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J. Phys. Chem. 1996, 100, 1950-1956
Application of Circularly Polarized Luminescence Spectroscopy to Tb(III) and Eu(III) Complexes of Transferrins Sohrab Abdollahi and Wesley R. Harris* Department of Chemistry, UniVersity of MissourisSt. Louis, 8001 Natural Bridge Road, St. Louis, Missouri 63121
James P. Riehl* Department of Chemistry, Michigan Technological UniVersity, 1400 Townsend DriVe, Houghton, Michigan 49931 ReceiVed: July 20, 1995; In Final Form: October 3, 1995X
Circularly polarized luminescence (CPL) and total luminescence of Tb(III) and Eu(III) as substitutional replacements for iron in a series of Fe binding transferrins are reported. The measurement of the total emission intensity is a direct measure of binding of the lanthanide ions and illustrates that approximately 2 equiv of Tb(III) and Eu(III) is bound to these proteins. In agreement with previous work, circularly polarized luminescence from Tb(III) bound to the transferrins is quite large. Additional measurements show that the net CPL displays no dependence on concentration of the metal ion and shows little variation between the two binding sites. CPL from lactoferrin is very similar to serum transferrin, however, ovotransferrin shows differences in line shape and magnitude. These results are discussed in the context of the previously published crystal structure of the binding sites of the three proteins.
Introduction Circularly polarized luminescence (CPL) spectroscopy has been used for almost 20 years to study the structure and structural changes in luminescent optically active molecular systems.1,2 In this technique one measures the usually small net circular polarization in the luminescence from chiral molecular systems. Due to the inherent problem in measuring absolute emission intensities, it is common to report CPL results in terms of the ratio of the differential emission intensity, ∆I ()ILeft - IRight), to the average total emission intensity, I/2 ()ILeft + IRight)/2, either measured at a specific wavelength or integrated over an emission band. This ratio is usually referred to as the emission or luminescence dissymmetry factor and denoted by gem or glum. Advances in instrumentation in the past 5 years have resulted in significant increases in the selectivity and sensitivity of this chiroptical technique. These advances have allowed for the study of more dilute and more weakly luminescent systems than the early applications of CPL spectroscopy. Many of the pioneering applications of this technique involved studies of luminescent biomolecules or fluorescent labels attached to biomolecules, and this continues to be one of the more promising areas of research for CPL measurements. In our laboratory we have emphasized the measurement of CPL from luminescent lanthanide ions. For example, recently we have reported CPL measurements from Tb(III) and Eu(III) as substitutional replacements for Ca(II) in several calcium binding proteins.3-5 It was demonstrated in this previous work that the CPL spectrum for several EF-hand type proteins were very similar and that conformational changes induced by the addition of metal ion could be followed by variation in the magnitude of the CPL signal. The use of lanthanide(III) ions as substitutional replacements for Ca(II) and other metal ions is well known.6,7 In our applications we make use of the fact that Tb(III) and Eu(III) * Authors to whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, December 15, 1995.
0022-3654/96/20100-1950$12.00/0
are luminescent when excited with a medium-power Ar ion laser, and that the intraconfigurational f T f transitions involved in both the excitation and the emission are often associated with large optical activity.8 In this present work we extend the applications of this technique to a series of complexes in which Tb(III) or Eu(III) is bound to the Fe binding sites of the proteins transferrin (Tf), lactoferrin (Lf), and ovotransferrin (ovoTf). These proteins are distinguished by the unique feature that metal binding entails the formation of a tertiary complex between the apoprotein, the metal, and a synergistic carbonate anion derived from the buffer.9-12 There is essentially no metal complexation in the absence of the synergistic anion, which is directly coordinated to the metal in the tertiary complex.11,13,14 Serum transferrin carries ferric ion in serum between sites of uptake, utilization, and storage. While its primary function is iron transport, it also serves as a bacteriostatic agent by sequestering essential iron away from invading microorganisms. Both lactoferrin, which is found in milk, tears, and other physiological fluids, and ovotransferrin, which is found in avian egg whites, are primarily bacteriostatic agents. The overall structures of all three proteins are very similar.11 Although each protein consists of a single polypeptide chain, this chain folds into two distinct lobes, each of which contains a single high-affinity metal binding site. The coordination geometry about the metal ion is almost identical for the two sites of transferrin and lactoferrin. However, there are subtle spectroscopic differences between the sites. Both show Fephenolate charge transfer bands centered at 465 nm, but the extinction coefficient of the C-terminal site is about 30% larger than that of the N-terminal site.15 In addition, there are distinct EPR spectra for the two sites in diferric transferrin,16 and the ovotransferrin sites can be distinguished by 27Al, 205Tl, and 45Sc NMR.17-19 The use of lanthanides as luminescent probes of metal ion binding sites in transferrins has also been a subject of considerable recent interest.20-26 In a very early CPL study, Gafni and Steinberg27 reported measurements of the CPL of Tb(III) bound © 1996 American Chemical Society
Application of Circularly Polarized Luminescence to human serum transferrin and ovotransferrin under varying metal:protein ratios. Broad band (30 nm) excitation of their samples at 300 nm was accomplished with a high-pressure mercury arc lamp, and the emission and circular polarization were measured at selected wavelengths. These authors reported that the total emissions of Tb(III) bound to serum transferrin and ovotransferrin were identical but that the CPL from these two proteins, although similar, did show small variations presumably due to differences in sequence and folding of the two proteins. These authors also examined the CPL under varying protein:metal ratios and saw no differences in the measured CPL. These results, along with some competitive binding studies, led the authors to conclude that the two metal ion sites have identical structures. In this work we examine Tb(III) and Eu(III) bound to serum transferrin under a variety of experimental conditions. In our experiments, direct electronic excitation of the lanthanide ions is accomplished with visible laser light. Comparison of the results is made to studies involving ovotransferrin and lactoferrin. Experimental Section Apotransferrin (apoTf). Human serum apotransferrin was obtained from two different sources: Sigma and Calbiochem. Solutions of apotransferrin were prepared by dissolving 100200 mg of protein into 1 or 2 mL of 0.1 M Hepes buffer, pH ) 7.4, containing 0.1 M sodium perchlorate. This solution was passed through a 1.6 × 30 cm column packed with Spectra/ Gel AcA 202 gel filtration beads in the same buffer. Eluent fractions containing the protein were identified using a Pharmica UV-1 monitor and concentrated using an Amicon Model 8010 ultrafiltration cell fitted with an XM-50 membrane under 70 psi of nitrogen gas. The concentrated apoTf solution was eluted through a second Spectra/Gel column with 0.1 M Hepes and concentrated by ultrafiltration to the desired molarity. The concentration was determined from the absorbance at 278 nm using an extinction coefficient of 93 000 cm-1 M-1.28 Monoferric N-Terminal Transferrin (Tf-FeN). Monoferric N-terminal transferrin was prepared from diferric transferrin by the method reported by Baldwin and de Sousa.29,30 Diferric transferrin was dissolved in a solution of 0.134 M EDTA and 2.57 M NaClO4 in 0.1 M Hepes. The absorption of the solution was monitored at 465 nm every 10 min. When no further change in absorption was observed, the solution was passed through a column of 2 × 30 cm Spectra/Gel AcA 202 and washed 5 or 6 times with 0.1 M Hepes in an Amicon ultrafiltration cell to eliminate all traces of EDTA and perchlorate. The concentration of N-terminal transferrin was determined from the absorbance at 278 nm based on a molar extinction coefficient of 103 000 cm-1 M-1. Monoferric C-Terminal Transferrin (FeC-Tf). Monoferric C-terminal transferrin was prepared by adding exactly 1 equiv of freshly prepared bis(nitrilotriaceto)ferrate(III) solution at pH 4.0 to an apotransferrin solution in 0.1 M Hepes at pH 7.4.30 Free NTA was removed by passing the sample solution through a Spectra/Gel column and then washing 5 or 6 times with 0.1 M Hepes in an Amicon ultrafiltration cell. The concentration of C-terminal monoferric transferrin was determined from the absorbance at 278 nm based on an extinction coefficient of 103 000 cm-1 M-1. Lanthanides Stock Solutions. Solutions of Tb(III) and Eu(III) were prepared from reagent grade chloride salts. TbCl3 and EuCl3 were dissolved in a small volume of water which had been adjusted to pH 2 through the addition of HCl. After dilution to volume, the pH of the stock solutions was ap-
J. Phys. Chem., Vol. 100, No. 5, 1996 1951 proximately 2.8. A second stock solution of Eu(III) was prepared by dissolving the chloride salt in a D2O solution which had been adjusted to the proper pD through the addition of HCl. The pD was measured with a pH meter using the correction term pD ) pH + 0.4.31 The Tb(III) and Eu(III) stock solutions were standardized by complexiometric titration with EDTA, using Xylenol Orange as the indicator in an pH 5.5 acetate buffer.32 Eu(III):Transferrin CPL Solutions. Eu(III):Tf samples were prepared in D2O to enhance emission intensity.33 A 0.1 M Hepes buffer was prepared by dissolving the acid form of Hepes directly into D2O. The pD was then adjusted to 7.4 by the addition of small amounts of solid NaOH. Solid apotransferrin was dissolved in the Hepes/D2O buffer, and solid NaHCO3 was added to bring the bicarbonate concentration to 1.5 mM. Instrumentation. CPL and total luminescence (TL) spectra were recorded on an instrument constructed in our laboratory operating in a photon-counting mode.2 Laser excitation of Tb(III) and Eu(III) was accomplished at 488 and 465 nm, respectively, with a Coherent Innova-70 argon ion laser. The emitted light was collected at 90° to the direction of excitation and was passed through a 50 kHz photoelastic quarter-wave modulator (Morvue), which together with a linear polarizer acts as a circular analyzer. The 50 kHz modulation in the emission beam corresponds to alternately left then right circularly polarized emitted light. The polarization-modulated beam was then focused onto the entrance slits of a 0.22 m double monochromator (SPEX) and detected by a cooled EMI-9558QB photomultiplier tube. Output pulses were passed through an amplifier/discriminator and counted by a specially built differential gated photon counter which was phase referenced to the 50 kHz modulator frequency. The ratio of the differential count (corresponding to an up/down counter) to the total count (a separate up counter) was input to a personal computer after a fixed number of total counts were collected. Quartz fluorescence sample cells were used throughout the experiments. Stray excitation light was eliminated by a long pass filter which was located between the circular analyzer and the sample. Results Difference UV Absorption. The binding of lanthanides to transferrin may be monitored by recording the differences in absorption between cuvettes containing equal amounts of protein and buffer, one of which contains metal ion.20,34-36 In this procedure, acidic solutions of the lanthanide ion are added to the sample cuvet, and equal volumes of water are added to the reference cuvet. A set of difference ultraviolet spectra produced by the addition of aliquots of Tb(III) to 2 mL of apotransferrin at pH 7.4 and room temperature are plotted in Figure 1. The difference bands at 247 and 295 nm are characteristic of metal ion binding at the two transferrin binding sites. Results for corresponding measurements using Eu(III) look very similar. In order to normalize the absorption results and to correct for dilution effects, the absorbance data are converted to apparent absorptivities by dividing the absorbance at 245 nm by the analytical concentration of transferrin. In Figure 2 the resulting absorptivity is plotted versus the ratio of total lanthanide to total transferrin concentration. In this figure we show results for both titration of transferrin by Tb(III) and Eu(III). The titration curves shown in Figure 2 are similar to previous results.34 The initial slopes for both ions are linear indicative of complete binding of each aliquot. Beyond 1 equiv of metal ion, less than 100% binding is indicated by the curvature seen in both sets of data. The lower value for absorptivity obtained for the slightly larger Eu(III) ion is consistent with previous results.34
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Figure 1. Difference UV absorption spectra for the addition of Tb(III) to an aqueous solution of apotransferrin at pH ) 7.4
Figure 3. Circularly polarized luminescence (upper curve, ∆I) and total luminescence (lower curve, I) for a solution of 1.0 × 10-3 M transferrin containing 2.0 equiv of Tb(III). pH ) 7.4; λexc ) 488 nm; [HCO3-] ) 3.0 mM.
In CPL spectroscopy one commonly reports the quantity glum, which is defined as follows
Figure 2. Absorptivity versus the ratio of total lanthanide concentration to transferrin concentration (see text).
Luminescence Results. In Figure 3 we plot total luminescence (I) and circularly polarized luminescence (∆I) for a solution of apotransferrin containing 2 equiv of Tb(III). The spectral region displayed corresponds to the 5D4 f 7F5 transition of Tb(III). This is the most intense emissive transition for this ion. Direct excitation of Tb(III) was accomplished by using the 488 nm line of an Ar ion laser. The total emission spectrum was recorded with a band pass of 0.4 nm, whereas the circularly polarized luminescence was recorded at 1 nm intervals with a band-pass of 0.9 nm. Since no significant additional structure in the CPL spectrum is seen using higher spectral resolution and since a complete CPL spectrum taken even under these conditions requires more than 3 h of data collection, it was decided to record all spectra using the parameters given above. The use of 488 nm laser excitation avoids problems with photodegradation of samples when using UV radiation to excite the Tb(III) ions via intermolecular energy transfer from neighboring aromatic residues. In this study no photodegradation of the samples was observed even after more than 3 h of 1.2 W of 488 nm radiation. As can be seen in Figure 3, the total emission shows some structure due to the complex crystal field splitting expected for the J ) 4 and J ) 5 states in a low-symmetry site. The circularly polarized emission shows a number of peaks of varying sign and magnitude that correspond to several of the peaks seen in the total luminescence spectrum. This and all subsequent spectra were obtained using an optimal bicarbonate concentration (3 mM) which was determined by titration of TbTf-Tb with 0.1 M bicarbonate solution while monitoring the total emission intensity.
glum )
Ileft - Iright ∆I ≡ I/2 (Ileft + Iright)/2
(1)
The factor of 1/2 in this equation is added to make this definition consistent with that of the absorption equivalent gabs.1 In our instrumentation, as described above, it is the ratio glum that is determined on an absolute scale. It should be noted that the concentrations of apoTf used for these luminescence studies are at least a factor of 10 larger than in the difference UV measurements, in order to increase the emission intensity. Nevertheless, the concentrations of Tb(III) are quite small, and a considerable amount of laser power must be used to generate sufficient emission intensity. As a result, measurements at the lowest concentrations may be contaminated with background noise, Iback. glum is then given by the following
glum(measured) )
(Ileft + Iback) - (Iright + Iback) ∆I ≡ I/2 [(Ileft + Iback) + (Iright + Iback)]/2 (2)
where we have simply added Iback to all measured intensities. The background noise cancels from the numerator of eq 2, and thus, the presence of such noise has the effect of reducing the measurement of glum.
glum (measured) )
Ileft - Iright ∆I ≡ I/2 (Ileft + Iright)/2 + Iback
(3)
As seen below, for the low concentrations in which the background signal is not negligible, one can either measure the intensity for a reference sample which has no added Tb(III) or, if titrating aliquots of Tb(III), extrapolate the intensity measure-
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Figure 5. Plot of measured glum (filled symbols) and corrected glum (open symbols) as a function of equivalents of Tb(III) for a 1.0 × 10-3 M solution of transferrin at pH ) 7.4. Squares correspond to λemission ) 545.5 nm and circles to λemission ) 540 nm (see text). Figure 4. Plot of glum (filled circles) and total emission intensity (open circles) measured at 545.5 nm (filled circles) as a function of equivalents of Tb(III) for a 1.0 × 10-3 M solution of transferrin at pH ) 7.4.
ments back to zero concentration. In either case, Iback may be estimated and used to determine the corrected value for glum. Aqueous solutions of Tb(III) and Eu(III) are extremely weak emitters, and thus, the total emission intensity is a direct measure of metal ion binding. In Figure 4 we plot the emission intensity (open circles) measured at the positive peak of the CPL spectrum (545 nm) versus equivalents of Tb(III). The total emission data are in reasonably good agreement with the difference UV results and show a linear increase in intensity until approximately 2 equiv of Tb(III) has been added. Also given in this figure is the value of glum measured at the same wavelength. As can be seen in this figure, the glum values are small upon addition of the first few aliquots of Tb(III) but reach a “limiting” value of approximately +0.06 after addition of more than 0.5 equiv. The error in glum (shown by the representative error bar) is equal to x2/N, where N equals the total number of photon counts,37 and is the same for all points plotted. As discussed above, for these low concentration solutions, the measurement of glum may be contaminated with background noise. In Figure 5 we plot the measured glum versus the “corrected” glum for the data presented in Figure 4 as well as the values measured at the (negative) peak of the CPL spectrum at 540 nm. The corrected values were obtained by using eq 3 and setting Iback equal to 350 counts/s. As can be seen, the correction has more of an effect on the peak with the lower total emission intensity. Except for the very smallest concentrations, for both wavelengths, accounting for the signal background has the effect of making the value of glum constant during the addition of Tb(III). As discussed below, glum is an intrinsic measurement of the chirality of the emitting chromophore and, as such, should be independent of concentration if only one type of emitter contributes to the measurement. The possibility that variations in glum at low concentrations were being caused by effects due to local saturation of apotransferrin when adding small amounts of Tb(III) was examined by repeating the measurements described above on a sample prepared by adding 2.083 equiv of Tb(III) and then diluting the solution with buffer so that the total Tb(III) concentration corresponded to that used for the measurement reported in Figure 4, i.e. 0.25 equiv. The glum values that were obtained from this dilution experiment were within the limits of experimental error of the previous
results. The most likely explanation for the low CPL intensity for the first points in the titration of Tb(III) is the presence of a small amount of residual complexing agent. Emission from such a species would not be circularly polarized and, therefore, would result in a lower measurement for glum. It should be pointed out that, in general, care must be taken in interpreting glum results at one wavelength in a CPL spectrum as complex as that obtained here and as shown in Figure 3. Relatively small changes in coordination geometry may result in large changes in glum (and ∆I) due to crystal field effects. Furthermore, by simply recording results at a fixed wavelength, one might completely miss other changes in spectral line shape and magnitude. In these experiments and other experiments of this type, it is common practice in our laboratory to record an entire CPL spectrum at several points during the titration. For the systems studied here, these complete spectra involved more than 3 h of data collection. However, no significant changes in line shape were observed. In order to investigate whether or not CPL may be used to probe differences in the chiral environment of the two metal ion sites in transferrin, samples of transferrin were prepared in which Fe(III) was selectively bound to the N-terminal (FeNTf) or C-terminal (FeC-Tf) metal ion binding site. Total emission and CPL spectra for these two samples after addition of 1 equiv of Tb(III) are given in Figure 6. As can be seen, the set of spectra are almost identical and illustrate the similarity of the metal ion environment of the two sites in this protein. As discussed above, due to the complex nature of the states involved in this transition, one would expect that even relatively small changes in coordination geometry would have a significant effect on the CPL spectra. Such changes in line shape and intensity have been extensively used to probe the solution structure of Tb(III) complexes.1 In Figure 7a we plot CPL and total emission spectra for 2 equiv of Tb(III) bound to human lactoferrin, and in Figure 7b are results for chicken ovotransferrin. These data were collected using the same experimental parameters as described previously. Comparison of Figure 7a with the spectra given in Figure 3 illustrates the close similarity of these related proteins, whereas significant changes in magnitude and small changes in line shape are seen for ovotransferrin. Due to the complex nature of the states involved, CPL from Tb(III) often displays the complex structure displayed in Figure 3. For this reason, Tb(III) is the ion of choice when one is interested in monitoring structural changes. The ground state
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Figure 8. Circularly polarized luminescence (upper curve, glum) and total luminescence (lower curve, I) for a solution of 0.34 × 10-3 M transferrin in D2O containing 3.0 equiv of Eu(III) (pH ) 7.4, λexc ) 488 nm, [HCO3-] ) 3.0 mM). Figure 6. Circularly polarized luminescence (upper curve, ∆I) and total luminescence (lower curve, I) for (a) a solution of 1.0 × 10-3 M monoferric N-terminal transferrin containing 1.0 equiv of Tb(III) (pH ) 7.4, λexc ) 488 nm, [HCO3-] ) 3.0 mM) and (b) a solution of 1.0 × 10-3 M monoferric C-terminal transferrin containing 1.0 equiv of Tb(III) (pH ) 7.4, λexc ) 488 nm, [HCO3-] ) 3.0 mM).
attainable by the use of D2O in place of H2O due to the difference in vibrational frequency.31 In Figure 8 we plot the total emission in the spectral region corresponding to the 5D0 f 7F2 transition of Eu(III) and glum results for 2 equiv of Eu(III) added to transferrin in a D2O buffer solution. The corresponding measurement in aqueous solution resulted in extremely weak luminescence; however, the difference UV results for the D2O solution were identical to the aqueous solution, indicating similar binding constants in the two solvents. Even in D2O the luminescence was very weak, and as a result glum was measured only at the points shown in this figure. More than 8 h of data collection was necessary to obtain the results given in Figure 8 compared to less than 1 h for the Tb(III) results at similar concentrations. Discussion
Figure 7. Circularly polarized luminescence (upper curve, ∆I) and total luminescence (lower curve, I) (a) for a solution of 1.0 × 10-3 M lactoferrin containing 2.0 equiv of Tb(III) (pH ) 7.4, λexc ) 488 nm, [HCO3-] ) 3.0 mM) and (b) for a solution of 1.0 × 10-3 M ovotransferrin containing 2.0 equiv of Tb(III) (pH ) 7.4, λexc ) 488 nm, [HCO3-] ) 3.0 mM).
of Eu(III), on the other hand, is nondegenerate (7F0) as is the emitting state (5D0), and as a result, the luminescence spectra from this ion are much more amenable to detailed interpretation and, potentially, the development of quantitative spectra: structure correlations. As a general rule, however, Eu(III) species are not as luminescent as the corresponding Tb(III) complex, particularly in aqueous solution where deactivation of the emitting state by coordinated water is more efficient. Significant enhancement of the emission of Eu(III) is often
As displayed in Figures 3, 6, and 7, the total emission spectra in the 5D4 f 7F5 transition of Tb(III) as a substitutional replacement for Fe(III) in transferrins are not a very sensitive measure of minor structural differences, although the emission intensity may be used as a measure of metal ion binding as shown in Figure 4. It is also important to note that, even though all of the spectra shown were measured with a band pass greater than 1.5 nm, no significant changes in line shape were observed when collecting data under conditions of higher spectral resolution. This general result is consistent with previous measurements of Tb(III) luminescence from Ca binding proteins. Total emission from Tb(III) complexes in aqueous and nonaqueous media, on the other hand, often shows considerable well-resolved crystal-field splitting,1 and therefore, the lack of observable splittings in these protein systems reflects, at least to some extent, the lack of rigidity in the lanthanide coordination sites. The circularly polarized luminescence from Tb(III) bound to the series of transferrins described above does show considerable structure as evidenced by the various positive and negative peaks seen in the spectra. The source of this fine structure is the crystal field splitting associated with the ground and excited state terms for this ion, with various combinations of crystal field transitions exhibiting preferences for emission of left or right circularly polarized light. However, the extremely complex nature of this transition (in C1 symmetry there may be 99
Application of Circularly Polarized Luminescence different crystal field transitions!) prohibits any detailed assignments or structural correlations. One is thus limited to observing similarities and differences and relating these to structural similarities or changes. All of the CPL spectra shown in this work are, in fact, very similar, and one concludes that the metal ion sites within the individual transferrins and between the different transferrins are structurally very similar. The similarity in CPL spectra from Tb(III) between related proteins has also been seen in previous work on Ca binding EF-hand proteins. However, for the few classes of proteins that have been studied so far, the CPL spectra are all different. As seen previously,27 the magnitude of the CPL for Tb(III): transferrin is extremely large [glum(540 nm) ) -0.20]. This value is, in fact, comparable to the values seen in certain higher symmetry lanthanide complexes and represents the largest value so far measured for Tb(III):protein systems. For Ca binding proteins the largest glum values so far reported are a factor of 10 smaller in magnitude. It is not clear, however, whether or not this large value is a consequence of a particularly “chiral” stable site for the lanthanide ion or is just due to accidental superposition of crystal-field transitions having large individual glum values. The crystal structure of diferric serum transferrin shows that the ferric ion coordination geometries are very similar for the N- and C-terminal binding sites. The degree of similarity between the two binding sites for other metal ions has been assessed from NMR data on the transferrin complexes of 45Sc, 27Al, 205Tl, and 113Cd.17,18,38-40 For the transferrin complexes of Sc3+, Al3+, and Cd2+, essentially identical NMR spectra are seen for the C- and N-terminal binding sites. However, distinct signals for each site are observed in the spectrum of the dithallium transferrin complex. As shown in Figure 6a,b, only very small differences are seen in the CPL spectra of Tb(III) bound at the individual C- and N-terminal sites. Thus, we conclude that Tb3+ is another metal ion for which there is no significant difference in the coordination geometries between the two transferrin binding sites. This is, in fact, the same conclusion drawn by Gafni and Steinberg27 in their lowresolution studies. The CPL spectra of Tb-Tf and Tb-Lf are very similar to each other, but the spectrum for Tb-ovoTf is quite different, as shown in Figure 7b. Crystal structures are available from the Brookhaven Protein Data Bank for the ferric complexes of the isolated N-lobe of all three transferrins.41 Stereoviews of all three N-terminal binding sites are shown in Figure 9. The sites have all been oriented to place the single histidine ligand in an axial position along the +z axis. This orientation puts both tyrosines and the aspartic acid in the equatorial plane. The bidentate carbonate occupies the fourth equatorial site and the axial position trans to the histidine. The positions and bond distances for the four ligating groups of the protein are similar in all three complexes. One significant deviation is that the Fe-ligand bond distance to tyr-192 in ovotransferrin is only 1.77 Å, compared with values ranging from 1.92 to 2.10 Å for the other Fe-tyrosine distances. However, the largest variation among the three proteins appears to be related to the orientation of the bidentate carbonate anion. Since the bicarbonate forms only a four-membered chelate ring, it cannot fit into a regular octahedral geometry. This is shown by the O-Fe-O bond angles of only about 64° for the carbonate chelate ring in all three proteins. In the transferrin and lactoferrin structures, the carbonate ligand sits roughly midway between the equatorial and axial sites it would occupy in a regular octahedron. Thus, the his-Fe-CO3 (equatorial) bond angles are larger than the 90° expected for cis positions, and
J. Phys. Chem., Vol. 100, No. 5, 1996 1955
Figure 9. Stereoscopic views of the N-terminal binding sites of transferrin, lactoferrin, and ovotransferrin.
the his-Fe-CO3 (axial) angle is smaller than the 180° expected for trans positions. However, in the ovotransferrin structure, this carbonate ligand shifts up toward the histidine and over toward the aspartate. Thus, the cis his-Fe-CO3 angle drops to only 72°, and the trans his-Fe-CO3 angle drops to only 135°. This distortion involves an enlargement of the triangular face of the coordination octahedron involving the two tyrosine residues and the trans carbonate oxygen. One can quantify this distortion by measuring the average L-M-L bond angle for the three cis bond angles associated with this face on the octahedron. The average bond angle is 101.9° for Tf, 97.3° for Lf, and a much larger 114.3° for ovoTf. To characterize the overall symmetry of the ferric-protein binding sites from the crystallographic data, we have calculated the root-mean-square deviation between the 12 cis L-Fe-L bond angles and the 90° angle expected of a regular octahedron. The resulting rms deviations are 11.0° for Lf, 14.5° for Tf, and 19.1° for ovoTf. Thus, for the ferric complexes, the ovoTf N-terminal binding site appears to be the most distorted from a regular octahedral geometry. Since the Tb3+ ion is thought be retain one water ligand when it binds to apoTf,20 the Tb-Tf complex is presumably 7-coordinate. Thus, the ferric ion crystal structure is only a guide to what one might expect for the Tb-Tf complex. Because of the relatively high molar absorptivities of the lanthanide-Tf complexes in the difference UV spectra, there has been
1956 J. Phys. Chem., Vol. 100, No. 5, 1996 speculation that the aquo ligand might be hydrogen bonded to a nearby noncoordinating tyrosine residue.34 If this were true, the aquo ligand would have to bond to the face of the ferric complex defined by the two tyrosines and the histidine. One can speculate further that the distortion in the ovotransferrin complex might make it more favorable for the aquo ligand to move into the open area between the two tyrosines and the axial carbonate. Thus, the differences observed in the CPL spectra of transferrin and ovotransferrin could reflect different orientations of the aquo ligand. The overall symmetry of the binding sites in transferrin, ovotransferrin, and lactoferrin has also been evaluated from measurement of the quadrupole coupling constants in the 27Al NMR spectrum.37 Based on these coupling constants, the overall symmetry appears to vary as
ovoTF (N site) > Tf (N and C sites) > ovoTf (C site) > Lf (C and N sites) Thus in the Al NMR, the ovoTf N site appears to be the most symmetrical. A similar ordering of the ovoTf and Tf binding sites is given by the quadrupole coupling constants of the 45Sc complexes.18 The NMR results showing larger differences in coupling constants for the two binding sites of ovotransferrin suggest the possibility that the unusual CPL for ovoTf actually consists of two distinct but overlapping spectra, one for each ovotransferrin binding site. Acknowledgment is made to the American Chemical SocietyPetroleum Research Fund (JPR) and to the National Institutes of Health (WRH #5R01-DK3553) for partial support of this work. References and Notes (1) Riehl, J. P.; Richardson, F. S. Chem. ReV. 1986, 86, 1. (2) Riehl, J. P.; Richardson, F. S. Methods Enzymol. 1993, 226, 539553. (3) C¸ oruh, N.; Riehl, J. P. Biochemistry 1992, 13, 7970-7976. (4) Riehl, J. P.; C¸ oruh, N. Eur. J. Solid State Inorg. Chem. 1991, 28, 263-67. (5) Riehl, J. P; C¸ oruh, N., Collect. Czech. Chem. Commun. 1991, 56, 3028-31. (6) Martin, R. B.; Richardson, F. S. Q. ReV. Biophys. 1979, 12, 181. (7) O’Hara, P. B. Photochem. Photobiol. 1987, 46, 1067. (8) Richardson, F. S. Inorg. Chem. 1980, 19, 2906. (9) Brock, J. H. In Metalloproteins, Part II; Harrison, P., Ed.; Macmillan: London, 1985; pp 183-262.
Abdollahi et al. (10) Bates, G. W.; Graybill, G.; Chidambaram, M. V. In Control of Animal Cell Proliferation; Boynton, A. L., Leffert, H. L., Eds.; Academic Press: New York, 1987; pp 153-202. (11) Baker, E. N. In PerspectiVes on Bioinorganic Chemistry; Hay, R. W., Dilworth, J. R., Nolan, K. B., Eds.; JAI Press: London, 1993; pp 161205. (12) Aisen, P. In Iron Carriers and Iron Proteins; Loehr, T. M., Ed.; VCH: New York, 1989; pp 353-371. (13) Anderson, B. F.; Baker, H. M.; Norris, G. E.; Rice, D. W.; Baker, E. N. J. Mol. Biol. 1989, 209, 711-734. (14) Sarra, R.; Garratt, R.; Gorinsky, B.; Jhoti, H.; Lindley, P. Acta Crystallogr. 1990, B46, 763-771. (15) Bali, P. K.; Harris, W. R. Arch. Biochem. Biophys. 1990, 281, 251256. (16) Harris, D. C.; Aisen, P. In Iron Carriers and Iron Proteins; Loehr, T. M., Ed.; VCH: New York, 1989; pp 239-351. (17) Aramini, J. M.; Krygsman, P. H.; Vogel, H. J. Biochemistry 1994, 33, 3304-3311. (18) Aramini, J. M.; Vogel, H. J. J. Am. Chem. Soc. 1994, 116, 19881993. (19) Aramini, J. M.; Vogel, H. J. J. Am. Chem. Soc. 1993, 115, 245252. (20) Luk, C. K. Biochemistry 1971, 10, 2838-2843. (21) Meares, C. F.; Ledbetter, J. E. Biochemistry 1977, 16, 5178-5190. (22) O’Hara, P.; Yeh, S. M.; Meares, C. F.; Bersohn, R. Biochemistry 1981, 20, 4704-4708. (23) Yeh, S. M.; Meares, C. F. Biochemistry 1980, 19, 5057-5062. (24) O’Hara, P. B.; Bersohn, R. Biochemistry 1982, 21, 5269-5272. (25) Martin, D. M.; Chasteen, N. D.; Grady, J. K. Biochim. Biophys. Acta 1991, 1076, 252-258. (26) O’Hara, P. B.; Gorshki, K. M.; Rosen, M. A. Biophys. J. 1988, 53, 1007-1013. (27) Gafni, A.; Steinberg, I. Z. Biochemistry 1974, 13, 800-803. (28) Chasteen, N. D. Coord. Chem. ReV. 1977, 22, 1-36. (29) Baldwin, D. A.; de Sousa, D. M. R. Biochem. Biophys. Res. Commun. 1981, 99, 1101-1107. (30) Bali, P. K.; Harris, W. R. Arch. Biochem. Biophys. 1990, 281, 252256. (31) Mikkelsen, K.; Nielsen, S. O. J. Phys. Chem. 1960, 64, 632-637. (32) Lyle, S. J.; Rahman, Md. M. Talanta 1963, 10, 1177-1182. (33) Horrocks, W. DeW., Jr.; Sudnick, D. R. Acc. Chem. Res. 1981, 14, 384. (34) O’Hara, P. B.; Koenig, S. H. Biochemistry 1986, 25, 1445-1450. (35) Harris, W. R.; Chen, Y. Inorg. Chem. 1992, 32, 5001-5006. (36) Zak, O.; Aisen, P. Biochemistry 1988, 27, 1075-1080. (37) Schippers, P. H. Ph.D. Dissertation, University of Leiden, The Netherlands, 1988; Chapter 1. (38) Aramini, J. M.; German, M. W.; Vogel, H. J. J. Am. Chem. Soc. 1993, 115, 9750-9753. (39) Sola, M. Inorg. Chem. 1990, 29, 1113-1116. (40) Bertini, I.; Luchinat, C.; Messori, L. J. Am. Chem. Soc. 1983, 105, 1347-1350. (41) The crystallaographic data on the isolated N-lobes were obtained from the Brookhaven Protein Data Bank as the following files: serum transferrin ) 1TFD, lactoferrin ) 1LCT, and ovotransferrin ) 1NNT.
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