Linear electric field effect and electron spin-echo studies of uteroferrin. Evidence for iron coordination by a nitrogen-containing ligand.

Uteroferrin and semimethemerythrin, proteins possessing spin-coupled binuclear iron centers, exhibit large linear electric field effects in their mixed-valence, EPR-active states. This indicates that the paramagnetic center of each protein is noncentrosymmetric and suggests that charge may be localized on one of the iron atoms. The magnetic field dependence of the linear electric field effects for both proteins demonstrates that the direction of most facile polarization of the binuclear iron centers is near the orientation giving rise to gmin. Electron spin-echo studies of uteroferrin reveal that its magnetic electron interacts with at least one and possibly two classes of nitrogen nuclei. Furthermore, comparison of echo envelope spectra for uteroferrin with that of ferric bleomycin suggests that one of these nuclei is from a histidine ligand.

Uteroferrin and semimethemerythrin, proteins possessing spin-coupled binuclear iron centers, exhibit large linear electric field effects in their mixed-valence, EPR-active states. This indicates that the paramagnetic center of each protein is noncentrosymmetric and suggests that charge may be localized on one of the iron atoms. The magnetic field dependence of the linear electric field effects for both proteins demonstrates that the direction of most facile polarization of the binuclear iron centers is near the orientation giving rise to g , , , . Electron spin-echo studies of uteroferrin reveal that its magnetic electron interacts with at least one and possibly two classes of nitrogen nuclei. Furthermore, comparison of echo envelope spectra for uteroferrin with that of ferric bleomycin suggests that one of these nuclei is from a histidine ligand.
Uteroferrin is a two-iron, basic glycoprotein with acid phosphatase activity and a molecular weight close to 35,000 (1). It is isolated from the uterine fluids of pregnant or pseudopregnant, hormone-treated sows (2). The protein can exist in either of two interconvertible redox states: purple (oxidized) which is enzymatically inactive and EPR-silent, or pink (reduced) which is enzymatically active and exhibits an intense, rhombic, g' = 1.74 EPR signal, the expression of which is controlled by the binding of phosphate and other tetrahedral anionic inhibitors (3). Quantitative EPR, magnetic susceptibility (4), and 'H-NMR studies (5) indicate that uteroferrin's pair of iron atoms are arranged in a spin-coupled, binuclear cluster, a property the protein shares with bovine spleen purple acid phosphatase (l), ribonucleotide reductase (6), twoiron ferredoxins (7) and hemerythrin, the respiratory protein of certain marine invertebrates (8). Mossbauer studies of 57Fesubstituted uteroferrin definitively established the presence of the exchange-coupled binuclear cluster and further suggest that the reducing electron of the pink form is mainly localized on one of the iron atoms (9).
The NMR detection of solvent-exchangeable resonances with appropriate chemical shifts further suggests that histidine may be coordinated to each iron atom of the reduced cluster ( 5 ) .
To elucidate further the electronic structure of the spincoupled binuclear cluster in uteroferrin and help identify nitrogen-containing ligands, we have carried out LEFE' and electron spin-echo studies on the paramagnetic pink form.
Complementary LEFE studies were also done on semimethemerythrin, a protein with a spin-coupled binuclear iron center with an EPR spectrum remarkably similar to that of uteroferrin (12) and with a structure elucidated by x-ray crystallographic analysis (13).

MATERIALS AND METHODS
Preparation of Uteroferrin-Uteroferrin was isolated from the uterine flushings of pseudopregnant, hormone-treated sows according to published procedures (14).

All samples used in these studies had an optical purity index ( A z~/ A h m e x )
= 15.0. Iron analysis by wet digestion (1.8 iron atoms/molecule of protein) was carried out as previously described (4). Protein concentrations were estimated from the previously reported extinction coefficient at 280 nm: 50 mM-' cm" (4). Purple uteroferrin, which typically bears one tightly hound phosphate/molecule (4), was reductively stripped of its phosphate by treatment with 0.2-0.3 M 8-mercaptoethanol, followed by passage through a Sephadex G-15 column equilibrated with 0.1 M acetate buffer, pH 4.9. The phosphate content of the protein, measured according to procedures in Ref. 4, was less than 0.1 phosphate/ molecule of protein.
Preparation of Semimethemetythrin-Oxyhemerythrin, a gift from R. Wilkins, was prepared by described methods (15) from live Phascolopsis gouldii worms purchased from Marine Biological Laboratories, Woods Hole, MA. Methemerythrin was prepared by dialyzing oxyhemerythrin against ferricyanide and then several times against the appropriate buffer system. Methemerythrin was reduced to the semimet form by the anaerobic addition of 1 electron eq of dithionite (16).

LEFE and Electron Spin-Echo Studies-LEFE measurements at
X-band were made on pink uteroferrin and semimethemerythrin at the magnetic field settings given in Fig. 1, using apparatus and methods described elsewhere (17,18). Experiments were carried out with the electric field, E, parallel to Ho, the applied magnetic field (EIHo), and with the electric field perpendicular to the applied magnetic field (ELH0). Shift parameters, u, were calculated by the half-fall method described earlier (19).
Three pulse electron spin-echo decay envelopes (20) for pink uteroferrin were recorded at 3810 and 3903 G , respectively, at temperatures of 1.8 and 4.2 K. Pulse I to pulse I1 intervals, T , of 182, 240, and 362 ns, which are approximately integral multiples of the free proton precession period, were chosen to suppress the modulation of the echo decay envelope arising from weakly coupled protons (17). In order to eliminate glitches in the envelope caused by the passage of unwanted two-pulse echoes through the boxcar gate, the microwave phase of the first two transmitter pulses was reversed in alternate spin-echo cycles as suggested by Dr. M. K. Bowman (Argonne National Laboratories).' The echo envelope was Fourier transformed using the envelope reconstruction method (21) in order to yield the required frequency spectrum.
The abbreviation used is: LEFE, linear electric field effect.
Pulsed EPR Studies of Uteroferrin 4513

RESULTS AND DISCUSSION
As shown in Fig. 1, the antiferromagnetically spin-coupled two-iron centers of pink uteroferrin and semimethemerythrin exhibit a large LEFE; the EiHo and ElH0 curves are similar to one another, but are distinctly different from the LEFE curves of reduced two-iron ferredoxins, proteins that also possess spin-coupled Fe(I1)-Fe(II1) clusters (7). The observation of a LEFE demonstrates unequivocally that the S = % paramagnetic centers of both proteins are noncentrosymmetric (19). This conclusion is in accord with recent 57Fe-Mossbauer studies indicating that the reducing electron for the binuclear iron centers of both proteins resides primarily on just one of the iron atoms (9). The structural inequivalence of the two iron-binding sites in hemerythrin, revealed by xray crystallography, is also in accord with this conclusion (13). A large deviation from centrosymmetry for uteroferrin is consistent with data indicating that tyrosyl residues coordinated to the paramagnetic center are asymmetrically distributed between its two iron atoms, with one iron apparently binding two and the other none ( 5 ) .
Although a complete analysis of the LEFE requires single crystal data, certain inferences can be made from our powder studies. The largest effects, which probably indicate the direction of maximum polarization for both binuclear centers, is obtained when the electric field is aligned along the gmi, principal axis, i.e. the maximal electric field-induced shifts occur when the Zeeman field is essentially parallel to the gmin axis and E is parallel to Ho (E,,H,). It may be argued then, as for the two-iron ferredoxins (7), that the LEFE arises primarily from the electric field-induced displacement of the largely localized reducing electron away from one iron atom and toward an electron accepting center, possibly the other iron atom.
Previous 'H-NMR studies of pink uteroferrin detect three sets of solvent-exchangeable resonances with sizable isotropic shifts ( 5 ) . Model studies indicate that these resonances arise from labile protons attached to metal-coordinated nitrogen ligands (5). Such ligands may be candidates for detection by electron spin-echo spectroscopy through their modulation of the spin-echo decay envelope. Fig. 2 depicts the three-pulse echo decay envelope and the corresponding spectrum obtained by Fourier transformation for pink uteroferrin. For T = 182 ns the echo envelope spectrum is complex, containing six peaks at 1.1, 2.0, 2.5, 3.8, 5.3, and 7.4 MHz. Spectra derived from data with T = 240 and 362 ns have the same frequencies as the spectrum obtained with T = 182 ns, save for an additional narrow line at 2.9 MHz. From the similarity of the spectra obtained at these differing T values (and hence differing dead times) it may be inferred that the peaks in the frequency spectrum are genuine and are not artifacts of the  I  I  I  I  I  I  I  I  I   I  I  I  I  I  I  I  I  I  I  envelope reconstruction and Fourier transformation procedure.
Comparison of peak positions derived from data at two magnetic field settings, namely, 3810 and 3903 G, shows that frequencies of 3.0 MHz or below shift less than 0.1 MHz with change in applied field, while those with higher frequencies shift slightly more than 0.1 MHz. Lines attributable to protons would be expected to show a considerable magnetic field dependence because the Zeeman energy of protons is characteristically greater than the hyperfine energy. For example, the peak due to a strongly coupled proton would move approximately 0.42 MHz for a 100 G change in field. For 31P, a similar shift in frequency with magnetic field would be somewhat smaller ( i e . 0.17 MHz for 100 G change in magnetic field), but the protein was rendered free of phosphate prior to the spin-echo experiments. In contrast, lines arising from 14N should be relatively weakly dependent on field. It is likely, therefore, that all seven peaks in the echo envelope spectrum arise from coupling between 14N of metal-coordinated ligands and the unpaired electron spin of uteroferrin. However, without suitable model compounds a detailed interpretation of electron spin-echo data is not possible.
There can be no more than six distinct transitions within the superhyperfine level system associated with a single 14N nucleus (22), and of these transitions no more than four have been observed to give rise to resolved lines in frozen solution samples. It seems likely, therefore, that the seven lines observed in the frequency spectrum of uteroferrin are due to interaction with more than one I4N-containing ligand. This conclusion is in accord with 'H-NMR data which also suggest coordination by more than one nitrogen-containing ligand ( 5 ) . Furthermore, the pair of narrow peaks in the echo envelope spectrum at 2.0 and 2.5 MHz closely resemble the highfrequency components of a three-line pattern found for ferric bleomycin (23). In bleomycin, this pattern has been tentatively assigned to the zero-field quadrupolar frequencies of nitrogen of a ferric-coodinated imidazole, and we feel that a similar assignment is reasonably applied to uteroferrin.
The source of the remaining four peaks in the spectrum invites speculation. Consideration of the origin of the solventexchangeable peak a t -25 ppm in the 'H-NMR spectrum provides a clue ( 5 ) . An isotropically shifted solvent-exchangeable resonance of this magnitude implicates a nitrogen-containing ligand. Furthermore, its upfield position probably excludes histidine with its unusual downfield shift. At pH 4.9, iron coordination by arginine via an unprotonated guanidinium group, with its usual pK greater than 11, also seems unlikely. The intriguing possibilities of iron coordination to an amine or an amide through its carbonyl oxygen remain.