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Langmuir 1988,4,580-583
Magnetic Resonance Studies of Cu(I1) Adsorbed on the Surface of the Bacterium Klebsiella pneumoniae W. Moh1,t H. Motschi,t and A. Schweiger*t Swiss Federal Institute of Technology Zurich, Physical Chemistry Laboratory, ETH-Center, CH-8092 Zurich, Switzerland, and Swiss Federal Institute for Water Resources and Water Pollution Control ( E AWAG), CH-8600 Dubendorf, Switzerland Received October 27, 1987 Magnetic resonance studies (EPR, ESEEM, and ENDOR) of copper(I1) ions adsorbed on deactivated bacteria are discussed. EPR data are consistently interpreted in terms of a strong surface complex formation of the Cu(I1) ion with functional groups attached to the outer cell membrane of Klebsiella pneumoniae. Elucidation of the metal-binding sites involved in the primary coordination sphere is accomplished by combining the complementary resonance techniques of ESEEM and ENDOR. Modulations of the electron spin-echo envelope are interpreted in terms of a remote nitrogen hyperfine interaction, whereas a large isotropic 14Ncoupling observed in the ENDOR spectrum is unraveling a direct copper-nitrogen coordination. From proton ENDOR data it is concluded that both axial and equatorial water bind as a ligand to the Cu(I1) surface complex. Introduction The fate of trace constituents, especially of metal ions, in natural water systems is mediated by the interactions of the solid/solution interface. Equilibrium models have been extensively applied to calculate the partition of a chemical species between solution and solid phase. Active surfaces are constituted mainly of colloidal suspended materials derived from hydrous oxide minerals and from organic origin. Surface coordination have contributed much to the understanding of adsorption isotherms which can be determined by titration and other analytical methods (atomic adsorption spectroscopy, electrochemical methods). However, thermodynamic models do not emerge from molecular structure descriptions, and inversely the bonding of an adsorbed species cannot directly be deduced from equilibrium concepts. This can be achieved by applying spectroscopic techniques amenable to resolve interactions of the bonding between the surface and adsorbate in a hydrous state. Recently we have demonstrated the potential of paramagnetic resonance spectroscopy to obtain structural information of surface complexes on hydrous oxides, e.g., 6-A1203.4 It has been pointed out that EPR may serve as a useful supplement to the thermodynamic modeling of surface complexation. EPR measurements generally do not allow the direct resolution of hyperfine interactions between the paramagnetic metal center and the surface functional groups. By the application of complementing techniques such as electron-nuclear double resonance (ENDOR) or electron spin-echo envelope modulations (ESEEM), inner-sphere coordination of ternary copper(I1) complexes with different ligands and oxovanadium(1V) adsorbed on 6-A1203has been established by observing the corresponding hyperfine c o ~ p l i n g . ~ ? ~ Recent studies at the Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG) have demonstrated that the role of biological debris in natural water systems is most prominent. Therefore we have initiated spectroscopic studies on organic material for which we have chosen as a model surface deactivated bacteria of the strain Klebsiella pneumoniae. This material interacts with dissolved trace metals (Cd, Zn, Cu) by the formation of surface-complexed species and coordination Swiss Federal Institute of Technology Zurich. *SwissFederal Institute for Water Resources and Water Pollution Control.
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compounds, which diffuse to the electrode during the period of a voltametric experiment.' Differential pulse polarographic (DPP) measurements have shown a high complex formation constant of Cu(I1) at a single potential to the surface functional site. EPR measurements indicate that Cu(I1) is bound to the bacteria in natural abundance, possibly to protein parts of the outer membrane.s An increased loading in the range 10-5-10-3 M Cu(II)/g of suspended solid material at pH 5-9 apparently continues to fill sites of a rather uniform functional nature. It is for this reason that we have attempted to investigate metal adsorption on an entire system (a bacterium) rather than on isolated fragments of biological material with the aim of obtaining answers about the heterogeneityjuniformity of the surface functional group and the chemical nature of the ligating moiety. Copper(I1) was chosen not only because it is an important transition-metal ion in biological systems but also because of its unique features for the application of EPR spectroscopy. The d9 configuration of the Cu(I1) ion has maximum spin density of the unpaired electron directed toward ligand positions (d,z-yz is the ground-state orbital in a prevalently tetragonal geometry). It is for that reason that EPR parameters of Cu(I1) complexes are very sensitive indicators of changes in the ligand field. Yet, ligand hyperfine interactions are scarcely resolved in EPR spectra of powderlike samples; therefore it is indicated to apply resonance techniques such as ENDOR and ESEEM to resolve weak hyperfine interactions. Materials and Methods Biologically deactivated cell material of K. pneumoniae was doped with Cu(I1) ions. To an aliquot (5.1 mL) containing 250 mg of dry suspended material was added 5 mL of water, 5 mL of 0.1 M KC104, and 5 mL of M C U ( N O ~ and ) ~ , the mixture was adjusted to pH 8. After dilution to 50 mL with H20 and 6 h of stirring, the solid material was deposited by slow centrifugation and dried at room temperature. Cu(I1)-histidine 1:2 (1)Stu", W.; Huang, C. P.; Jenkins, S. R. Croat. Chim. Acta 1970, 42, 223.
(2)Schindler, P.;Furst, B.; Dick, B.; Wolf, P. U. J. Colloid Interface Sei. 1976, 55, 469. (3)Westall, J. C.;Hohl, H. Adu. Colloid Interface Sci. 1980, 12, 265. (4) Motachi, H. Colloids Surf. 1984, 9,333. (5)Motschi, H.; Rudin, M. Colloid Polym. Sci. 1984, 262, 579. (6)Rudin, M.; Motachi, H. J. Colloid Interface Sci. 1983, 98,385. (7)S i m h s Goncalves, M. L.; Sigg, L.; Stumm, W. Enu. Sci. Technol. 1985, 19,141. (8) Motschi, H. In Aquatic Surface Chemistry; Stumm, W., Ed.; Wiley-Interscience: New York, 1987;p 111.
0 1988 American Chemical Society
Langmuir, Vol. 4, No. 3, 1988 581
Magnetic Resonance Studies of Cu(II)
Table 1. g Values and Hyperfine Parameters (in MHz) for Cu(I1) Adsorbed on K.pneumoniae and the Model Complexes Cu(His). C U ( H ~ S )and ~, CU(H~O)~*+ EPR ENDOR
pneumoniae
Cu(His)L in frozen solution 2.25 175 L=l 2.22 183 L = 2e CU(H~O),~+ 2.44 138
1 p J I
260
I
I
280 300 Magnetic field
,
L'
320 (mT)
1
340
30.6
35.3
35.9
37.9
2.4
4.7
3.3
8.1
n14Ncoupling along gll. * "N coupling along g, (average value). Coupling of the axial protons along g, (average value). Coupling of the equatorial protons along g, (average value). e S-band EPRZ1 (abbreviations: ax, axial; eq, equatorial).
Figure 1. X-band EPR spectrum of Cu(I1) adsorbed on the surface of the bacterium K. pneumoniae: 5 X lo4 M Cu(II), pH 7, temperature 77 K, microwave frequency 9.525 GHz. complexes were prepared as 2 X M solutions of a 1:2 mixture of glycerol/water to assist homogeneous glass formation a t low temperature. The sample volume for EPR experiments was about 180 pL.
E P R and ENDOR spectra were obtained on a Varian E-line spectrometer, equipped with a self-made ENDOR accessory with a TE012 probehead. Radio frequency (RF) fields were generated by means of a dedicated system consisting of a broad-band R F amplXer connected to an improved version of a tunable resonance circuit (frequency range, 3-40 MHz; R F field amplitude, B, 5 10 G (rotating frame)g). ENDOR signals were doubly coded modulation of the static magnetic field, 35 Hz; amplitude modulation of the RF, 3.7 kHz. The details of the pulsed ESR spectrometer have been published elsewhere.l0 ESEEM patterns were measured by using the two-pulse echo sequence, T/~-T-T-T-~c~o, with a microwave pulse length of 10 (r/2)and 20 ns ( T ) and T increments of AT = 10 ns. The deadtime of the spectrometer was about 150 ns. The experiments have been carried out at 77 K (EPR) and between 10 and 15 K (ESEEM, ENDOR) using a Helitran gascooling system from Air Products Limited.
Results Cu(I1) ions generally form tetragonal complexes. Immobilized Cu(I1) is characterized by an anisotropic EPR spectrum,which is typical for systems with axial symmetry. In frozen solutions (randomly distributed species) the magnetic parameters for the static magnetic field Bo oriented parallel (A,?, gll) and perpendicular (gl) to the complex normal can be evaluated from turning points in the first derivative EPR spectrum. The X-band EPR spectrum of Cu(I1) adsorbed on the surface of K. p n e u m o n i a e is shown in Figure 1. It is typical for monomeric copper chemisorbed on a surface." Only three copper hyperfine transitions are clearly visible in the low-field range of the spectrum. Ligand hyperfine structure is not resolved. The gll and A,,values of copper are listed in Table I. To resolve magnetic interactions with ligand nuclei ENDOR and ESEEM experiments were performed. ENDOR spectra for t w o Bo field orientations are shown in Figure 2. The spectrum in Figure 2a occurs from species with their molecular planes aligned perpendicular to Bo and, thus, represents a single crystal-like spectrum. Apart from a strong peak at the proton Larmor frequency v p and (9)Forrer, J.; Schweiger, A.; Ghthard, Ha.H. J.Phys. E 1977,113,470. (10)Fauth, J.-M.; Schweiger, A.; Braunschweiler, L.; Forrer, J.; Emst, R. R.J. Magn. Reson. 1986,66, 74. (11)von Zelewski, A.;Bemtgen, J. M. Inorg. Chem. 1982,21, 1771.
nI b 10
\
I
I
15
20 y (MHzI
25
Figure 2. (a) Single-crystal-like ENDOR spectrum (Boparallel to the complex normal) of Cu(I1) adsorbed on K. p n e u m o n i a e . Signals near 18 MHz are due to strongly coupled 14Nnuclei (two peaks separated by 2vN); T = 20 K. (b) Two-dimensional ENDOR (Boperpendicular to the complex normal). The peaks of axially and equatorially coordinated water as well as the signal of strongly coupled I4N are clearly visible; T = 20 K.
a proton peak typical for coordinated water, two features separated by twice the nuclear Larmor frequency of 14N are visible. The spectrum in Figure 2b is due to molecules with Bo in the molecular plane. It has the character of a two-dimensional ENDOR spectrum.12 Symmetric to the peak at v,,, signals of axially and equatorially coordinated water13 are observed. The broad line between 16 and 30 MHz indicates nitrogen interactions typical for directly coordinated 14Nligands with Bo in the complex plane.6 The ENDOR data are collected in Table I. The Fourier transformation (F'T) of an ESEEM pattern to the frequency domain contains in principle the same information as the one obtained from an ENDOR experiment, with the advantage of much higher sensitivity for weakly coupled n ~ c l e i . ~ The ~ J ~two-pulse ESEEM trace ~~
(12)Schweiger, A. In Structure and Bonding; Clarke, M. J., Goodenough, J. B., Hemmerich,P., Ibers, J. A., Jmgensen, C. K., Neilands, J. B., Reinen, D., Weis, R., Williams, R. J. P., Eds.; Springer-Verlag: Berlin, 1982;Vol. 51, p 1. (13)Atherton, N. M.; Horsewill, A. J. Mol. Phys. 1979,37, 1349. (14)Mims, W. B. In Electron Paramagnetic Resonance; Geschwind, S., Ed.; Plenum Press: New York, 1972;p 263. (15)Kevan, L.In Time Domain Electron Spin Resonance; Kevan, L., Schwartz, R., Eds.;Wiley-Interscience: New York, 1979;p 279.
582 Langmuir, Vol. 4, No. 3, 1988
Mohl et al.
maining two transitions give lines which are generally too broad to detect. I t is not possible to infer unambiguously the nature of the surface functional groups of the Cu(I1) coordination from these data. For that reason we studied the magnetic resonance parameters of different suitable model compounds. As representative examples, the data of histidine (His) coordinated to Cu(II) (1:l and 2:l) and of Cu(H20)2+ are included in Tables I and 11.
Discussion EPR parameters of Cu(I1) loaded to the cell material are in good agreement with data obtained for Cu(HieJL, L = 1, 2. As will be explained in more detail below, a histidine-containing peptide is the only possible unit of all amino acids to account for the spectroscopic data. The data in Table I are collected for comparison of ESR and ENDOR results of Cu(I1) on sites of the cell surface with Cu(I1)-histidine complexes. From the analysis of the ENDOR spectrum it is concluded that the high-frequency part is to be assigned to the imidazole nitrogen whereas the low-frequency domain is typical for coordination of water molecules in axial and equatorial positions. Furthermore, the EPR data (g , Allcu) 0 2 4 6 8 10 indicate a strong stabilization of the Cu(I1) complex,il which v IMHz) is also supported by thermodynamic (electrochemical) Figure 3. (a, top) Two-pulse electron spin-echo envelope modinvestigations.' Bidentate chelate formation (i.e., bonding ulation (ESEEM)trace (time domain) of Cu(I1) adsorbed on K. pneumoniae. Bo perpendicular to the complex normal: temvia the nitrogen of the glycine and imidazole part of His) perature 20 K, pulse length 10 ( ~ / 2 and ) 20 ns (T).(b, bottom) provides a mechanism which would explain the high staFourier transformation of the modulation trace in a (frequency bility of the complex formed. It still remains an open domain). The transition frequencies at 4.18, 1.52, and 0.59 MHz question whether the glycine-nitrogen ligand remains correspond to a weakly coupled (remote)nitrogen. linked to the peptide chain or whether it would be scissored off as the amino acid when complexed to copper. The Table 11. Nuclear Frequencies (MHz) of the Remote 14Nof latter situation would account for the determined stability the Imidazole Ring Obtained from a Fourier of the complex and is further corroborated by voltametric Transformation of the Two-Pulse ESEEM Patterns of Cu(I1) Adsorbed on K.pneumoniae (See Figures 3a,b) and experiments, which indicated the existence of a correCu(His)* and NQR Data of His (in MHz) sponding complex in solution.' Yet a third binding mechanism could be postulated from the interaction with ESEEM NQRb the two functional groups (glycine and imidazol) stemming CdHisL histidine . from two separate peptide chains or a second histidine of (frozen (diamagnetic) Cu(I1) on the same chain. The nitrogen hyperfine splitting of the K . pneumoniae solution) NHB >NH >Namino acid is considerably weaker than that of the imid4.18 3.77 3.92" azole N and is probably obscured by the strong proton 1.52 1.52 1.47" 1.067 1.424 2.477 signals around 12-16 MHZ.~O Thus from the ENDOR 0.59 0.44 0.63" 0.802 0.738 2.156 0.265 0.686 0.321 spectra of Cu(I1) complexes with functional groups on the bacterial surface and His as ligand it is well established "Reference 23. *Reference 18. that at least one nitrogen is directly coordinated. The hypothesis as postulated above is supported by and the corresponding FT-ESEEM spectrum for BoperESEEM results, which give evidence of a weakly coupled pendicular to the complex normal are shown in Figure 3. (remote) nitrogen. Below 5 MHz the spectrum is characterized by three Due to steric constraints22the tridentate His ligand acts prominent peaks (Table 11). These frequencies are typical like for a remote nitrogen in ligands of Cu(I1) c ~ m p l e x e s . ' ~ ~ ~ ~ a bidentate ligand for cupric square-planar complexes. Possible donor sites are the amino nitrogen and/or the The transitions at 0.59 and 1.52 MHz correspond to two sp2-coordinated nitrogen sites of the imidazole ring, reof the zero-field nuclear-quadrupole resonances.18 This spectively. For comparison, the ESEEM frequencies of is due to the fact that at X-band frequencies the nuclear Cu(II) on the cell material and Cu(His), are shown in Table Zeeman term of 14Nnearly coincides in magnitude with 11. The data of Cu(His), are very similar to those obtained the hyperfine coupling of the remote nitrogen. Thus, by Freedman and Mims,= who attributed their resonances depending on the sign of AN, Zeeman and hyperfine terms to the remote nitrogen of the imidazole ring, not directly cancel each other in one m, manif01d.l~ The third line at coordinated to the metal center. - 4 MHz corresponds to a transition between the outer Table I1 also contains the NQR data of His.ls Comtwo 14Nsuperhyperfine levels (AmI = 2 transition), where parison with the FT-ESEEM data leads to the conclusion Zeeman and hyperfine terms add to each other. The rethat of the two imidazole nitrogens only the sp2-hybridized nitrogen acts as donor site. We have to state that by (16) Burger, R. M.; Adler, A. D.; Horwitz, S.B.; Mims, W. B.; Peisach, J. Biochemistry 1981,20, 1701. (20) Iwaizumi, M.; Kudo, T.; Kita, S. Znorg. Chem. 1986, 25, 1546. (17) Freedman, J. H.; Pickart, L.; Weinstein, B.; M h , W. B.; Peisach, (21) Basosi, R.; Valensin, G.; Gaggelli, E.; Froncisz, W.; PasenkiewJ. Biochemistry 1982,21,4540. icz-Gierula, M.; Antholine, W. E.; Hyde, J. S.Znorg. Chem. 1986,25,3006. (18) Hunt,M. J.; Mackay, A. L.; Edmonds, D. T. Chem. Phys. Lett. I-
~
1976, 34, 473. (19) Mims,
W. B.; Peisach, J. J. Chem. Phys. 1978, 69, 4921.
(22) Sigel, H. Met. Ions B i d . Syst. 1973, 2, 73. (23) Mims, W. B. J.Magn. Reson. 1984,59, 291.
Langmuir 1988,4, 583-588
H2°
I "L
["
HP' I HO
2
Figure 4. Schematic representation of the local environment of the Cu(I1) ion on the surface of K. pneumoniae. The suggested
structure involves a bidentate chelate coordination with histidine bound to a surface peptide chain.
FT-ESEEM on paramagnetic species the exact zero-field NQR frequencies are not obtained as, for instance, in the corresponding diamagnetic metal complexes. Two reasons might be responsible for these deviations. Firstly, the proton on the remote 14N of metal-ligated His may be hydrogen bonded to the protein structure; in the case of model compounds there can be a hydrogen bond to the solvent, which is H20 in our case. It should be noted that the two lower NQR frequencies (yo, u-) are shifted by more than the high-frequency value (v+) in relation to the values obtained for systems when there is no substitution on the tricoordinated nitrogen site.18 Secondly, and presumedly, the most striking deviation stems from some difficulties of which ESEEM spectroscopy suffers in disordered systems: anisotropic broadening leads to destructive interference of the echo modulation,
583
which resulta in distorted line shapes in the frequency domain.24 Reijerse and K e i j ~ e r sshowed ~~ by spectra simulation that intense low-frequency components may arise, caused by anisotropic hyperfine interactions in combination with an anisotropy of the modulation intensity, which yield somewhat shifted quadrupole values. This might also be the case in our system. Furthermore, it has ale0 been pointed out%that the high-frequency component provides helpful information about the validity of the assumption that contact and nuclear Zeeman interaction are of comparable magnitude. For U l 4 ~N 1 MHz, this transition should occur at about 4 MHz, which is sufficiently confirmed by our experimental data (cf. Table 11). Summarizing all results leads to the assumption that the Cu(I1) ion is coordinated bidentally to histidine with evidence of remaining H20molecules occupying axial as well as equatorial positions. The tentative structure of the complex formed by Cu(I1) on the bacterial cell surface is shown in Figure 4. In this work it is shown that by means of complementary magnetic resonance data it is possible to elucidate the local environment of adsorbed metal ions (e.g., Cu(I1)) on unknown biological surfaces. On the basis of the available data an assignment of the most likely surface functional group was achieved.
Acknowledgment. This research has been supported by a grant of the Board of the Swiss Federal Institutes of Technology. Registry No. Cu(His), 77280-83-2; Cu(Hi&, 13870-80-9; C U ( H ~ O ) 14946-74-8; ~~+, His, 71-00-1. (24) Astashkm, A. V.; Dikanov, S. A.; Tsvetkov, Yu. D. Chem. Phys. Lett. 1987, 136,204. (25) Reijerse, E. J.; Keijzers, C. P. J. Mugn. Reson. 1987, 71,83.
Absorption and Fluorescence Properties of Rhodamine B Derivatives Forming Langmuir-Blodgett Films M. Van der Auweraer,* B. Verschuere, and F. C. De Schryver Chemistry Department KULeuven, Celestijnenlaan 200F, 3030 Leuuen, Belgium Received June 4, 1987. In Final Form: November 20, 1987 Surface pressurearea isotherms for mixed films of diodadecylrhodamine B and arachidic acid with varying mixing ratios were recorded. Monolayers and multilayers were deposited on hydrophylic glass slides. Absorption spectra, fluorescence spectra, and relative quantum yields of fluorescence were examined as a function of the mixing ratio in the film. The type of aggregates formed in homogeneous solution was not observed, and the relative quantum yield of fluorescence decreases with increasing amount of dye in the layer. From these results a packing in the Langmuir-Blodgett monolayer can be proposed. Two dye molecules form a dimer in which the transition dipoles and the vector linking the centers of the molecules form an angle of 55O, leading to a dimer without spectral shift. The similarity between the photophysical properties of adsorbed rhodamine B molecules at high coverages and of the monolayers investigated in this contribution could point to a similar packing of the chromophores.
Introduction Although the aggregation of rhodamine' in solution has been observed several years ago, the nature of the aggregates and their photophysical properties are still a controversial topic.2 FBrster and KBnig3showed that in an (1) Avnir, D.; Levy, D.; Reisfeld, R. J. Phys. Chem. 1984, 88, 5956. ojeda, R. P. Chem. Phys. Lett. 1982,87,656. (2) Lopez-Arbeloa,I.; 0.;
aqueous solution of rhodamine B Beer's law is valid up to a concentration of 5 X lo* M; at higher concentrations a new band at the hypsochromic side was observed. The band at low concentrations is due to absorption of the monomers, while the hypsochromic band was attributed to the absorption of dimers. Dimerization leads to a blue (3) Ffster, Th.; KBnig, E. 2.Electrochem. 1967, 61,344.
0743-7463/88/2404-0583$01.50/00 1988 American Chemical Society
