Comparison of the Electron Spin Echo Envelope Modulation (ESEEM) for Human Lactoferrin and Transferrin Complexes of Copper(II) and Vanadyl Ion

Copper(I1) and vanadyl ions were bound to human milk lactoferrin or serum transferrin with carbonate or oxalate as the synergistic anion. Electron spin echo envelope modulation (ESEEM) due to nitrogen of a coordinated histidine imidazole was observed for both the copper and vanadyl complexes. For both metals, the modulation frequencies in the Fourier transforms of the data were similar for the two proteins and were weakly dependent on anion. When data in DzO/glycerol-d3 were compared with data in HzO/glycerol, the deep  deuterium  modulation  indicated  multiple exchangeable protons in the vicinity of tbe metals with at most one proton within about 2.9 A of the metal. The distribution of exchangeable protons around the metals as probed by ESEEM was the same, within experimental uncertainty, for the copper or vanadyl complexes with either carbonate or oxalate as the anion. When “C-labeled oxalate was used as the synergistic anion, ”C-ESEEM was observed for both the copper and vanadyl complexes of lactoferrin and transferrin. The deeper “C modulation for copper and vanadyl transferrin [13C]oxalate than for vanadyl transferrin [13C]carbonate suggests that both ends of the oxalate are bound to the metal in the transferrin and lactoferrin complexes.

wide range of metals has been shown to bind to the siderophilins in vitro. Spectroscopic techniques that permit evaluation of the metal binding sites of lactoferrin and transferrin may help to elucidate the biological activity of these proteins.
The electron spin echo studies of lactoferrin and transferrin described in this paper were designed to compare three aspects of the metal binding sites in lactoferrin and transferrin: 1) coordination by nitrogen from a histidine imidazole, 2) interaction of the metal ion with exchangeable protons, and 3) coordination of oxalate as the synergistic anion. Previous ESEEM' studies of copper transferrin had demonstrated coordination of a histidine imidazole and binding of the synergistic oxalate ion to the metal (11, 12). Similar studies had not been reported for lactoferrin. No ESEEM results had been reported for the vanadyl complexes of either transferrin or lactoferrin.

RESULTS AND DISCUSSION
Nitrogen Modulation in Copper Complexes-The two-pulse ESEEM for CuLfCO3 is shown in Fig. la. The data are similar to those reported for CuTfCOs and CuOTfC03 by Zweier et al. (11,12). The Fourier transform of the data and a simulation are shown in Fig. 1, b and c, respectively. Corresponding three-pulse data are shown in Fig. 1, d-f. The modulation frequencies and values obtained from the simulations are summarized in Table 2 [3][4][5][6] are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

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This is an Open Access article under the CC BY license.   lation of the ESEEM data for VOLE03 are shown in Fig. 2, b and c. The peak at about 14 MHz is due to proton modulation, and the lower frequency peaks are due to nitrogen modulation. The modulation frequencies are similar for the four vanadyl protein complexes ( Table 2), but these frequencies are quite different from those observed for the copper complexes.
Since there are few data in the literature concerning ESEEM due to nitrogen bound to vanadyl ion, data were obtained for two nitrogenous-base complexes of vanadyl bis(hexafluoroacetylacetonate), VO(hfac)z. The two-pulse ESEEM for VO(hfac)z.L, L = pyridine and imidazole, are shown in Fig. 3. The nitrogen modulation in these echoes is shallow, analogous to the modulation in Fig. 2u. Fourier transforms and simulations are shown in Figs. 4 and 5. The nitrogen modulation frequencies obtained for VO(hfac),py in the perpendicular region of the EPR spectrum ( Table 2) are similar to those reported for vanadyl bis(acety1acetonate) pyridine, VO(acac)2py, (4.45 and 8.1 MHz) in the perpendic-ular region of the EPR spectrum (40). The value of ulN reported for VO(acac),py on the basis of an approximate analysis was 5.6 MHz (40).
The analysis of the ESEEM for the carbonate and oxalate complexes of vanadyl transferrin and lactoferrin indicated nitrogen hyperfine coupling constants of 6.6 and 7.0 MHz, respectively. These values are similar to the hyperfine coupling constants obtained by ESEEM in this study for VO(hfac)zL, L = pyridine, imidazole, and obtained by EN-DOR for nitrogen bound equatorially to vanadyl ion (41-43). The nitrogen hyperfine coupling constants for the vanadyl transferrin and lactoferrin complexes are consistent with coordination of nitrogen, presumably a histidine imidazole, in the equatorial plane of the vanadyl ion.
Vanadyl-nitrogen coupling constants are generally too small to resolve in CW EPR spectra. The absence of resolved nitrogen hyperfine coupling in the CW EPR spectra of VOTfanion and VOLf-anion complexes (17, 26-28) could not be used to determine if there is a nitrogen bound to the vanadyl ion. Thus, the ESEEM studies provide information that is not accessible in the CW EPR spectra of these complexes.
Interaction with Exchangeable Protons-Several studies

ESEEM of Copper(II) and Vanadyl Lactoferrin and Transferrin
have demonstrated the utility of comparing ESEEM data for proteins in DzO and HzO solutions (44-46). The ratio of twopulse data in D20 to data in HzO separates the deuteron/ proton modulation from other modulation frequencies. The deuteron modulation is deeper than the proton modulation, so the ratio is dominated by the deuteron modulation. The ESEEM for CuTfC03 in 1:l D20:glycerol-d3 divided by the ESEEM in 1:l HzO:glycerol is shown in Fig. 6a. The analogous ratio for CuLfCO3 is shown in Fig. 6b. Similar data were obtained for CuTfox and CuLfox. The ratios of the ESEEM for VOTfC03 and VOLfCO3 in 1:l D20:glycerol-c13 and 1:l H20:glycerol are shown in Fig. 7, a and b. The deep deuterium modulation is similar for the four copper complexes and the two vanadyl complexes and indicates similar interaction with exchangeable protons. Although there is not a unique set of distances that is consistent with the data, several conclusions can be reached. 1) There is at most one exchangeable proton within about 2.9 8, of the metal that contributes to the deuterium modulation. terons would be about 1.5 to 2.7 MHz. If the isotropic interaction is a substantial portion of the coupling observed in the ENDOR experiment, the coupling to these deuterons would be too large for detection by ESEEM. D20 molecules coordinated to copper that have been detected by ESEEM have had isotropic coupling constants of 0.1 to 0.2 MHz (46). Thus, the ENDOR and ESEEM data for the copper transferrin complexes seem to provide complementary information: the EN-DOR-detected protons are in the first coordination sphere of the copper and the ESEEM-detectedprotons are in the second coordination sphere. Since the unpaired electron for vanadyl ion is an orbital that does not participate in u bonding to coordinated ligands, isotropic couplings are smaller for vanadyl than for copper(I1) (50). An OD group, from water or a protein side chain, bound directly to vanadyl would be expected to have an impact on the ESEEM. The absence of such an interaction in the vanadyl ESEEM indicates that the OD group which the ENDOR experiment indicated was bound to copper(I1) is not present in the vanadyl complex. This coordination site may already be occupied by the vanadyl oxygen.
Coordination of Oxalate-The observation of 13C modulation in the ESEEM data for 13C-labeled oxalate bound to CuTf and CuOTf (11, 12) indicated that the oxalate was bound to the copper. Since the 13C modulation is a small perturbation on the deep nitrogen modulation, the modulation was observed by taking the ratio of the ESEEM for the ['TI-and ['2C]oxalate complexes (11, 12). Similar 13C modulation was observed in this study for the '%-labeled oxalate complexes of copper lactoferrin and transferrin. The data for CuTf are in good agreement with the literature (11). The modulation frequency is shifted away from the free carbon frequency for the complexes of all three proteins due to the isotropic coupling, a = 1.0 MHz. Nonzero values of a arise when there is delocalization of the metal unpaired electron into orbitals of the atom that gives rise to the modulation, which occurs when there is a bonding pathway between the metal and the nucleus.
The 13C/12C ratio data for VOTfox and VOLfox are shown in Fig. 8, a, c and b, d, respectively. In the vanadyl complexes, the 13C modulation is at the free carbon frequency which indicates that a = 0.0 MHz. Simulated spectra were obtained for one 13C at 2.5 to 2.7 8, from the vanadyl (Fig. 8, a and b) and for two 13C at 2.8 to 2.9 A from the vanadyl (Fig. 8, c and   d). The simulations with one 13C at a shorter distance agreed slightly better with the data at short values of 7 than the simulations with two 13C at a longer distance. Due to uncertainties concerning the assumptions inherent in the simulations, these small differences in the agreement between the calculated and observed data may not permit a distinction between one and two 13C nuclei interacting with the vanadyl. However, much weaker 13C modulation was observed for VOTf( [13C]carbonate) (Fig. 8 e ) than for VOTf( [13C]oxalate) (Fig. 8a). Since   peak at 170 ppm was obtained in the 13C NMR spectrum of ZnTf(["C]oxalate) (51). It was proposed that the oxalate was again bound to the divalent metal through one end, but that the interaction of the other end with the protein resulted in a chemical shift that was similar to that for the end that was coordinated to the metal (51). Our results suggest another interpretation for these results. Both ends of the oxalate could be coordinated to the metal, but one end of the oxalate also interacts with the positively charged protein residue. The additional interaction with the protein could make the two carbons nonequivalent. A chemical shift difference between two coordinated ends of the oxalate could also arise from the different trans ligands. These effects may be different for the trivalent and divalent metals, resulting in a larger shift difference for the Ga(II1) and Al(II1) complexes than for the Zn(I1) complex.