Novel Biological Copper Proteins through Anion Addition to the Mutant Met121Gly of Pseudomonas aeruginosa Azurin.код для вставкиСкачать
COMMUNICATIONS 3-methylaltropyranoside (2.0 Hz; 4C, chair conformation) and that of the x-altropyranose unit in 2A(S),3A(R)-/Gcyclodextrin (6.6 Hz;"] mainly ' C , chair conformation) .[81 The major product of acidic hydrolysis of 3 was a mixture of altrose and altrosan (1,6-anhydroaltropyranose),which is produced by acid treatment of a l t r ~ s e . [ ~Further I studies on the conformation, the inclusion properties, and the chemical modification of 3 are currently in progress. Experimental Procedure 3: A solution of100 mg of 2, prepared according to ref. [ 5 b], in water (50 m L ) was refluxed for five days 191. The mixture was concentrated in vacuo and chromatographed on a reversed-phase column (Merck Lobar column RPI 8. size B) with water to give 3 (S2.0mg. 72.9%), m.p. 227 'C (decomp.); [zlb'' = +63.4 ( c = 0.41 in H,O): ' H N M R (500 MHz, D,O, 40 C . CH,CN): 6 = 4.73 (d, 'J(H.H) = 4.5 Hr. 7 H . H-l).4.19 (m, 7 H ; H-5). 3.93 (dd, ,J(H.H) = 3.7, 5.8 Hr. 7 H ; H-4). 3.86 (dd, 'J(H,H) = 3.7, 7.8 Hr, 7 H ; H-3). 3.80 (dd. 'J(H,H) = 4.5. 7.8 Hz, 7 H : H-2). 3.77 (dd, 'J(H,H) = 6.3, 12.5Hz. 7 H ; H-6). 3.72 (dd. ,J(H,H) = 3.6. 12.5 Hz, 7 H ; H-6); I3C N M R (125 MHz, D,O, 40 C, CH,CN). 6 =103.1 (C-I). 77.2 (C-4). 73.7 (C-5). 71.2 (C-2). 70.0 (C-3). 61.3 (C-6): FAB-MS: m!:. 1135 [iM H'], 1157 [ M N a i l . + + Received: December 13. 1994 Revised version. April 27, 1995 [Z 7546 IE] German versioii: Angew. Clieni. 1995. 107, 1783- 1784 Keywords: altrose . 8-cycloaltrin cyclodextrins . oligosaccharides [ I ] G . Wenz. Anpm. Cheni. 1994, 106.851 -870; A n g r w . C/ie,rn.I n / . Ed. EngI. 1994. 33, 803-822, and references therein. (21 M. Sawada. T. Tanaka. Y. Takai. T. Hanafusa, M. Kawamura. T. Uchiyama. CurhoIij.rlr. Rrs. 1991, 217, 7- 17.  M. Mori, Y. Ito. T. Ogawa, Tetru/!edroti Lert 1989. 30, 1273-1276: M. Mori. Y Ito. J. Uzawd. T. Ogawa, ;bid. 1990, 3I. 3191-3194. (41 F. W. Lichtenthaler, S. Immel, Telrahetlron: Asymtnefrl. 1994, 5. 2045 -2060.  a) A. W Coleman, P. Zhang, C.-C. Ling. J. Mahuteau, H. Parrot-Lopez. M. Miocque. Siiprunid. Clzcm. 1992, I , 11-14; h) A. R Kahn. L. Barton, V. 7. D'Souza. J Clirm. Soc. C I I P ~Comniun. . 1992, 1 I12 11 14. The conformational energy of 'C, conformation of r-altropyranose wab reported to be clote to that of the 4C, conformation; S. J. Angyal. Angew. Clieni. 1969, X I . 172 182; A n ~ e r r Chmi. . Int. Ed. Enpl. 1969.8. 157-166;s. J. Angya1.V A. Pickles, A w r . J. Cliem. 1972, 25. 1695- 1710. (71 K. Fujita. K. Ohta, Y. lkegami, H. Shiinada. T. Tahara, Y . Nogami. T. KO%&,K . Saito. T. Nakajima. T?trahet/ron Lett. 1994. 35, 9577-9580. (81 For a pyranoid IC, conformation '-1, should be around 8 H7. Therefore. the pyranose rings in 3 may be closer to a ' C , rather than the 'C, form.  The purity of 2 is very important for the reaction; conramii~ationby even a small amount of benzenesulfonates, which are intermediates in the epoxidation leading to 2. is very harmful for the present reaction, since 3 is hydrolyzed by sulfonic acid produced during the reaction Novel Biological Copper Proteins through Anion Addition to the Mutant Metl21Gly of Pseudomonas aevuginosa Azurin"" Momcilo Vidakovic and Juris P. Germanas* Blue copper proteins are widely distributed in nature, where they function as electron-transfer mediators in important biochemical processes such as photosynthesis and metabolism.['] The unique spectrocheniical properties of these proteins have long attracted the attention of bioinorganic chemists.i21For proteins in the oxidized state [copper(~r)],these characteristics include an intense absorption band centered around 600 nm ( E = 2000-6000 M - l c m - ' ) , an EPR spectrum with an unusually small copper hyperfine splitting ( I = 3/2, A ,, 2 7 0 x cm-'). and anomalously high redox potentials for the Cu"/Cu' couple [180-680 mV versus the normal hydrogen electrode (NHE)] . Crystallographic analysis has revealed that the copper(I1) ion in these proteins is coordinated to two histidine residues and one cysteine residue in an approximately trigonal planar g e ~ m e t r y . ' ~Besides ] these three strong interactions, weaker axial interactions between the copper ion and other ligands are generally present. F o r example, in azurin and plastocyanin, the thioether sulfur atom of a methionine residue interacts with the copper center, while in stellacyanin, a glutamine carboxamide oxygen is thought to coordinate to the metal.[41 The nature of the axial interaction is believed to significantly influence the spectrochemical characteristics of metal centers of the blue copper proteins.[51To probe the effect of various axial ligands on the spectroscopic and electrochemical properties of these copper sites, we have prepared by site-directed mutagenesis a variant of Pseuc~oomonasaeruginosu azurin, Met121Gly,[61 in which the weakly coordinating (R, -cu = 3.1 A) thioether side chain of Met121 is replaced by a hydrogen atom. This creates a cavity that can be occupied by exogenous l i g a n d ~ . ' ~In] this communication we report the preparation and characterization of unique biological copper species, obtained through addition of the anions N,, SCN-, and C N - to Metl21Gly azurin. Their spectroscopic properties are distinct from those of any known naturally occurring copper proteins.['' The UV-Vis spectra of blue copper proteins display a characteristic absorption band at about 600 nm, due to a Scys(7z)to Cu" charge transfer (LMCT) transition, along with another band at about 450nm from a SCYh(pseudo-o) to Cu" LMCT.['' The relative intensities of the two bands vary, depending on the protein. A correlation between the relative intensity of the band at 450 nm and the axial displacement of the copper atom from the plane formed by the His,Cys ligand set has been reported for a number of blue copper proteins.["] Upon titration of Metl2lGly azurin with azide, thiocyanate, or cyanide, the intense absorption band at 614 nm of the uncornplexed protein disappeared, and a new, weaker band at approximately 420 nm arose, along with a feature a t about 590 nm (Table 1) .[' The wavelengths of the transitions at 420 nm were dependent on the specific anion, indicating that the anion ligated directly to the [*] Prof. Juris P Germanas, Momcilo Vidakovic Department of Chemistry University of Houston Houaton, TX 77204-5641 (USA) Telcfax: Int code 713-743-2709 e-mail' germanas(!< uh-edu + [**I 1622 ,$-', VCH Vrriag.s,o~~s.sellsclzrrfl m h H . 0-69451 Weoihrim./YYS Thir work was supported by the University of Houston and rhc American Cyanamid (Faculty Research Award to J. P G.). The authors express their gratitude to Prof. R . S. Czernuszewicz and G. Fracrkiewicz (University of Houston) for the resonance Raman spectra, and Mr. V. Kurchev for assistance in acquiring the EPR spectra. 057/)-)-0833:Y5: 15/5-16225 10.00+ .25,'0 An,oriv. Clicm. Inr. Ed. EnxI. 1995, 34. N o . I S COMMUNICATIONS Tablc I Spcctrorcopic and electrochemical properties of P . trerrrgC1o.str Met 121Gly ( M I 21G) azurin and its amon adducts LA [a1 [nm ( ~ - ' c m- ' I ] Protein Anion 628 (5300) 614 (3500) 590 (950) 5x2 (1100) 5x0 (950) Wild type a ~ u r i i i M121G iiiurin MI?l(i + N, M121Ci + SCN M121Ci + C'& L,, ( c ) [;I R , [cl K, Ibl [v - 7 [nm ( W c m - l ) ] (560) (2000) (2020) (14190) ~ 30 30 1x00 (niV vs. NHI;) 293 261 239 234 nd [d 58 [fl 53 [f] 2 259 1.270 1.247 2.205 2.216 ~ 456 420 416 432 E"' [d] A I [cl (IO-"cm-') 101 89 106 .Av [el (cin-') 40x 40X 35X 353 338 [ a ] Optical :ihsor-ption inaxima and extinction coefficients of S,,,-Cu CT bands centered around 600 and 400 nm. [b] Protein ~ligandassociation constant at 23 C. [c] X-band EPR pammctcrs (,y, = y , ; A , = A Vcir splitting due to copper nuclear spin, I = 3:2). [d] Midpoint redox potential at 23 C. 0.1 cf NaCI. 0.3 M Iigand. 20 m\f Tr-is. HCI. pH X . 0 [el 1-i-cqtieiicyo1'inost intense peak ol' resonance Raman spectrum. [f] Determined by computer simulation of spectrum. [g] Not determined copper(i1) ion. From the greater intensities of the 420 nm bands relative to those of the 590nm bands in the spectra of the protein -1igand adducts, we inferred a larger separation between the copper(i1) ion and the plane of the protein's His,Cys ligand set than in the uncomplexed form.["] The formation constants of the 1 : 1 complexes, determined spectrophotometrically, revealed the cyanide adduct to be substantially more stable than the azide or thiocyanate adducts (Table I). Rcsonance Raman spectra of Metl21Gly azurin and its anion adducts were recorded with excitation wavelengths of 568 nm (without exogenous ligand) or 41 3 nm (with exogenous ligand). The set of four strong bands in the spectrum of the uncomplexed protcinl"' was replaced in the spectrum of the anion adducts by a pair of peaks at lower energy (Fig. 1 ) . Since the most intense band in the resonance Raman spectra of blue copper proteins results from a predominantly Cu-S stretching vibrational rnode.l"] the shift of the intense band in the spectra of the anion adducts to lower wavenumber indicated that the anion complexes possessed weaker, and consequently longer, Cu-S bonds than the uncomplexed protein (Table 1 ) . The appearance of bands attributable to bond-stretching modes of azide and thiocyanate in the spectra of the specific adducts further confirmed that these anions coordinated direct11 to the copper(i1) ion of Metl2lGly azurin (Fig. 1). The EPR data of spectra of the 1 : 1 complexes also reflected a change in coordination environment at the copper site in the protein-anion adducts. The copper(i1) centers of the adducts displayed EPR spectra with axial symmetry, in contrast to the uncomplexed protein, which displayed an EPK spectrum with rhombic symmetry (Fig. 2). Strikingly, the values for the copper 4) M121G Ae;568nm 0 T I 4, M121G+CNA =413nm 3480 M I I I 3500 3520 BIG ex 3540 Fig. 2 X-band EPR spectra <it 77 K of the MetlZlGl\ n i i i t m t of P oerzigitioxi azuriii and its adducts with anions. The spectra are (l'roiii top) LIet121GIy azurin: MetlZlGIy iizurin cyanide: Met121Gly azurin + thiocy'iiinte, and MctlZIGIy azurin azide. + + 4, M121G+N3a :413nm ex ~ M M121G+SCNA :413nm hyperfine coupling constant ( A of the complexes (Table 1) were well above the range of values observed for typical blue copper sites ( 570 x cm- ').[*I Furthermore the A values for the adducts were considerably smaller than those seen for common tetragonal, or type 2, copper compounds (160-220 x 10 cm - I ) .[21 Plots of g versus A for copper complexes often produce linear correlations for compounds of similar structural or electronic origin.['31When the values of g for the Metl21Gly-anion adducts were plotted against the values of A . a linear correlation was obtained ( R = 0.95), which was distinct from the correlations seen for blue or type 2 (tetragonal) copper centers. Differences in the precise values of the adduct's EPR parameters reflected small structural variations in the ligand environment of the copper atom of each a d d ~ c t . [ ' ~ l The redox potentials of Metl21Gly azurin and its anion complexes were determined by potentiometric titration. Binding of thiocyanate or azide to the copper(i1) ion of the protein pro- VI , M ex , 250 - , , 300 350. A; / c m-' , 400 4 I b-ig. 1 Resondnce Rainan spectra of (from top) MetlZlGly azurin: Metl2lGIy iizurin + cyaiiidc. Metl21Gly azurin azide; MetlZlGIy azurin + thiocyanate + Excitation w,iwlengths i,,for each protein are given. I = intensity COMMUNICATIONS duced species with lower redox potentials than the free protein (Table 1). The midpoint potential of the cyanide adduct could not be determined by this method because of competitive oxidation of cyanide. The lower potentials of the two anion adducts relative to Met121 Gly azurin could be rationalized by inferring electrostatic stabilization of the higher copper oxidation state by the negatively charged ligands. The spectroscopic properties of the Metl 21 Gly azurin-anion adducts indicate the presence of a novel form of protein-bound copper having spectroscopic properties distinct from those of either blue or type 2 copper proteins. According to a computergenerated model of the Metl21Gly active site.[I4] coordination of external ligands to the copper(1r) ion in the space previously occupied by the Met121 side chain should result in a distorted tetrahedral environment for the metal center. Binding of anionic species to the copper(r1) ion should also result in lengthening of the other metal-ligand bonds. Absorption and resonance Raman spectra of the Metl21Gly azurin-anion complexes reflect substantial axial displacement of the copper atom from the plane of the His,Cys hgand set along with elongation of the S,,,-Cu bond.[", The values for the parallel hyperfine splittings in the EPR spectra of the 1 : 1 complexes are consistent with diminished covalency in the S,,,-Cu bond relative to the uncomplexed form, resulting from S-Cu bond lengthening.[I5] In natural blue copper proteins with distorted tetrahedral metal centers and short axial interactions with neutral ligating atoms, absorption spectra display intense bands at about 450 nm, and EPR spectra suggest a rhombic g tensor.['] Absorption spectra with i,,,, at about 420 nm and EPR spectra with axial symmetry for the Met121 Gly azurin-anion adducts signify a unique electronic structure of the copper centers in these proteins as a result of the anionic character of the axial ligands. Further support for the proposed model of the Metl21Gly azurin-anion adduct copper centers comes from properties of structurally characterized, pseudo-tetrahedral copper complexes. which display spectroscopic characteristics remarkably similar to those of the adducts reported here.['61 A variety of unusual copper proteins can be easily created through addition of anions to the azurin mutant Metl 21Gly. The ability to create ligand-accessible metal centers in coordinatively saturated metalloproteins by directed mutagenesis holds promise for the preparation of metalloproteins with selective ion-sensing capabilities. Further studies aimed at detailed characterization of the electronic and geometric structure, as well as the electron-transfer properties of the active sites of these unusual proteins are in progress. Received: January 24, 1995 [Z 7656 IE] German version: Aiigcw Clicm. 1995, 107. I773 I776 ~ Keywords: copper compounds . EPR spectroscopy . metalloproteins . mutagenesis . Raman spectroscopy [I] a ) E. T. Adam. A h . Protrin C/imz. 1991. 42, 145-197; h) E T. Adman in 7iipic.v in Moliwrlar cind Strritrciral B ; o / o I ~ JMrfal/~i/)rorrin,s . (Ed.: P. M. Harrison) MacMillan. New York. 1986, pp. 1-42.  E. I Solomon, M. J. Baldwin. M . D. Lowery, Clicni. Rev. 1992. 92. 521 542.  a) P. arruginosa azurin: H. Nar. A. Messerschmidt. R. Huber, M. van de Kamp. G. W. Canters. J M d . B i d 1991, 221. 765-772: E. T. Adam. L. H. Jensen. f s r . J. Chem. 1981, 2 1 , 8-12; b) A . dmitrificans azurin: G. E. Norris. B F. Anderson, E. N. Baker, J Mol. B i d 1983, 165, 501 - 521; c) poplar plastocyanin: J. M. Cuss, H. C. Freeman, ;hid. 1983. 169, 521 -563: d ) hasic blue protein from cucumber J. M. Guss. E. A. Merritt. R. P. Phizackerley, B Hedman. M. Murata. K. 0.Hodgson. H. C. Freeman, Scimcr 1988.24/,806-X11; e) ascorhate oxidase: A. Messerschmidt, A. Rossi. R. Ladenstein. R Huber. M Bologncsi, G Gatti. A. Marcheslni. R. Pctruzzelli, A . Finuzrl-Agro. J. Mol. B i d . 1989. 206. 513-529; f) A . cjr/oclri.rrc.\ nitrite rcductase: J W. Godden. S. Turley, D. C . Teller, E. T, Adman. M Y, Liu. W J. Payne. .I. LeGall. S&iicc 1991. 253. 438 442. 141 a ) Three-dimensional model of stellacyanin. R. A. Fields. J. M. Guss. H. C. Freeman. J Mol. B i d 1991. 222. 1053- 1065: b) ENDOR evidence of the coordination of Gln in stellacyanin: H. Thomann, M. Bernardo, M . J. Baldwin. M. D. Lowery. E. I. Solomon. J Aim Climi. Soc. 1991. 113, 5911 5913; c ) Akoli,qrnc,.\ drwirrifrcoiis azurin mutant with spectroscopic similarities with stellncqaniii: A. Romero. C. W. G Hoitink. H . Nar. R Huher. A. Messerschmidt. G. W. Canters. J. M o l . 5ioi. 1993. 2 3 . 1007 1021 IS] a ) A. A. Gewirth. S L. Cohen, €1. J. Schugar, E. 1. Solomon. Inorg. Chem. 1987. 26. 1 1 3 3 ~1146; b) H. B. Gray. B. G. Malmstrom. Coninienrs InorR. cilpiil. 1983. 2. 203 209.  a) T. K. Chang. S A. Iverwn. C. N. Kiser. A. N . Lew. C. Rodrigues, J. P. Germanas. J. H . Richards, Pror,. i v c i / l . Acrid S L ~L'SA . 1991, 88, 1325--1329: b) B. G. Karlsson, M . Nordling. T Pascher, L:C. Tsai, L. Sjolin, G. Lundberg, Pi'fJ/t& Enx. 1991, 4 . 343-349.  Regeneration of copper centers 01' blue copper proteins through exogenous lisand addition to a type 2 azurin mutant. T. den Blaauwen. G . W. Canters. J A m Chrin. SJC. 1993. 1 / 5 . 1121-1129.  This work WJS first presented at the Symposium "Copper Coordination Chemistry- Bioinorganic Perspectives". August 3 -7. 1992 (presentation P56). As this work war being carried out. Pascher et al. reported UV-Vis and EPR data for forms existing at high pH of twci a u r i n mutants. Metl2lGlu and Mctl21Lys (T. Paschcr, B. G. Karlsson. M. Nordling, B. G. Malmstrom, T. Vaniigird, Eui-. J B i o c h n 1993.212.289- 296). which resemble those of the Metl 21 Gly anion complexes reported here; the similarities of the spectroscopic data imply that the position 121 side chain in the former proteins coordinates to the copper(i1) ion.  A. A. Gewirth, E. 1. Solomon. J Am. Chw?i. Soc. 1988, i l f i . 831 1 - 8320. [lo] a) J. Han. T M. Loehr. Y Lu. J. S. Valentine. B A. Averill. J. Sanders-Loehr, J. Am. C'iimr. Sor 1993. 115. 4256 -4263: h j Y. Lu, L. B. LaCroix, M D. Lowery, E. I . Solomon, C.J. Bender. J. Peisach, J. A. Roe, E. Gralla. J. S. Valentine. ;hid 1993, 115, 5907 ~5918. [ I I ] N o changes in the UV-vis spectrum of MetlZlGly azurin were detected in the presence of ruhstantial concentrations (c > 2 M ) of the following anions at p H 8.0- acetate, bromide, chloride. cyanate. fluoride. nitrite, or phosphate.  B. C Dave. J. P. Germanas. R. S Czernuszewicz, J Am. C/iem. Soc. 1993. l15. 12 175-12 176. [l 31 a) A . W. Addison in Coppi,i- Coordinulioii Chiwiistrj. BiociirmienirindInor~~iiiic Ptmpruirc.s (Eds.: K D. Karlin. .I. Zuhieta) Adenine, New York. 1985, pp. 109- 128; h j J. Peisach. W E. Blumberg. A d 7 Biochon. Binphw. 1974. 16s. 691 -708. 1141 Carried out using the Quanta Package with the structure described by Nar et al. (ref. [ 3 a ] ) a s a framework. 1151 S. E. Shade. J. E. Penner-Hahn, H. J. Schugar. 9. Hedman. K. 0. Hodgson. E. I. Solomon, J. Am. C%am Soc. 1993, 115. 767-776. 1161 0. P. Anderson. J. Becher. H Frydendahl. L. F. Taylor. H. Toftlund. J. Cheni. S w C%eni.Conimun.1986, 699- 701 ' A Redox-Switchable Hemilabile Ligand: Electrochemical Control of the Coordination Environment of a Rh' Complex** Elizabeth T. Singewald, Chad A. Mirkin,* and Charlotte L. Stern We report the synthesis and characterization of a redoxswitchable hemilabile ligand (RHL), FcOCH,CH,PPh, (1, Fc = (y5-CsH,)Fe($-C,H,), and its complexation to Rh' to form the square-planar, cis-phosphane, cis-ether Rh' complex 2 (Scheme 1). This new ligand type can yield electrochemical con[*] Prof. C. A. Mirkin, E. T. Singewald. C . L. Stern Department of Chemistry. Northwestern University Evanston, lllinoii 60208-31 13 (USA) Telefax: Int. code + (708)491-7713 [*'I This work wits supported in part by the National Science Foundation ( N S E CHE-9121859), the Dow Chemical Company, and Johnson -Matthey (gift of RhCI,). C . A M . acknowledges n Dreyfus Foundation New Faculty Award (1991 1996). ii Beckinan Young Investipator Award (1992-1994). a Naval Young Investigator Award (1994- 1997). a National Science Foundation Young Investigator Award (1993 - 1998). and an A. P. Sloan Fellowship (1995- 1997) for partial support of this research.