nm, absorption and CD spectra of solutions at pH 11.6 are as those of complex A. When pH is raised from 11.6 to l___, 3 q the values of Ae at 520 run and 320 run and of E at 550 nm increase, approsimately doubling those of complex A (Fig. Z), and a new negative bands appears at 360 nm; simultaneously the 390-nrn absorption band disappears_ At pH higher than 13.3 the values of E and Ae level off, indicating that a new complex (labeled B) has been fully formed. As in the case of complex A, the amount of complex B that is formed increases as the molar ratio is raised from 2 to 4 and then
becomes function reported L,
independent of molar ratio. The plots of Ae3ao and &e510 at pH 13.2 as a of [LyTy] /[Cu] are illustrated in Fig. 3. Spectral data of complex B are in Table I_
Transition. In the absence
rise to a positive
of copper.
the L,
transition
CD band at 227 nm with Ae/LyTy
of the tyrosyl
moiety
= +3.6 in the protonated
gives
form and
to a positive band at 245 nm with Ae/LyTy = i-3.9 in the deprotonated fomr [19] _ In the presence of cupric ions at [LyTy] /[Cu] = 4 and at pH 6.3. the CD spectrum dis= +3.7. As the pH is raised, the 345-m-n plays a positive band at 228 nm with &/LyTy
A. Gamier and L. Tosi
600 500 400
\; cl)
300 200 100
300
TABLE
1. Optical I
--__cu(11J-I
400
and
CD
Spectral
L-Tyr.
L-Lys),
A
II
Ab
h frm~) 2xc
Xb
hfnm)
CD
h&l
.lE L-Lys),
B
Ab CD
B
OpticA
h (nm) E
CD
L-Lys),
. Cu( II)-
and CD Spectral
550fsh)
390
1-w
6007
600 i-o.15
510 -0.90
Data
100 310 +0.55
290 -0.20
530 1’0 590
320 790 io.55 -0.45
510
+0.10
-0.95
h (nm) E X(nm)
600
520
360
315
AE
+0.50
-7--- 70
-0.10
+i 20
370 -0.7
325 io.95
520 ‘30
Ab
h (run)
520
CD
E Xtnm) &
230 525 -2.1
C The mohr rtbzorprion coeffickn: apressed in rcrms of the molar circular dichroism. sh = shoulder.
L-Tyr,
Complexes=
CornpIe:\
----
CUf 11)-C L.-L?-&
Cu(IIJ-(L-Tyr),,
of Cut 11)-t
L-Lys), . and Cu(iI)_(L-Tyr),,
Sva2rn
CutiI)-(I_-Tyr.
data
600
500
nm
290
790 - 1.40
(E) and the molar circular dichroism coriticienr (AE) art’ concenrrrtGon oi the cupric ions; Ab = absorption. CD =
Cupric Complexes
of Poly(L-Lysine,
L-Tyrosine)
153
band appears: at pH S-5 the CD spectrum cm be resolved into two positive gaussian curves lying at 231 nm and 345 nm (Table 2). Ar pH higher than 12.3. when complex B is fully formed. the CD spectrum diwlays a strong positive band at 235 nm that can be resolved into two positive Gaussian curves at 215 nrn and 230 nm (Table 2).
TABLE 2_ Circular Dichroism Spectral Data of (L-Tyr, L-LYS), in Both Absence and Presence of Copper at [LyTyl/[Cul = 4, [LyTyl = 4x 10-a M. PH
h (nm)
QTYr
3.30
0.0
8.50
0.4
Ae/LyTy
2wl&(cm-l)
2
+3_6 -4.0 +I.6
3100
-3.0 +3x +3_7
3000 2500
1.0
6.36
0.0
8.sb
0.4
+‘.6 +1.7
1600 3200
I >_zb
1.0
+4.6 +3.&x
4000 3200
b
cm-1
‘700
12.20
a
100
In the absri.cc of copper. In the presence of copper.
c Due to n - n* peptide transition. d Due to L, transition of the deprotonated tyrosyl residue. f Due :o L, transition of the protonsted tyrosyl residue_ f Band width at half of the peak height.
Resommce obtained
RamanSpecrra. on excitation
contour
of the absbrption
displayed
by complex
shops the Raman Kaman excitation lustrated cx11- 1 .
and
laser
band
A using
The
at 390 the 457.9
bands
line approaches 1172
cm-’
whose
Xmas
Raman spectra the wavelengths
nm.
Curve
of complex
of which
a in Fig. 5 shows
nm exciting
intensities
lie at 1602
_ X11 the other
rise to Ramsn bands on the spectra 12 12 cm-‘. S5S CIII-~. S36 cm-l. trum of complex A.
Cu( II)_( L-Tyr),
radiation.
A can be
fall tvithin
the
the RR spectrum
line of an Ar ion Iaser:
curve
b
spectrum of the polymer at pH 4 in the absence of copper_ The part of the absorption band tire ilprofile rend the corresponding
in the insert.
the exiting
Resonance-enhanced
with
are enhanced
cm-l.
vibrations
as the wavelengrh
cm-l.
of the tyrosine ( L-Tyr.
of the polymer
and 6-16
1501
cm -l
1260
cm-l.
of
1320
side chain that give
L-Lys),
at pH Li. lying
at
are not observed on the RR spec-
System
The polymer (L-Tyr), becomes soluble a.t pH higher than 12.‘. At pH higher than 9 Cu(l1) binds to (L-Tyr), and a complex similar to complex I3 of the Cu(II)-(L-Tyr. L-Lys),
system
shown
in Table
is formed.
The
1_ The amount
spectral
of complex
data
of wavelengths
formed
depends
higher
than
on [Tyr]
/ [Cu]
300
nm are
molar
ratio
A.
I54
Gamier and L. Tosi
, 1600
1400
a00
1000 cm -3
1200
FIGURE 5. Curve a resonance Raman spectrum of Cu(II)_(L-Lys. L-TM, system ot [ LpTyJ /[Cu J = -I. [Cu j = 0.83 X 10-3, pH 7.9, using 457_9-nm exciting line from 1 lO-mW .-Ir* laser: curve b. Raman spectrum of (L-Lys. L-Tyr), HCI; [ LyTy j = 8 X 10-Z M, pH 4, excitation line 513.5 nm, 500 m\V_ Instrumenta conditions: spectral slit width 4 cm-l; scan rate 50 cm-l!minr time constant. 3 sec. Insert: Raman excitation profiIe of the Cu(II)-fL-Lys, L-Tyr) complex !Uolx Roman intensity ratio (I Rc) of band in question rehtive to the 936-cm--1 band of ClO,--. corrected for sampie self-absorption. instrumental spectral response, and v dependence. Experimental points are scaled to unity at 472.7 nm: (0) 1601. (2) 1502, (s) 1260, (o) 1176 cm-l. (*) CiOa- band. Laser wavelengths are indicated.
and becomes
independent
L,
lies at 245
transition
this band disappears
of it at molar
ratios higher than S. The CD band of the pH higher than 11.7: when the pH is lowered to 11.2, in favor of the band af 227 nm. nm at
DISCUSSION Complex
A At moiar ratios [LyTyJ/[CuJ higher or equal to 4 znd at pH higher than 7.8, which corresponds to the departure of five protons per copper. all metal ions are bound to the copolymer forming complex A. The (L-Lys. L-Tyr), system contains three potential metal binding sites: the amino-nitrogen of the Iysine residue. the phenolate oxygen of the tyrosyl one. and the peptide nitrogen_ The origin of the five protons released by complex A formation and, therefore, the nature of the Lands
of Cu(lI)
this
complex
with
the
Cu(iI)-(L-Lys),
can be inferred that
[21]_
of
the
by comparison
complexes
Cu(II)-(L-Arg),
we
of the CD spectral obtained
[33] _ and
in
our
data of
studies
Cu(II j(L-Om),
on
[23]
Cupric Complexes of Poly(L-Lysine, L-Tyrosine)
155
[23] systems at pH 10, which were labeled “complex II.” In the three cases the binding of two amino nitrogens of lateral chains and two adjacent peptide nitrogens to Cu(II), forming a square planar complex of nearly Dab symmetry, has been established on grounds of potentiometric, absorption, and CD measurements. The CD spectral data of all these complexes are very similar. We have assigned the bands at 590 nm and 520 run to two d-d transitions: the first to 3d,, + 3d,2 _ ,,2 and the second, to 3d,,, 3d,, + 3d,2 _ .2 . These transitions are magnetic-dipole allowed but electric-dipole forbidden, and their energies are essentially determined by the four ligands in the coordination square and are not much affected by the Iigand in apical position [24] _ On the contrary, the energy of the 3d,2 + 3d,z _ .2 transition is sensitive to the presence of axial ligands which interact directly with the d,2 metal orbital. However, this transition is both magnetic- and electric-dipole forbidden so, as a rule, it gives rise to, at most, very weak bands in the CD spectrum. Accordingly, we can interpret our data in the following way: two amine nitrogens from lysine residues and two adjacent peptide nitrogens are bound to the cupric ion in the coordination square. This account for four of the five protons released at pH 7.8 as complex A is formed. It must be recalled that, unlike complex II in Cu(II)-(L-Arg), , Cu(II)-(L-Om), and Cu(II)-(L-Lys), systems, where binding of lateral ammo groups and peptide nitrogens occur in two well-defmed steps, the four nitrogens of complex A bind simultaneously. Moreover, complex A is formed at pH 7.8, whereas complex II begins to appear at this pH approximately and is fully defmed only at pH 10-10.5. Looking now at the absorption spectral data of complex A of the Cu(II)-(L-Tyr, L-Lys), system and of complex II of the Cu(II)-(L-Lys), system (Table 1), we notice that the first one displays a shoulder at 520 nm that corresponds to the d-d transition band at 550 nm of the second system. In addition the tyrosine containing system exhibits a new band at 390 nm that strongly suggests the involvement of the phenolate oxygen in Cu(I1) binding, and this accounts for the fifth proton titrated_ Since the four planar positions have been occupied by four nitrogens, the only coordinating site available for the oxygen is an apical one. The value of the extinction coefficient (e/Cu = 600) and the energy of the transition at 390 run are too high to assign the corresponding band to a d-d transition; rather, they should be accounted for by a chargetransfer transition between the phenolate oxygen and Cu(I1). The presence of such an absorption band has already been observed by Boggess and Martin [16] in the spectra of the Cu(II)-( ~~-orrho-tyrosine) system at pH 10.5, but they did not observe such a band on complexation of Cu(I1) with para-tyrosine. The position and the intensity of the 390~run band are consistent with a rather high metal-oxygen distance and, therefore, with fmation of a phenolate oxygen in apical position; in fact, in copper complexes the ligands in apical position are generally at a greater distance from copper that those lying in the coordination square 1241. According to Barnes and Day [25] ~ the charge-transfer band energy is a function of the metal-ligand bond length (for the same ligand and the same metal), with higher frequency as the bond length increases. Absorption spectra of transferrins bound to different metal ions aiso exhibit a strong band at this wavelength range: at 440 run for Cu(II), 465 nm for Fe@), and 405 nxn for Co(III) 1261. This band has been assigned to a charge-transfer transition between a phenolate oxygen and the metal ion [26], or more precisely, to a chargetransfer transition from the psr orbital of phenolate to a do* metal orbital [12] _ Taking into consideration Barnes and Day’s statement 1251, Prados et al. [26] assume
156
A_ Gamier
and L. Tosi
that in the case of the cupric transferrin complex, the phenolate oxygen is bound to the copper at a relatively short distance from it, specifically, occupying one of the corners of the coordination square. It has to be noticed that in the Cu(Ii)-(L-Tyr, L-LYS), system the 390-nm bard of complex A is not optically active_ Inasmuch as this transition is optically active in the case of Cu(II)-transferrins, this behavior most probably reflects the presence of a longer Cu(II)-0 bond in complex A. Curve a in Fig_ 5 shows the RR spectrum of complex A_ As can be noticed, the band at 1602 cm-1 that is assigned primarily to a ring C-C stretching mode 112, 131 lies at 1619 cm-1 in the Raman spectrum of (L-Lys, L-Tyr), (curve b) when aU the tyrosine side chains are protonated (pH 4)_ This peak corresponds to the 1615-cm-1 band of the fully protonated tyrosine groups in the ir spectra of (L-Lys. L-Tyr), in Da0 solution that shifts to 1603 cm-l on deprotonation [ 191. The band at 1501 cm-1 is also largely due to a symmetric ring stretch [ 12, 131, although considerable contribution from a C-O stretching coordinate is present [13]. This band is not observed in the Raman spectra of the polymer at Iow pH (Fig_ 5, curve b), but is present in the ir spectra of (L-Lys, L-Tyr), in D20 solution. lying. at 1517 cm-l for the fully protonated tyrosine residue and shifting to 1502 cm-l on deprotonation of phenolic groups [ 19]_ A broad band at 1250 cm-l appears in the Raman spectrum of (L-Lys. L-Tyr), when the tyrosine side chains are fully protonated and shifts to 1260 cm-’ in the spectrum of complex A. The C-O stretching mode contributes predominantly to this band [ 1 2, 13]_ The peak appearing at 1180 cm-l in the spectrum of the fully protonated polymer (curve b) shifts to lower frequencies after complexation (1172 cm-l in curve a), which is consistent with the assignment of this band to a C-H bending mode of the benzene ring [ 13]_ The RR spectral pattern of complex A is characteristic of metaI-transfer&s, with the peak frequencies practically identicrd to those of Cu(II)-ovotransferrins [12, 131. There are some small differences, however_ In the spectrum of Fig. 5 (curve a) the band at 1260 cm-’ is less intense relatively to the others than in the spectra of metaltransferrins. As a consequence_ the shoulder at 1320 cm-l in the spectra of the latter appears as a we%resolved band in curve a. We can qualitatively explain this behavior on the basis of a longer phenolate oxygen-copper bond in complex A than in metaitransferrms. The modes which are expected to be enhanced by the RR process are those that couple significantly with the electronic transition_ The C-O stretching mode should be less strongly involved in comples A. since the electronic transition probability would be less altered by the vibration as a consequence of the smaller metaloxygen orbital overlap. Circular dichroism spectra at wavelengths lower than 250 nm can be used to obtain infomrstion on the secondary structure of the copolymer. As we have shown earlier [ 191. at pH 8.5 and in the absence of copper. the copolymer is in the P conformation: this is corroborated by the presence of a negative band at 220 nm due to ?I + rr* transition of the peptide group (Table 2). We have also observed the presence of the positive band at 245 nm due to the L, transition of deprotonated tyrosine (from the data of Table 2 it follows that about 40% of tyrosine is deprotonated). In addition, the positive band at 227 nm of the still protonated tyrosine is masked by the 220-nm negative band. On the other hand, at the same pH and in the presence of copper at [LyTy] / [Cu] = 4, the 215-n~1 band is still present and the positive one at 227 nm is now clearly
Cupric Complexes of Poly(L-Lysine, L-Tyrosine)
157
apparent, due to the absence of the negative band at 220 nm. The absence of this 220~run negative band indicates that complex A maintains the copolymer inthe nonperiodic conformation_ At molar ratios higher than 4 and as the pH is raised to 8.5, some p structure is apparent, in much larger proportions as the molar ratio [LyTy]/ [Cu] ishigher_ We have already observed such a behavior in the case of Cu(II)-(L-Arg), [9-l], Cu(II)-(L-Orn), [22], and Cu(iI)-(L-Lys),, [20] systems. Complex B. At a further stage, and by raising the pH from 11.6 to 12.2, we observe the formation of a second complex_ Insofar as the absorption and CD bands due to d-d transitions lie nearly at the same wavelengths in the spectra of complexes A and B, we can assume that the cupric ion is still bound to four nitrogens lying at the corners of the coordination square. The values of Ae5ao and Aeaao are twice as strong, a result that can be interpreted as indicating the substitution of two amino nitrogens of lysine residues by two adjacent peptide nitrogens. In this case the cupric ion should be bound to two chelates, each of which would induce the same optical activity on the d-d transitions at 520 run. Other investigators 127,281 have already noticed such an additivity of the optical activity induced by two chelates in the case of Cu(II)-dipeptide complexes_ The same trend observed in the 320-m-n CD band corroborates our assignment of this peak to a peptide nitrogen-to-metal charge-transfer transition [L l-23,29] _ We have already observed the formation of such a complex at pH higher than 12, and at residue to copper molar ratios higher than 8 in the Cu(II)-(L-Arg), system (to be published). As complex B is formed, the 390~run-absorption band disappears, which suggests that the phenolate oxygen is no longer bound to Cu(I1). This compares with the result obtained by Prados et al. [26] on the Cu(II)-conaIbumin system: the absorption at 440 nm increases to a maximum intensity near pH 8.6 and then decreases as the pH is raised further_ This incompatibihty between phenolate oxygen and peptide binding when four peptide nitrogens coordinate to Cu(I1) is corroborated by the study of theCu(II)-(L-Tyr), system. In this system the complex formed at pH higher than 9 exhibits the same spectral patterns of complex B in the Cu(II)(L-Lys, L-Tyr), system (see Table I), and no band is detectable at 390 nm. At wavelengths lower than 260 nm, the CD spectra of complex B in the Cu(II)(L-Lys, L-Tyr), system exhibits a strong positive band, which can be resolved into two Gaussian curves (Tabie 2): the 245 nm band is due to L, transition of tyrosyl residues, which are almost all deprotonated at this pH; the 230 run band for which Ae/LyTy = i4.6 is more difficult to assign. It most probably originates from a polypeptide backbone transition_ This band does not appear in the Cu(II)-(L-Tyr), system: at pH higher than 11.7, one observes the 245-m-n band due to the deprotonated tyrosyI residue, which decreases as the pH is decreased, and the band at 227 mn appears. At pH lower than 11.2, only the 227-nm band is observed, which indicates that all the tyrosyl residues are protonated and that the polypeptide is in a nonperiodic conformation_ CONCLUSIONS The present report describes the characterization of two complexes obtained by interaction of cupric ions with two poly(o-amino acids): random (L-Lys, L-Tyr) 1: 1 and (L-Tyr),. Up to pH 7.8, a first complex (complex A) can be identified spectroscopically when the first polymer coordinates to the metal. In this complex the cupric ion
A.
158
Gamier
and L. Tosi
is bound to four nitrogens (two from amino groups from lateral chains and two from peptide groups) and a pehnolate oxygen. At pH higher than 11.6 in the case of (L-Lys. LTyr),
1 I 1, and higher than 9 in that of (L-Tyr),
a second comples
is formed (com-
to the cupric ion_ It appears
plex B) that contans four peptide nitrogens coordinated
that the binding of four peptide nitrogens to the metal to form the second comples precludes phenolate coordination_ It has been observed, in addition, that the formation of both complexes maintain the two polymers in the unordered structure_
REFERENCES
3. 3_ -I. 5_ 6_
R. C. Warner and L Weber. J_ .-lm. CXem. Sot_ 75.5094-5101 (1953)_ S. K;. Komatsu and R. E. Feeney, Biochemisrry 6, 1136-l 141 (1967). X_ Vishni;l. 1. Weber. and R. C_ Warner. J. Am. Chem. SOC. 83, 2071-2080 (1961). X. T. Tnn and R. C. Woodworth, Biochemistry 8. 37 II-37 17 (1969)_ S. S. Lehrer.J. BioL Cfwm_ 244. 3613-3617 (1969). R. C. Woodworth. Ii. G. Jfornllee, ;Ind R. J. P. Williams. Biochemisfr_v 9. 839-842 (1970).
7. S.
C. R. Luk, Biochemiqs 10. 2838-2813 ( 1971). R_ E. I-eeney- rend S. K;. Komatsu, Structure and Bonding.
1.
9. IO.
Vol.
1, Springer Verhg.
( !966), p_ 149. P. Xrsen. R. Artss. and A. G_ Redfield. J. Biol. Chcm. 244.46283633 (1969). Y_ Tomimrttsu. S. Kent. and J_ R. Scherer. BiocI~ern. BiopllJx Res_ Commun.
Berlin,
53. 1067-1074
(1973). Ii_ P. R. Carey and N. %I. Young. Can. J. Biochem. 52.773-280 (1974). 11. B. P. Gtlber. V. Misko\vski, rend T. G. Spiro. f. ,Im_ CIiem. Sot. 96.6868-6873 (1974). l3_ Y. Tomimatsu, S. Kent, and J_ R. Scherer, Biochemisrv 15.4918+924 (1974). I-4. R. Y_ Wang, K;. J. Palmer. and Y. Tomimrttsu,iicm Cr_vsraliogr_. Seer. B 32.567-571 (1976). 15. J_ E_ Letter and J_ E_ Bauman. Jr_, 1. Am_ Cltem. Sot. 92.443147 f 1970).
17.
16.
R. K. Boggess and R. B. blxtin. J. Am. Chem. Sot. 97, 3076-3081 (1975). H. 31. Irving. hi. C. Miles. and L. D. Pettit, Anal. Chim. ALYU 38.475-488 (1967).
18. 19. 20.
Instruction Manual of Johin- Y~OII Dichro.graph. A. Garnier and L. Tosi, Biopolymers 17, 199-211 (1978). L. Tori and A. Garnier, 2nor.g. Cirim. ACM Letr. 29, L261-L263
21.
.L\.Grtrnier nnd L. Tosi. Biochem.
72. 23. 24_
A. Garnier and L. Tosi. BiopoQmers
C. V. Phxr. L. Tosi, nnd .L\.Gamier, Bioirror.g_ Chem. 8, 21-31 ( 1978). B_ J. Hrtthawzy, in Srrncrure and Bonding Vol. 14, Springer Veriag. Berlin (1973).
25_ 16.
J. C. Barnes tind P. Drly,J. Chem. Sot.. 3886-3892 (1970). R. Pmdos, R. 6. Boggess, R. B. Martin, nnd R. C. Woodworth,
27.
18. 29.
Biophys. Rex Commun. 14.2247-2261
(1978).
74, 1280-1285
(1977).
(1975).
BioinorR. Chem. 4, 135-142
(197.5). J. M. Tsnngaris and R. B. !Uartin, J. Am. Chem. Sot. 92.42553260 (1970). G. F. Bryce and F_ R. N. Gurd.1 Biof. Chem. 241. 1439-1448 (1966). X. Gsrnier nnd L. Tosi, Bioinorg_ Chem. 8,493-501 (1978).
Received
i@ July I Y 7S
pp_ 49-67.
ARCRACT The formation and structure of two CucII)-(L-Lys, L-Tyr), complexes have been investigated using potentiometric. absorption, circular dichroism (CD), and resonance Raman measurements. Two complexes
have been detected. The first, which is fully defined at
pH 7.8, contains four nitrogens-two from amino groups of lateral chains and two from peptide groups-and a phenolate oxygen, presumably in apical position, bound to the metal. The second complex that forms at pH 11.6-12.2 contains a cupric ion coordinated to four peptide nitrogens.
INTRODUCTION The invo1vemer.t of the phenolate oxygen of tyrosine residues at the metal binding sites of Fe(W), Cu(II), and several trivalent metal transferrins have been inferred from physicochemical studies [ 1, 2]_ Several spectrometric techniques, such as difference spectra [3-5 ] , nmr [6] , and fluorescence spectra [7], have supported this assumption. ‘Ihe visible absorption spectra of various metal transferrins in water solution present a moie--l - cm-l - 1) centered at 410470 ~TI strong absorption (E - 2000-8000 18, 9]_ Resonance-enhanced Raman spectra (RR) on excitation in the metal-transferrin absorption band has been applied to further elucidate the nature of ligands intervening in complexation. Tomimatsu et al. [lo] obtained the fir& RR spectrum of Fe(W) transferrin and by comparison with the Rarnan spectra of qrosine and histidine, Carey et al_ [ 1 l] assigned the RR-enhanced bands to tyrosine and histidine vibrational modes. Subsequently, in a RR study on Fe(II1) and Cu(I1) transferrin and on the Fe(II1) complex of ethylendiamine bis(o-hydroxyphenylacetate), Gaber et al. 1123 were able to demonstrate that all the enhanced Raman bands originate from phenolate vibrational modes only. More recently, the compound bis(2,4,6-tri-ehlorophenolato)diimidazole Cu(I1) was prepared as a possible model for Cu(I1) binding to transfer&s [ 131, and its crystal and molecular structure has been determined [ 141. The absorpJournalof InorganicBiochemistry10, 147-158 0 Elsevier North Holland, Inc., 1979
(1979) 0162-0134/79/020147+12$01.75
147
A_ Gzunier and L. Tosi
148
tion spectrum of this complex in acetone solution is very similar to that of Cu(II)transferrins presenting the strong b3nd referred to above_ By studying the RR spec-
trum of this complex 3nd the Raman spectra of the ligands. the specific vibrational modes of the pftenol3te moiety were characterized and 3 norm31 coordinate analysis enabled their unambiguous rtssignment. These data confirmed Gaber et al. [ 121 results clearly identifying tyrosine as 3 ligand in transferrins, and the strong band in their visible absorption spectrrt 3s arisin, 0 form 3 ligand-metal charge-transfer transition [I;] _ The Fe(II1) and Cu(I1) model compounds mentioned ertrlier do not contain the tyrosine moiety_ In f3ct. from the three isomers of tyrosine. only the phenolate group of orrlro-tyrosine psrticipes in bondin g to copper [ 15. I6]_ In mera- and para-tyrosine. the phenolate osygen is unable to bind to the metal. presumably due to steric constraints [ IS]_ In the case of transferrins. however. it tlppears tflat tfie engagement of 05 amino and carbonyl groups in the peptide bond enrlbles the free access of the tyrosine phenol3te oxygen at the met31 binding site. In this article we report the identification of 3 comples contrtining 3 para-tyrosine phenolate osygen bound to copper. The results were obtained by studying the interaction of cupric ions with two poly (arunino acid;): !L-Tyr. L-L_vs), and (L-Tyr),. using potentiometric, absorption. CD, and resommce Rrtmsn me3surements.
EXPERIXlEi\;TAL Poly( L-Tyr, L-Lys) I IBr 1: 1 and poly( L-Tyr) were purchased from Miles-Yeda Ltd. atid were used without further purification The average molecular weight of the L(Tyr. L-Lys) copolymers obtained by the Archibald method using 0.1-N KC1 3s supporting electrolvte was 25,000 at pf1 6. Sedimentation velocity was measured in a Beckmrtn Spin& Model E ultracentrifuge equipped with Schlieren optics. The average molecular weight of (L-Tyr), was 2 i 200, as indicated by the manufacturers. Potentiometric titrations were performed on a Tacussel ~11 meter. type ISIS AdAgCl-glass Methrom electrode, Model El47. The 20.000, using 3 combined electrode calibration ~3s made following Irving et al. procedure [17] : NaOI-1 WCIS rzsgent gmde (hlerck Co). Concentmtions of solutions were detemlined by 3 micro Kjeldahl method_ Absorption specrrrt of 185-900 nm were recorded on 3 Can; 13 spectrometer_ Circular dichroism specrirt of 185-800 nm were obtained by means of3 Jobin Yvon Dichrograph Model Mark III; it ~3s crtlibrated with 3 solution of epiandrosterone in dio.xane 3s indic3ted by tfte manufacturers [IS] . For CD measurements. the solutions were prepared to give values of optic31 density not exceeding 1.5. For messurements under 200 nm. 311 spectrometers were purged from oxygen prior to recording. Results are reported in terms of E (molar ttbsorption coefficient) and .k = ei - E, (molar circular dic!troism coefficient ). Tile values of E 3nd & are expressed either in temls of [Cu] or in terms of [LyTy] or of [Tyr] _ whiclr represent the molar residue concentration of tyrosine in eiich polymer. Kesommce Rrrm3n spectm were recorded with 3 Coderg PI10 spectrometer equipped with holographic gratings. using the escitation lines of 3 Spectra Physics 3rgon ion frtser. Samples vtere conmined in 3 specially designed Hellma R3m3n cuvette using 90” viewing optics. Rrtmrtn intensities were mettsured using tire vr mode of
Cupric Complexes
of Poly(L-Lysine,
149
L-Tyrosine)
cm-l) as internal standard. Potentiometric and spectroscopic meas(936 urements were made at 25°C under argon atmosphere_ All solutions were prepared with doubly distilled water. Gaussian analysis was performed by the use of a tenchannel DuPont de Nemours curve analyzer_ ClO*-
RESULTS Cu(II)-(L-Tyr,
L-Lys), System
Porerzriomerric Tirrafions. The copolymers (L-Tyr, L-Lys), HBr 1: 1 is soluble in water of the order of 10e3 M [19] I it redisand precipitates at pH 9.2 at concentrations solves at pH 11.6 giving clear solutions. In the presence of Cu(II) ions a complex is formed from pH 6 and is fully formed at pH 7.8 before precipitation occurs (at pH 8.5). The results of potentiotnetric titrations are shown in Fig_ 1. where curve a gives the number of protons neutralized per LyTy residue as a function of pH and curve b, the number of protons neutralized per copper ion at molar ratio [LyTy]/ [Cu] = 4. As can be noticed, when (L-Tyr. L-Lys), is titrated in the absence of cupric ions, the releasing of protons starts at about pH 7 and approsimately at pH 6 in the presence of copper: up to pH 7-S, rive protons per cupric ion are removed. yielding complex A.
FIGURE 1. Potentiometric titration curves of (L-Tyr. L-Lys), in absrncc (curve a) and presence (curve b) of cupric ions. Curve ;i gives number of protons neutralized per LyTp residue, [ LyTy] = 2 X lo-3 31; curve b gives number of protons neutralized per cupric ion at [!_yTy]/[c~] = 4, [ LyTy] = 4 X IO-3 M. __._region of polymer precipitation.
5
6
7
8 P"
9
10
150
A. Gamier and L. Tosi
Absorption
and Circular DichrotXm
Complex A. TIlis complex is formed at molar ratios higher than 3. At wavelengths higher than 300 nm, absorption spectra show one band and two shoulders at 390 nm, 320 nm_ and 520 nm. respectively_ The molar absorption coefficient per cupric ion (e!Cu) and the moIar CD coefticient per cupric ion (&/Cu) of these bands increase 3s the pH is raised from 6 to 7.8; at pH 7.8-8.5 (where precipitation occurs), no further variation is noticeabIe. The plots of e/Cu at 390 nm and &z/Cu at 520 run and 320 nm as a function of pH at [LyTy] /[Cu] = 4 are reported in Fig. 2. The amount of compies A that is formed depends on [LyTy] /[Cu] molar ratio. At pH higher than 7.8 the amplitudes of the absorption and CD bands increase as the molar ratio is raised from 2 to -I and then become independent of molar ratio. Figure 3 gives the plots of &/Cu at 520 and 320 nm as a function of [LyTy]/[Cu] at pH 8. From these data we can conclude that at pH higher than 7.8 (and lower than S-5) and at [LyTy]/[Cu] higher than 4. ali the cupric ions are bound to the copolytner forming complex A. Figure 4 shows the absorption and CD spectra of this complex. Spectral data are reported in Table
1 _ A preliminary
FIGURE
report on this complex
has been published
[20] -
II). Variations of mohr absorption and moIar CD coeffiat [ LpTy]/(Cu] = 4, [Cuj = IO-3 M. Figureson curves indicrtte wavelengths of the different bands; . . . . region of polymer precipitation. cients
2. Systsm ( L-Tyr, L-Lys),-Cu(
of different
+I
bands
3s pH function
I I
to.5 [ 01 <
----_-_------AE 320
/
1
.
E390 --------
600
;ii+ 500
_-_--------AE 520
-1 ;
400
! I
300
-1.5t I
200
-2c
'I S
6
100
I 7
8
?W
9
lo
11
12
< w
Cupric Complexes
of Poly(L-Lysine,
L-Tyrosine)
151
+l PH 8
GO ZJ
y\--??_-_
pH8
-1
-2
pH12.2
12
7
3
a
~W&J]~ FIGURE 3. System (L-Tyr, L-Lys),-Cu(II). Variations of molar CD coefficients of different bands as function of molar ratio at pH 8 and 12.2. [Cu] = 10-a M. Figures on curves indicate wavelengths of the different bands.
Complex
B. Whatever
the
value
of
the
molar
ratio
[LyTy]
/
[Cu], a
precipitate
occurs at pH 8.5 and redissolves at pH higher than 11.6. At wavelengths higher than 300 nm, absorption and CD spectra of solutions at pH 11.6 are as those of complex A. When pH is raised from 11.6 to l___, 3 q the values of Ae at 520 run and 320 run and of E at 550 nm increase, approsimately doubling those of complex A (Fig. Z), and a new negative bands appears at 360 nm; simultaneously the 390-nrn absorption band disappears_ At pH higher than 13.3 the values of E and Ae level off, indicating that a new complex (labeled B) has been fully formed. As in the case of complex A, the amount of complex B that is formed increases as the molar ratio is raised from 2 to 4 and then
becomes function reported L,
independent of molar ratio. The plots of Ae3ao and &e510 at pH 13.2 as a of [LyTy] /[Cu] are illustrated in Fig. 3. Spectral data of complex B are in Table I_
Transition. In the absence
rise to a positive
of copper.
the L,
transition
CD band at 227 nm with Ae/LyTy
of the tyrosyl
moiety
= +3.6 in the protonated
gives
form and
to a positive band at 245 nm with Ae/LyTy = i-3.9 in the deprotonated fomr [19] _ In the presence of cupric ions at [LyTy] /[Cu] = 4 and at pH 6.3. the CD spectrum dis= +3.7. As the pH is raised, the 345-m-n plays a positive band at 228 nm with &/LyTy
A. Gamier and L. Tosi
600 500 400
\; cl)
300 200 100
300
TABLE
1. Optical I
--__cu(11J-I
400
and
CD
Spectral
L-Tyr.
L-Lys),
A
II
Ab
h frm~) 2xc
Xb
hfnm)
CD
h&l
.lE L-Lys),
B
Ab CD
B
OpticA
h (nm) E
CD
L-Lys),
. Cu( II)-
and CD Spectral
550fsh)
390
1-w
6007
600 i-o.15
510 -0.90
Data
100 310 +0.55
290 -0.20
530 1’0 590
320 790 io.55 -0.45
510
+0.10
-0.95
h (nm) E X(nm)
600
520
360
315
AE
+0.50
-7--- 70
-0.10
+i 20
370 -0.7
325 io.95
520 ‘30
Ab
h (run)
520
CD
E Xtnm) &
230 525 -2.1
C The mohr rtbzorprion coeffickn: apressed in rcrms of the molar circular dichroism. sh = shoulder.
L-Tyr,
Complexes=
CornpIe:\
----
CUf 11)-C L.-L?-&
Cu(IIJ-(L-Tyr),,
of Cut 11)-t
L-Lys), . and Cu(iI)_(L-Tyr),,
Sva2rn
CutiI)-(I_-Tyr.
data
600
500
nm
290
790 - 1.40
(E) and the molar circular dichroism coriticienr (AE) art’ concenrrrtGon oi the cupric ions; Ab = absorption. CD =
Cupric Complexes
of Poly(L-Lysine,
L-Tyrosine)
153
band appears: at pH S-5 the CD spectrum cm be resolved into two positive gaussian curves lying at 231 nm and 345 nm (Table 2). Ar pH higher than 12.3. when complex B is fully formed. the CD spectrum diwlays a strong positive band at 235 nm that can be resolved into two positive Gaussian curves at 215 nrn and 230 nm (Table 2).
TABLE 2_ Circular Dichroism Spectral Data of (L-Tyr, L-LYS), in Both Absence and Presence of Copper at [LyTyl/[Cul = 4, [LyTyl = 4x 10-a M. PH
h (nm)
QTYr
3.30
0.0
8.50
0.4
Ae/LyTy
2wl&(cm-l)
2
+3_6 -4.0 +I.6
3100
-3.0 +3x +3_7
3000 2500
1.0
6.36
0.0
8.sb
0.4
+‘.6 +1.7
1600 3200
I >_zb
1.0
+4.6 +3.&x
4000 3200
b
cm-1
‘700
12.20
a
100
In the absri.cc of copper. In the presence of copper.
c Due to n - n* peptide transition. d Due to L, transition of the deprotonated tyrosyl residue. f Due :o L, transition of the protonsted tyrosyl residue_ f Band width at half of the peak height.
Resommce obtained
RamanSpecrra. on excitation
contour
of the absbrption
displayed
by complex
shops the Raman Kaman excitation lustrated cx11- 1 .
and
laser
band
A using
The
at 390 the 457.9
bands
line approaches 1172
cm-’
whose
Xmas
Raman spectra the wavelengths
nm.
Curve
of complex
of which
a in Fig. 5 shows
nm exciting
intensities
lie at 1602
_ X11 the other
rise to Ramsn bands on the spectra 12 12 cm-‘. S5S CIII-~. S36 cm-l. trum of complex A.
Cu( II)_( L-Tyr),
radiation.
A can be
fall tvithin
the
the RR spectrum
line of an Ar ion Iaser:
curve
b
spectrum of the polymer at pH 4 in the absence of copper_ The part of the absorption band tire ilprofile rend the corresponding
in the insert.
the exiting
Resonance-enhanced
with
are enhanced
cm-l.
vibrations
as the wavelengrh
cm-l.
of the tyrosine ( L-Tyr.
of the polymer
and 6-16
1501
cm -l
1260
cm-l.
of
1320
side chain that give
L-Lys),
at pH Li. lying
at
are not observed on the RR spec-
System
The polymer (L-Tyr), becomes soluble a.t pH higher than 12.‘. At pH higher than 9 Cu(l1) binds to (L-Tyr), and a complex similar to complex I3 of the Cu(II)-(L-Tyr. L-Lys),
system
shown
in Table
is formed.
The
1_ The amount
spectral
of complex
data
of wavelengths
formed
depends
higher
than
on [Tyr]
/ [Cu]
300
nm are
molar
ratio
A.
I54
Gamier and L. Tosi
, 1600
1400
a00
1000 cm -3
1200
FIGURE 5. Curve a resonance Raman spectrum of Cu(II)_(L-Lys. L-TM, system ot [ LpTyJ /[Cu J = -I. [Cu j = 0.83 X 10-3, pH 7.9, using 457_9-nm exciting line from 1 lO-mW .-Ir* laser: curve b. Raman spectrum of (L-Lys. L-Tyr), HCI; [ LyTy j = 8 X 10-Z M, pH 4, excitation line 513.5 nm, 500 m\V_ Instrumenta conditions: spectral slit width 4 cm-l; scan rate 50 cm-l!minr time constant. 3 sec. Insert: Raman excitation profiIe of the Cu(II)-fL-Lys, L-Tyr) complex !Uolx Roman intensity ratio (I Rc) of band in question rehtive to the 936-cm--1 band of ClO,--. corrected for sampie self-absorption. instrumental spectral response, and v dependence. Experimental points are scaled to unity at 472.7 nm: (0) 1601. (2) 1502, (s) 1260, (o) 1176 cm-l. (*) CiOa- band. Laser wavelengths are indicated.
and becomes
independent
L,
lies at 245
transition
this band disappears
of it at molar
ratios higher than S. The CD band of the pH higher than 11.7: when the pH is lowered to 11.2, in favor of the band af 227 nm. nm at
DISCUSSION Complex
A At moiar ratios [LyTyJ/[CuJ higher or equal to 4 znd at pH higher than 7.8, which corresponds to the departure of five protons per copper. all metal ions are bound to the copolymer forming complex A. The (L-Lys. L-Tyr), system contains three potential metal binding sites: the amino-nitrogen of the Iysine residue. the phenolate oxygen of the tyrosyl one. and the peptide nitrogen_ The origin of the five protons released by complex A formation and, therefore, the nature of the Lands
of Cu(lI)
this
complex
with
the
Cu(iI)-(L-Lys),
can be inferred that
[21]_
of
the
by comparison
complexes
Cu(II)-(L-Arg),
we
of the CD spectral obtained
[33] _ and
in
our
data of
studies
Cu(II j(L-Om),
on
[23]
Cupric Complexes of Poly(L-Lysine, L-Tyrosine)
155
[23] systems at pH 10, which were labeled “complex II.” In the three cases the binding of two amino nitrogens of lateral chains and two adjacent peptide nitrogens to Cu(II), forming a square planar complex of nearly Dab symmetry, has been established on grounds of potentiometric, absorption, and CD measurements. The CD spectral data of all these complexes are very similar. We have assigned the bands at 590 nm and 520 run to two d-d transitions: the first to 3d,, + 3d,2 _ ,,2 and the second, to 3d,,, 3d,, + 3d,2 _ .2 . These transitions are magnetic-dipole allowed but electric-dipole forbidden, and their energies are essentially determined by the four ligands in the coordination square and are not much affected by the Iigand in apical position [24] _ On the contrary, the energy of the 3d,2 + 3d,z _ .2 transition is sensitive to the presence of axial ligands which interact directly with the d,2 metal orbital. However, this transition is both magnetic- and electric-dipole forbidden so, as a rule, it gives rise to, at most, very weak bands in the CD spectrum. Accordingly, we can interpret our data in the following way: two amine nitrogens from lysine residues and two adjacent peptide nitrogens are bound to the cupric ion in the coordination square. This account for four of the five protons released at pH 7.8 as complex A is formed. It must be recalled that, unlike complex II in Cu(II)-(L-Arg), , Cu(II)-(L-Om), and Cu(II)-(L-Lys), systems, where binding of lateral ammo groups and peptide nitrogens occur in two well-defmed steps, the four nitrogens of complex A bind simultaneously. Moreover, complex A is formed at pH 7.8, whereas complex II begins to appear at this pH approximately and is fully defmed only at pH 10-10.5. Looking now at the absorption spectral data of complex A of the Cu(II)-(L-Tyr, L-Lys), system and of complex II of the Cu(II)-(L-Lys), system (Table 1), we notice that the first one displays a shoulder at 520 nm that corresponds to the d-d transition band at 550 nm of the second system. In addition the tyrosine containing system exhibits a new band at 390 nm that strongly suggests the involvement of the phenolate oxygen in Cu(I1) binding, and this accounts for the fifth proton titrated_ Since the four planar positions have been occupied by four nitrogens, the only coordinating site available for the oxygen is an apical one. The value of the extinction coefficient (e/Cu = 600) and the energy of the transition at 390 run are too high to assign the corresponding band to a d-d transition; rather, they should be accounted for by a chargetransfer transition between the phenolate oxygen and Cu(I1). The presence of such an absorption band has already been observed by Boggess and Martin [16] in the spectra of the Cu(II)-( ~~-orrho-tyrosine) system at pH 10.5, but they did not observe such a band on complexation of Cu(I1) with para-tyrosine. The position and the intensity of the 390~run band are consistent with a rather high metal-oxygen distance and, therefore, with fmation of a phenolate oxygen in apical position; in fact, in copper complexes the ligands in apical position are generally at a greater distance from copper that those lying in the coordination square 1241. According to Barnes and Day [25] ~ the charge-transfer band energy is a function of the metal-ligand bond length (for the same ligand and the same metal), with higher frequency as the bond length increases. Absorption spectra of transferrins bound to different metal ions aiso exhibit a strong band at this wavelength range: at 440 run for Cu(II), 465 nm for Fe@), and 405 nxn for Co(III) 1261. This band has been assigned to a charge-transfer transition between a phenolate oxygen and the metal ion [26], or more precisely, to a chargetransfer transition from the psr orbital of phenolate to a do* metal orbital [12] _ Taking into consideration Barnes and Day’s statement 1251, Prados et al. [26] assume
156
A_ Gamier
and L. Tosi
that in the case of the cupric transferrin complex, the phenolate oxygen is bound to the copper at a relatively short distance from it, specifically, occupying one of the corners of the coordination square. It has to be noticed that in the Cu(Ii)-(L-Tyr, L-LYS), system the 390-nm bard of complex A is not optically active_ Inasmuch as this transition is optically active in the case of Cu(II)-transferrins, this behavior most probably reflects the presence of a longer Cu(II)-0 bond in complex A. Curve a in Fig_ 5 shows the RR spectrum of complex A_ As can be noticed, the band at 1602 cm-1 that is assigned primarily to a ring C-C stretching mode 112, 131 lies at 1619 cm-1 in the Raman spectrum of (L-Lys, L-Tyr), (curve b) when aU the tyrosine side chains are protonated (pH 4)_ This peak corresponds to the 1615-cm-1 band of the fully protonated tyrosine groups in the ir spectra of (L-Lys. L-Tyr), in Da0 solution that shifts to 1603 cm-l on deprotonation [ 191. The band at 1501 cm-1 is also largely due to a symmetric ring stretch [ 12, 131, although considerable contribution from a C-O stretching coordinate is present [13]. This band is not observed in the Raman spectra of the polymer at Iow pH (Fig_ 5, curve b), but is present in the ir spectra of (L-Lys, L-Tyr), in D20 solution. lying. at 1517 cm-l for the fully protonated tyrosine residue and shifting to 1502 cm-l on deprotonation of phenolic groups [ 19]_ A broad band at 1250 cm-l appears in the Raman spectrum of (L-Lys. L-Tyr), when the tyrosine side chains are fully protonated and shifts to 1260 cm-’ in the spectrum of complex A. The C-O stretching mode contributes predominantly to this band [ 1 2, 13]_ The peak appearing at 1180 cm-l in the spectrum of the fully protonated polymer (curve b) shifts to lower frequencies after complexation (1172 cm-l in curve a), which is consistent with the assignment of this band to a C-H bending mode of the benzene ring [ 13]_ The RR spectral pattern of complex A is characteristic of metaI-transfer&s, with the peak frequencies practically identicrd to those of Cu(II)-ovotransferrins [12, 131. There are some small differences, however_ In the spectrum of Fig. 5 (curve a) the band at 1260 cm-’ is less intense relatively to the others than in the spectra of metaltransferrins. As a consequence_ the shoulder at 1320 cm-l in the spectra of the latter appears as a we%resolved band in curve a. We can qualitatively explain this behavior on the basis of a longer phenolate oxygen-copper bond in complex A than in metaitransferrms. The modes which are expected to be enhanced by the RR process are those that couple significantly with the electronic transition_ The C-O stretching mode should be less strongly involved in comples A. since the electronic transition probability would be less altered by the vibration as a consequence of the smaller metaloxygen orbital overlap. Circular dichroism spectra at wavelengths lower than 250 nm can be used to obtain infomrstion on the secondary structure of the copolymer. As we have shown earlier [ 191. at pH 8.5 and in the absence of copper. the copolymer is in the P conformation: this is corroborated by the presence of a negative band at 220 nm due to ?I + rr* transition of the peptide group (Table 2). We have also observed the presence of the positive band at 245 nm due to the L, transition of deprotonated tyrosine (from the data of Table 2 it follows that about 40% of tyrosine is deprotonated). In addition, the positive band at 227 nm of the still protonated tyrosine is masked by the 220-nm negative band. On the other hand, at the same pH and in the presence of copper at [LyTy] / [Cu] = 4, the 215-n~1 band is still present and the positive one at 227 nm is now clearly
Cupric Complexes of Poly(L-Lysine, L-Tyrosine)
157
apparent, due to the absence of the negative band at 220 nm. The absence of this 220~run negative band indicates that complex A maintains the copolymer inthe nonperiodic conformation_ At molar ratios higher than 4 and as the pH is raised to 8.5, some p structure is apparent, in much larger proportions as the molar ratio [LyTy]/ [Cu] ishigher_ We have already observed such a behavior in the case of Cu(II)-(L-Arg), [9-l], Cu(II)-(L-Orn), [22], and Cu(iI)-(L-Lys),, [20] systems. Complex B. At a further stage, and by raising the pH from 11.6 to 12.2, we observe the formation of a second complex_ Insofar as the absorption and CD bands due to d-d transitions lie nearly at the same wavelengths in the spectra of complexes A and B, we can assume that the cupric ion is still bound to four nitrogens lying at the corners of the coordination square. The values of Ae5ao and Aeaao are twice as strong, a result that can be interpreted as indicating the substitution of two amino nitrogens of lysine residues by two adjacent peptide nitrogens. In this case the cupric ion should be bound to two chelates, each of which would induce the same optical activity on the d-d transitions at 520 run. Other investigators 127,281 have already noticed such an additivity of the optical activity induced by two chelates in the case of Cu(II)-dipeptide complexes_ The same trend observed in the 320-m-n CD band corroborates our assignment of this peak to a peptide nitrogen-to-metal charge-transfer transition [L l-23,29] _ We have already observed the formation of such a complex at pH higher than 12, and at residue to copper molar ratios higher than 8 in the Cu(II)-(L-Arg), system (to be published). As complex B is formed, the 390~run-absorption band disappears, which suggests that the phenolate oxygen is no longer bound to Cu(I1). This compares with the result obtained by Prados et al. [26] on the Cu(II)-conaIbumin system: the absorption at 440 nm increases to a maximum intensity near pH 8.6 and then decreases as the pH is raised further_ This incompatibihty between phenolate oxygen and peptide binding when four peptide nitrogens coordinate to Cu(I1) is corroborated by the study of theCu(II)-(L-Tyr), system. In this system the complex formed at pH higher than 9 exhibits the same spectral patterns of complex B in the Cu(II)(L-Lys, L-Tyr), system (see Table I), and no band is detectable at 390 nm. At wavelengths lower than 260 nm, the CD spectra of complex B in the Cu(II)(L-Lys, L-Tyr), system exhibits a strong positive band, which can be resolved into two Gaussian curves (Tabie 2): the 245 nm band is due to L, transition of tyrosyl residues, which are almost all deprotonated at this pH; the 230 run band for which Ae/LyTy = i4.6 is more difficult to assign. It most probably originates from a polypeptide backbone transition_ This band does not appear in the Cu(II)-(L-Tyr), system: at pH higher than 11.7, one observes the 245-m-n band due to the deprotonated tyrosyI residue, which decreases as the pH is decreased, and the band at 227 mn appears. At pH lower than 11.2, only the 227-nm band is observed, which indicates that all the tyrosyl residues are protonated and that the polypeptide is in a nonperiodic conformation_ CONCLUSIONS The present report describes the characterization of two complexes obtained by interaction of cupric ions with two poly(o-amino acids): random (L-Lys, L-Tyr) 1: 1 and (L-Tyr),. Up to pH 7.8, a first complex (complex A) can be identified spectroscopically when the first polymer coordinates to the metal. In this complex the cupric ion
A.
158
Gamier
and L. Tosi
is bound to four nitrogens (two from amino groups from lateral chains and two from peptide groups) and a pehnolate oxygen. At pH higher than 11.6 in the case of (L-Lys. LTyr),
1 I 1, and higher than 9 in that of (L-Tyr),
a second comples
is formed (com-
to the cupric ion_ It appears
plex B) that contans four peptide nitrogens coordinated
that the binding of four peptide nitrogens to the metal to form the second comples precludes phenolate coordination_ It has been observed, in addition, that the formation of both complexes maintain the two polymers in the unordered structure_
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Received
i@ July I Y 7S
pp_ 49-67.