1581
Copper( 11) and Nickel( 11) Complexes of Sulfhydryl and Imidazole Containing Peptides: Characterization and a Model for “Blue” Copper Sites Yukio Sugiura* and Yoshinobu Hirayama Contribution from the Faculty of Pharmaceutical Sciences, Kyoto Unicersity, Kyoto 606, Japan. Received August I O , 1976
Abstract: The Cu(I1) and Ni(I1) complexes of new sulfhydryl and imidazole containing peptides such as N-mercaptoacetyl-Lhistidine ( M A H ) and 2-mercaptopropionyl-~-cysteine, have been characterized by visible, C D , I H N M R , and ESR spectra. Potentiometric measurements showed that the formation constant (log KIKc) of their Cu(I1) and Ni(I1) complexes increases in the order NNpNl,,, < S N p O < S N p N l m < S N p S donor sets. The 1 : l MAH-Cu(I1) complex, which shows an intense absorption near 600 nm ( t 830) and a small copper hyperfine coupling constant ( A = 93 G), has similar unique spectral characteristics to “blue” copper proteins. The model complex strongly suggests that “blue” copper sites involve cysteine sulfhydryl d(Cu) charge transfer transiand histidine imidazole coordinations and that the intense 600-nm band is attributed to US tion. 11
“Blue” copper proteins, which occur widely in nature as an electron carrier, have attracted particular attention because of their unique properties. The distinctive features of “blue” copper centers are unusually high extinction coefficients near 600 nm ( t 1000-SOOO), anomalously small copper hyperfine coupling constants (All = 30-100 G), and markedly positive copper redox potentials (Eo’ = 0.2-0.8 V).’ However, the ligand environment of “blue” copper proteins has never been established. Recent resonance Raman2 and x-ray photoelectron3 studies suggested the presence of Cu(I1)-S(Cys) coordination for “blue” copper sites and S Cu(I1) charge transfer transition for -600-nm electronic absorption bands. Sulfur-copper bonding in “blue” copper chromophore has also been indicated from the experiments on model Cu( 11) complexes of sulfhydryl containing peptides4 and polythiaether~.~ On the other hand, ‘ H N M R spectra at 250 M H z of plastocyanins revealed that the imidazole groups of histidine residues are liganded directly to the copper.6 We report here the characterization of sulfhydryl containing peptide-Cu( 11) and -Ni( 11) complexes which involve sulfhydryl, deprotonated peptide nitrogen, and imidazole groups as the coordination sites. Of special interest is the N-mercaptoacetyl-L-histidine-Cu(I1) complex as a model for “blue” copper sites.
-
-
spectrometer equipped with a Gauss meter and frequency counter. Potentiometric p H titration was carried out as follows: exactly equimolar amounts (0.004 M ) of the ligand and Cu(I1) (or Ni(I1)) ion were mixed in 18 ml of water and to this was added 2 ml of 1 M KNO! to make the total volume of 20 ml. The mixture was titrated with 0.1 M K O H solution at 20 OC under a nitrogen atmosphere, and the pH measurements were carried out with a Radiometer titrator, Type TTT-I C. The formation and dissociation constants of the peptidemetal complexes were calculated according to the previously reported meth~d.~.~
Results Visible Absorption and CD Spectra. Sulfhydryl containing peptides react with Ni(I1) and Cu(I1) ions to form unique 1:l complexes in which the deprotonated peptide nitrogen group participates in coordination with the metal. Tables I and I1 summarize visible absorption and C D spectral data for Ni(I1) and Cu(I1) complexes of the sulfhydryl coqtaining peptides and the related ligands. The A,, of the 1:l Ni(I1) complexes shifts to a longer wavelength in the order 2-MPC > MAH > 2-MPG, consistent with the order of the formation constants. DMPG forms the 2:l complex in addition to the 1:l complex. The 3-MPH-Ni(II) complex gives A,, value similar to that of the 3-MPG-Ni(II) complex. The result suggests that the terminal carboxylate group rather than the imidazole group Experimental Section of 3-MPH participates in Ni(I1) coordination. In the 1:l near 600 nm 2,3-Dimercaptopropionylglycine,2-mercaptopropionyl-~-cysteine, Cu(I1) complexes, on the other hand, the A,, N-mercaptoacetyl-L-histidine, and 3-mercaptopropionyl-~-histidine shifts to a shorter wavelength in the order 2-MPC < MAH < 2-MPG. The DMPG-Cu(I1) complex has a similar visible were synthesized by the Schotten-Bauman reaction between 2,3dibromopropionic acid chloride (or 2-bromopropionic acid chloride, spectrum to that of the 2-MPG-Cu(II) complex. A striking bromoacetic acid chloride, 3-bromopropionic acid chloride) and feature is the high extinction coefficient of the MAH-Cu(I1) glycine (or L-cysteine, L-histidine) followed by a condensation with complex at -600-nm band. The diamagnetic Ni(I1) complex thiobenzoic acid and then hydrolysis in an ammonia s ~ l u t i o n The .~ of 2-MPC, which is composed of L-cysteine residue, typically peptide ligands were recrystallized with ethyl acetate and determined exhibits a negative C D extremum at 482 nm, whereas the by elemental analysis, iodometric titration, and I H N M R measurepositive C D extremum appears at 463 nm in the MAH-Ni(I1) ment. A freshly prepared solution of the ligands was used in each complex in which the histidyl residue is involved in the chelate experiment. The metal solution (0.01 M ) was prepared by dissolving ring. The MAH-Cu(I1) complex shows a unique CD spectrum cupric nitrate or nickel nitrate in water and standardized with 0.01 with the sign (-) above 440 nm. In general, the Ni(I1) and M EDTA solution. A carbonate-free potassium hydroxide solution Cu(I1) complexes of L-cysteine and L-histidine containing (0.1 M) was prepared by the method of ArmstrongBand standardized with potassium hydrogen phthalate. All other reagents used were of peptides give a CD curve which has a greater magnitude. commercial reagent grade, and deionized water was used throughPotentiometric Titration. The titration curve of M A H out. consists of three pH buffer zones, which give the values of Visible absorption and CD spectra were measured in an aqueous pK,(COOH, 3.43), pK2(imidazole, 7.14), and pK3(SH, 8.70). solution at 20 O C with a Shimadzu recording spectrophotometer, The acid dissociation constants of 2-MPC are pK](COOH, Model Double-40R, and a Jasco 5-20 spectropolarimeter, respectively. 3.66), pK*(>CHSH, 7.66), and pK3(-CH2SH, 10.69). The I H N M R spectra were recorded a t 100 M H z on a Varian HA- 100 titratiop curves of 1:l MAH (or 2-MPC)-Ni(II) and -Cu(II) N M R spectrometer. Sample concentration was 0.2 M in DzO (pD systems gave clearly an inflection at a = 4.0 ( a = moles of base 9.6) and chemical shifts were measured from internal TSP. X-band per mole of ligand). The data strongly indicate that a proton ESR spectra were obtained a t 7 7 and 293 K with a Jeol ME-3X
+
Sugiura, Hirayama
/ Cu(IIJand N i ( I I ) Complexes of Sulfhydryl Containing Peptides
1582 Table 1. Visible Spectral Data for Ni(I1) and Cu(I1) Complexes of Sulfhydryl Containing Peptides Absorption max, nm Ligand
(c)
Cu(I1) complex
Ni(I1) complex 575 (440) 605 (1 OO)a 567 (450) 543 (240) 5 15 (250) 475 (410) 510 (290) 610 (80)" 600 (670)'I
440 (1 300) 455 (150) 410(1200) 382 (770) 405 (850) 375 (2750) 400 (1 020) 475 (120) 452 (3100)
2,3-Dimercaptopropionylglycine 2- Mercaptopropion yl-~-cysteine N-Mercaptoacetyl-L-histidine 3-Mercaptopropionyl-~-histidine 2-Mercaptopropionylglycine 3-Mercaptopropionylgl ycine 2,3-Dimercapto- 1-propanolIo
' L/M
The composition of the metal complexes is L / M = 1:1, except for
= 2: 1 and
400 (sh)
605 (280)
405 (700) 438 (970)
580 (280) 598 (830)
400 (sh) 400 (sh)
605 (260) 630 ( I 00)
L / M = 3:2, respectively
Table 11. Circular Dichroism Data for Ni(I1) and Cu(I1) Complexes of Sulfhydryl Containing Peptides Comulex
Circular dichroism A,,,.
2-Mercaptopropionyl-~-cysteine-Ni(11) N-Mercaptoacetyl-L-histidine-Ni( 11) 2-Mercaptopropionyl-~-cysteine-Cu(11) N-Mercaptoacetyl-L-histidine-Cu( 11) Glycyl-L-histidine-Ni(I1) I Glycyl-L-histidine-Cu(II)'* L-Cysteine-Ni(I1) (2:l)l I L-Cysteine methyl ester-Ni(I1) (2:I)l I N-Acetyl-L-cysteine-Ni(I1) (2:I)l
nm (Ac)
390 (0.39), 440 (-2.16). 482 (-4.30), 565 (2.88) 385 ( - l . l 2 ) , 463 (4.21), 560 (-2.27) 395 (-0.82), 575 (0.59) 365 (-3.49). 405 (-l.67), 445 (-l.97), 545 (1.58), 630 (-0.64) 430 (2.08), 488 (- 1.92) 485 (-O.I6), 565 (0.12) 390 (-0.1 I ) , 455 (-0.62), 535 (0.45) 420 (-0.03), 500 (0.19), 580 (0.22) 400 (0.35). 460 (-0.07). 525 (-0.20), 610 (-0.04)
Table 111. Acid Dissociation and Formation Constants for Ni(I1) and Cu(I1) Complexes of Sulfhydryl Containing Peptides and the Related Ligands Acid dissociation and formation constants Ni(I1) complex Ligand
PK I
PK2
PK~
2,3-Dimercaptopropionylglycine
3.66
7.66
10.69
2-Mercaptopropionyl-~-cysteine
3.29 3.43 3.48 3.60 3.71 8.69 2.75 O.OOS) than is usually associated with the u charge transfer bands. Accordingly, the 600-nm band of the MAH-Cu(1I) complex may be assigned to the u(S) d(Cu) charge transfer transition. Solomon et al. have recently indicated that the 16000-cm-l band in “blue” copper proteins exhibits a y value well below 0.005, and such is attributed to a US d,:-).? charge transfer t r a n ~ i t i o n . How’~ ever, a and u charge transfer can be distinguished by A€/€
-
400
450
500
550
600
650
x, Figure 3. Visible and circular dichroism spectra of N-mercaptoacetyl-
1.-histidine-copper(I1) complex. The spectra were obtained by mixing the ligand ( 1 .O mM) and CuCIl ( I .0 mM) in borate buffer solution of pH 9.2. C D spectrum was recorded on a Jasco 5-20 spectropolarimeter at 20 OC.
Journal of the American Chemical Society
/ 99:5 /
March 2, 1977
-
-
-
Table V. Visible Spectral Data for Sulfhydryl Containing PeptideCo( 11) Complexes and Co(I1)-Substituted ‘‘Blue’’ Copper
tense 600-nm band is attributed to a aS d(Cu) charge transfer transition. All naturally occurring plastocyanins have a single cysteine residue and show a common amino acid sequence of C y ~ ~ ~ - X - y - H i sThe ~ ’ . ’sequence ~ includes CysSer-Pro-His in the broad bean and elder plastocyanins, CysAla-Pro-His in potato plastocyanin, and Cys-Glu-Pro-His in Chlorella plastocyanin. This can hardly be attributed t o natural concidence. Thus, the MAH-Cu(I1) complex, which has a N1,NpS donor set (NI, = imidazole nitrogen, N p = peptide nitrogen, S = sulfhydryl sulfur), is of interest with regard to the quite recent suggestion for the coordination core ( N l m z N p S ) of “blue” copper sites.i9
Proteinso
Ligand Co(1I) plastocyanin2* Co(I1) stellacyanin22 Co( 11) azurinZ2 N - Mercaptoacetyl-L-histidine 3-Mercaptopropionyl-L-histidine 2-Mercaptopropionyl-~-cysteine 2-Mercaptopropionylglycine 3-Mercaptopropionylglycine Glutathione Glycylglycine P-Alanyl-L-histidine
Visible absorption maxima A,, nm (€1 510 (380), 630 (280). 675 540 (320), 625 (380), 655 525 (220), 635 (340), 660 560 (360), 603 (370), 668 585 (300), 620 (430), 656
(390) (460) (290) (320) (410)
603 (450), 655 (500), 717 (470)
Acknowledgment. Gratitude is due to Professor H. Tanaka for helpful advice and M. Ohara for assistance with the manuscript.
610 (290), 675 (380), 730 (260) 610 (330). 685 (450), 720 (430) 620 (240), 680 (370), 720 (280) 495 ( 1 5) 490 (20)
References and Notes
The spectra were measured by mixing the ligand (4.0 mM) and CoCI2 (2.0 m M ) at pH 9.2 in a fully deaerated Tunberg tube. [I
values only in favorable cases, such as distorted tetrahedral D2d. In a square-planar geometry it is possible that the 600-nm band is aS d,z-yz. whereas the 440-nm peak could be as d,2-,2. One would expect both a-and US Cu(I1) charge transfer to be a t higher energies in square-planar complexes than in the blue copper proteins, if the latter possess nearly tetrahedral Cu(I1) sites. The more intense band in the MAH-Cu(I1) complex is a t 438 nm, whereas the reverse is true in “blue” copper proteins. This fact suggests the possibility that the geometrical difference between the model complex and “blue” copper center may be responsible for the difference observed in energy. From resonance Raman studies, the trigonal-bipyramida12a and flattened tetrahedralZbgeometries have been proposed for “blue” copper sites. The C D spectrum of the MAH-Cu(I1) complex shows three bands with the sign (-) above 440 nm. The C D spectra of laccase [Polyporus laccase: 469 ( A € -0.9), 532 (1.2), and 615 (-3.0) nm;20 Rhus vernicifera laccase: 446 (-2.4), 526 (3.0), and 633 (-5.6) nm2’] resemble more closely that of the MAH-Cu(I1) complex than those of the 2-MPC-Cu(II) complex [395 (-0.82) and 575 (0.59) nm] and glycyl-L-histidine-Cu(l1) complex [485 (-0.16) and 565 (0.12) nm]. As to the copper hyperfine coupling constant in the parallel region, the sulfhydryl containing peptide-Cu(I1) complexes such as M A H and 2-MPC have a close value to that of “blue” copper centers, where A 1 1 < 100 X cm-l. W e have already indicated that the high covalency of the Cu-S bonding contributes to a decrease in the unpaired electron density on the Cu(I1) atom.4a Furthermore, the 2:l MAH-Co(I1) complex showed absorption maxima a t 560 ( t 360), 603 (370), and 668 (320) nm, and the spectral feature resembles appreciably those of Co(I1) substituted “blue” copper proteins (see Table V). The MAH-Cu(I1) complex, as a model for “blue” copper sites, serves to verify the speculation that the blue copper center involves cysteine sulfhydryl and histidine imidazole coordinations and that the in-
-
-+
-
+
Sugiura, Hirayama
1585
/
(1) R. Malkin and B. G. Malmstrom, Adv. Enzymol., 33, 177 (1970). (2) (a) V. Miskowski, S. P. W. Tang, T. G. Spiro, E. Shapiro, and T. H. Moss, Biochemistry, 14, 1244 (1975);(b) 0. Siiman, N. M. Young, and P. R. Carey, J. Am. Chem. Soc., 98, 744 (1976). (3) E. I. Solomon, P. J. Clendening, H. B. Gray, and F. J. Crunthaner, J. Am. Chem. Soc., 97, 3878 (1975). (4) (a) Y. Sugiura, Y. Hirayama, H. Tanaka, and K. Ishizu, J. Am. Chem. Soc., 97, 5577 (1975); (b) Y. Sugiura and Y. Hirayama, lnorg. Chem., 15, 679 (1976). (5) T. E. Jones, D. B. Rorabacher, and L. A. Ochrymowycz, J. Am. Chem. Soc., 97, 7485 (1975). (6) J. L. Markley, E. L. Ulrich, S. P. Berg, and D. W. Krogmann, Biochemistry, 14, 4428 (1975). (7) The following abbreviations are used in this paper: DMPG = 2,3-dimercaptopropionylglycine; 2-MPC = 2-mercaptopropionyl-~-cysteine; MAH = Kmercaptoacetyl-L-histidine; 3-MPH = 3-mercaptopropionyl-~-histidine; 2-MPG = 2-mercaptopropionylglycine; 3-MPG = 3-mercaptopropionylglycine; CD = circular dichroism; ‘H NMR = proton nuclear magnetic resonance; ESR = electron spin resonance. (8) D. M. G. Armstrong, Chem. Ind. (London), 1405 (1955). (9) S. P. Datta and B. R. Rabin, Trans. Faraday SOC.,52, 1123 (1956). 10) D.L.Leussing, J. Am. Chem. SOC., 81, 4208 (1959). 11) J. W. Chang and R. B. Martin, J. Phys. Chem., 73, 4277 (1969). 12) G. F. Bryce and F. R. N. Gurd, J. Biol. Chem., 241, 1439 (1966). 13) (a) G. F. Bryce, R. W. Roeske. and F. R. N. Gurd, J. Biol. Chem., 240, 3837 (1965); (b) G. F. Bryce, R. W. Roeske, and F. R. N. Gurd, ibid., 241, 1072 (1966). 14) G. R. Lenz and A. E. Martell, Biochemistry, 3, 750 (1964). 15) Y. Sugiura, T. Takeyama, and H. Tanaka, Chem. Lett., 491 (1976). 16) T. Vanngard in “Biological Applications of Electron Spin Resonance”, H. M. Swartz, J. R. Bolton, andD. C. Borg, Ed., Wiley, New York, N.Y., 1972, p411. 17) The infrared spectrum of the 1: 1 MAH-Cu(II) complex isolated as the tetraethylammonium salt directly shows the coordination of sulfhydryl, deprotonated peptide nitrogen, and histidine imidazole groups. The disappearances of u (peptide NH) and u (SH) at 3270 and 2500 cm-’ in the free ligand reveal that the ligand coordinates through the sulfur and peptide nitrogen atoms. In addition, the shift of v (C=N) from 1640 (in the ligand only) to 1590 cm-’ (in the complex) suggests coordination of the imidazole nitrogen group in the Cu(ll) complex. The result of elemental analysis for the isolated complex shows the formation of approximately 1: 1 MAH-Cu(Ii) C.N42.14; . H * O :H, 6.63; complex. Anal. Calcd for C U ( C ~ H , ~ O ~ N ~ S X C ~ H ~ ) ~ N, 12.29; Cu, 13.93. Found: C, 42.40; H, 6.87; N, 12.03; Cu. 13.55. The MAH-Cu(lltimidazole complex showed absorption maxima at 435 ( I 1060) and 593 (910) nm. (18) (a) S. F. Mason, Q. Rev., Chem. SOC., 17, 20 (1963); (b) R. D. Gillard in “Physical Methods in Advanced Inorganic Chemistry”, H. A. 0. Hill and P. Day, Ed., Interscience, London, 1968, pp 167-213. (19) E. I. Solomon, J. W. Hare, and H. B. Gray, Proc. Natl. Acad. Sci. U.S.A., 73, 1389 (1976). (20) S.-P. W. Tang and J. E. Coleman, J. Biol. Chem., 243, 4286 (1968). (21) K. E. Falk and B. Reinhammer, Biochim. Biophys. Acta, 285, 84 (1972). (22) (a) D. R. McMillin, R. A. Holwerda, and H. B. Gray, Proc. Natl. Acad. Sci. U.S.A., 71, 1339 (1974);(b)D. R. McMillin, R. C. Rosenberg, and H. B. Gtay, ibid., 71, 4760 (1974).
Cu(II)and Ni(II) Complexes of Sulfhydryl Containing Peptides
