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Novel Biological Copper Proteins through Anion Addition to the Mutant Met121Gly of Pseudomonas aeruginosa Azurin.

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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.
[3] 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.
[5] 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.
[6]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.
[9] 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
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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
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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.
[2] E. I Solomon, M. J. Baldwin. M . D. Lowery, Clicni. Rev. 1992. 92. 521 542.
[3] 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.
[6] 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.
[7] 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.
[8] 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.
[9] 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.
[12] 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.
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aeruginosa, biological, protein, met121gly, additional, anion, mutant, novem, coppel, pseudomonas, azurin
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