A Purple Acid Phosphatase from Sweet Potato Contains an Antiferromagnetically Coupled Binuclear Fe-Mn Center

Abstract

A purple acid phosphatase from sweet potato is the first reported example of a protein containing an enzymatically active binuclear Fe-Mn center. Multifield saturation magnetization data over a temperature range of 2 to 200 K indicates that this center is strongly antiferromagnetically coupled. Metal ion analysis shows an excess of iron over manganese. Low temperature EPR spectra reveal only resonances characteristic of high spin Fe(III) centers (Fe(III)-apo and Fe(III)-Zn(II)) and adventitious Cu(II) centers. There were no resonances from either Mn(II) or binuclear Fe-Mn centers. Together with a comparison of spectral properties and sequence homologies between known purple acid phosphatases, the enzymatic and spectroscopic data strongly indicate the presence of catalytic Fe(III)-Mn(II) centers in the active site of the sweet potato enzyme. Because of the strong antiferromagnetism it is likely that the metal ions in the sweet potato enzyme are linked via a μ-oxo bridge, in contrast to other known purple acid phosphatases in which a μ-hydroxo bridge is present. Differences in metal ion composition and bridging may affect substrate specificities leading to the biological function of different purple acid phosphatases. A purple acid phosphatase from sweet potato is the first reported example of a protein containing an enzymatically active binuclear Fe-Mn center. Multifield saturation magnetization data over a temperature range of 2 to 200 K indicates that this center is strongly antiferromagnetically coupled. Metal ion analysis shows an excess of iron over manganese. Low temperature EPR spectra reveal only resonances characteristic of high spin Fe(III) centers (Fe(III)-apo and Fe(III)-Zn(II)) and adventitious Cu(II) centers. There were no resonances from either Mn(II) or binuclear Fe-Mn centers. Together with a comparison of spectral properties and sequence homologies between known purple acid phosphatases, the enzymatic and spectroscopic data strongly indicate the presence of catalytic Fe(III)-Mn(II) centers in the active site of the sweet potato enzyme. Because of the strong antiferromagnetism it is likely that the metal ions in the sweet potato enzyme are linked via a μ-oxo bridge, in contrast to other known purple acid phosphatases in which a μ-hydroxo bridge is present. Differences in metal ion composition and bridging may affect substrate specificities leading to the biological function of different purple acid phosphatases. Purple acid phosphatases comprise a family of binuclear metal-containing enzymes, the members of which have been identified in plants, animals, and fungi. The animal enzymes contain an antiferromagnetically coupled binuclear iron center. The active form of the enzyme (Fe(III)-Fe(II)) exhibits a distinctive EPR signal with principal g values of 1.93, 1.75, and 1.50 (1Doi K. McCracken J. Peisach J. Aisen P. J. Biol. Chem. 1988; 263: 5757-5763Abstract Full Text PDF PubMed Google Scholar), whereas the inactive (Fe(III)-Fe(III)) form is EPR-silent at X-band microwave frequencies (2Antanaitis B.C. Aisen P. Lilienthal H.R. J. Biol. Chem. 1983; 258: 3166-3172Abstract Full Text PDF PubMed Google Scholar, 3Averill B.A. Davis J.C. Burman S. Zirino T. Sanders-Loehr J. Loehr T.M. Sage T. Debrunner P.G. J. Am. Chem. Soc. 1987; 109: 3760-3767Crossref Scopus (187) Google Scholar). Plant purple acid phosphatases appear to be more diverse. Red kidney bean purple acid phosphatase, the best characterized plant enzyme, contains an Fe(III)-Zn(II) center (4Beck J.L. McConaghie L.A. Summors A.C. Arnold W.N. de Jersey J. Zerner B. Biochim. Biophys. Acta. 1986; 869: 61-68Crossref Scopus (139) Google Scholar). Replacement of Zn(II) by Fe(II) yields an enzyme with full activity and with spectral properties very similar to those of the animal enzymes (5Beck J.L. McArthur M.J. de Jersey J. Zerner B. Inorg. Chim. Acta. 1988; 153: 39-44Crossref Scopus (49) Google Scholar, 6Beck J.L. de Jersey J. Zerner B. Henrich M.P. Debrunner P. J. Am. Chem. Soc. 1988; 110: 3317-3318Crossref Scopus (67) Google Scholar). The sequences of three cDNAs encoding different isoforms of sweet potato purple acid phosphatase have recently been reported (7Durmus A. Eicken C. Sift B.H. Kratel A. Kappl R. Hüttermann J. Krebs B. Eur. J. Biochem. 1999; 260: 709-716Crossref PubMed Scopus (84) Google Scholar, 8Schenk G. Ge Y. Carrington L.E. Wynne C.J. Searle I.R. Carroll B.J. Hamilton S. de Jersey J. Arch. Biochem. Biophys. 1999; 370: 183-189Crossref PubMed Scopus (141) Google Scholar). Two of these isoforms have been purified. One form was shown by Durmuset al. (7Durmus A. Eicken C. Sift B.H. Kratel A. Kappl R. Hüttermann J. Krebs B. Eur. J. Biochem. 1999; 260: 709-716Crossref PubMed Scopus (84) Google Scholar) to contain one g-atom of iron and 0.77 g-atom of zinc/subunit. Comparison of its spectroscopic properties with those of the red kidney bean enzyme indicated that the metal ions are present as a binuclear Fe(III)-Zn(II) center (7Durmus A. Eicken C. Sift B.H. Kratel A. Kappl R. Hüttermann J. Krebs B. Eur. J. Biochem. 1999; 260: 709-716Crossref PubMed Scopus (84) Google Scholar). A different form exhibiting 66% sequence identity to the Fe(III)-Zn(II) enzyme has recently been characterized in our laboratory. It contains one g-atom of iron and 0.6–0.8 g-atom of manganese/subunit (8Schenk G. Ge Y. Carrington L.E. Wynne C.J. Searle I.R. Carroll B.J. Hamilton S. de Jersey J. Arch. Biochem. Biophys. 1999; 370: 183-189Crossref PubMed Scopus (141) Google Scholar). We now report enzymatic, magnetic susceptibility and EPR studies on this enzyme demonstrating that the enzyme contains a catalytically active Fe(III)-Mn(II) center that is strongly antiferromagnetically coupled, providing the first reported evidence for such a center in a protein. The enzyme was purified by a combination of juice extraction, acetone and ammonium sulfate fractionation, DEAE-cellulose chromatography at pH 7.0, and gel filtration on a Sephadex G-150 Superfine column at pH 4.90 as described elsewhere (8Schenk G. Ge Y. Carrington L.E. Wynne C.J. Searle I.R. Carroll B.J. Hamilton S. de Jersey J. Arch. Biochem. Biophys. 1999; 370: 183-189Crossref PubMed Scopus (141) Google Scholar). The enzyme was purple when concentrated and exhibited essentially the same visible absorption spectrum (λmax = 560 nm, εmax = 3207m−1 cm−1) as the red kidney bean enzyme (λmax = 560 nm, εmax = 3360 m−1cm−1) (4Beck J.L. McConaghie L.A. Summors A.C. Arnold W.N. de Jersey J. Zerner B. Biochim. Biophys. Acta. 1986; 869: 61-68Crossref Scopus (139) Google Scholar). Enzyme assays were performed at 25 °C using p-nitrophenyl phosphate (5 mm) as substrate in 0.1 m acetate buffer, pH 4.90. The specific activity of the sample used for magnetic susceptibility and EPR measurements was 675 units/mg. The protein subunit concentration was 0.556 mm (based on a subunit weight of 55 kDa and anA 1%1 cm at 280 nm of 27.03 (8Schenk G. Ge Y. Carrington L.E. Wynne C.J. Searle I.R. Carroll B.J. Hamilton S. de Jersey J. Arch. Biochem. Biophys. 1999; 370: 183-189Crossref PubMed Scopus (141) Google Scholar)). Metal ion content was determined by inductively coupled plasma mass spectrometry using a PerkinElmer SCIEX-ELAN 5000 spectrometer. Samples and standards were prepared in 0.1% HNO3. Separate standard curves were routinely prepared for iron, zinc, copper, and manganese. Samples were measured in quadruplicate. Metal ion analysis showed that the sample of enzyme used for magnetic susceptibility and EPR measurements contained 1.035 ± 0.108 iron, 0.582 ± 0.044 manganese, 0.183 ± 0.019 zinc, and 0.11 ± 0.028 copper/subunit of 55 kDa. Pig purple acid phosphatase was prepared as described previously (9Campbell H.D. Dionysius D.A. Keough D.T. Wilson B.E. de Jersey J. Zerner B. Biochem. Biophys. Res. Commun. 1978; 82: 615-620Crossref PubMed Scopus (89) Google Scholar). Protein concentrations for the oxidation and reduction experiments were 300 μm for the pig enzyme and 300 μm (subunit concentration) for the sweet potato enzyme. Oxidation was carried out by incubating the enzymes in 0.1 m acetate buffer, pH 4.90, containing 30 mm H2O2 at 4 °C for 45 min followed by removal of the peroxide by gel filtration. Reduction of oxidized pig purple acid phosphatase and untreated sweet potato enzyme was carried out by incubation in pH 4.90 buffer containing 140 mm β-mercaptoethanol at 25 °C for 150 min followed by gel filtration. The specific activities of the samples were determined using the assay described above. Sweet potato purple acid phosphatase (40 μm) in 0.1 m acetate buffer (pH 4.9) was mixed with pyridine-2,6-dicarboxylate (2 mm) at 20 °C (5Beck J.L. McArthur M.J. de Jersey J. Zerner B. Inorg. Chim. Acta. 1988; 153: 39-44Crossref Scopus (49) Google Scholar). An aliquot of freshly prepared sodium dithionite solution (10 mm) was added. After a short incubation (10–60 s) the mixture was separated on a Sephadex G-25 column, which was equilibrated with 0.1 m acetate buffer (pH 4.9). Multifield saturation magnetization (10Day E.P. David S.S. Peterson J. Dunham W.R. Bonvoisin J.J. Sands R.H. Que L.J. J. Biol. Chem. 1988; 263: 15561-15567Abstract Full Text PDF PubMed Google Scholar, 11Day E.P. Methods Enzymol. 1993; 227: 437-463Crossref PubMed Scopus (28) Google Scholar) data were collected with a Quantum Design MPMS SQUID magnetometer and corrected for the magnetization of the deuterated acetate buffer (pD 4.9, 0.1 m), the effects of mismatches in residual ferromagnetic impurities in the quartz sample holder (volume = 0.146 ml), background diamagnetism of the sample, and temperature-independent paramagnetism (11Day E.P. Methods Enzymol. 1993; 227: 437-463Crossref PubMed Scopus (28) Google Scholar). Fitting of the data employed the program WMAG (11Day E.P. Methods Enzymol. 1993; 227: 437-463Crossref PubMed Scopus (28) Google Scholar) and an S = 5/2 spin Hamiltonian. To reduce the number of variables, the g and E/D values were held at 2.0 and 0.1925, respectively, as deduced from the EPR spectrum. Reasonably good fits, as judged by the “goodness of fit”; parameter in the program, were obtained for ∣D∣ values between 0.8 and 1.2 cm−1. 1Slightly better fits were obtained using these g, D, and E values and an S = 2 Hamiltonian (e.g. Fe(II) or Mn(III)), but this does not agree with the EPR data. X-band (9–10 GHz, TE102rectangular cavity) EPR spectra were measured on Bruker EPR spectrometers (ESP300E and Elexsys E500). Low temperatures (1.5–2.5 K) were obtained with a flow-through Oxford instruments ESR910 cryostat in conjunction with an Oxford instruments ITC-4 variable temperature controller. Spectrometer tuning, signal averaging, and subsequent data manipulation were performed with either Bruker esp300e (v3.02) software for the ESP300E spectrometer or Xepr® (v2.0) for the Elexsys spectrometer. Instrument settings were as follows: microwave power, 20 mW; modulation frequency, 100 kHz, and the modulation amplitude was always less than one-tenth of the linewidth at half-height. The microwave frequency and magnetic field were calibrated using an EIP 548B microwave frequency counter and a Bruker ER035m gaussmeter, respectively. Computer simulation of the EPR spectra was performed using version 1.0 of XSophe© J. Scopus Google Scholar, Scopus Google Scholar, A. C.J. C. K. 1999; Scholar) on a was employed J. Scopus Google Scholar) for the of the which were used to the field and The EPR spectral was with the of the and spectra were performed with Xepr® on the of sweet potato purple acid phosphatase, the one used in the present contained essentially of iron, 0.6–0.8 of manganese, and of and of mass 55 kDa (8Schenk G. Ge Y. Carrington L.E. Wynne C.J. Searle I.R. Carroll B.J. Hamilton S. de Jersey J. Arch. Biochem. Biophys. 1999; 370: 183-189Crossref PubMed Scopus (141) Google Scholar). purple acid phosphatases from red kidney and one from sweet the active form of the enzyme contains an Fe(III)-Zn(II) the content of the metal ion is from to 0.8 (4Beck J.L. McConaghie L.A. Summors A.C. Arnold W.N. de Jersey J. Zerner B. Biochim. Biophys. Acta. 1986; 869: 61-68Crossref Scopus (139) Google Scholar, A. Eicken C. Sift B.H. Kratel A. Kappl R. Hüttermann J. Krebs B. Eur. J. Biochem. 1999; 260: 709-716Crossref PubMed Scopus (84) Google Scholar, 8Schenk G. Ge Y. Carrington L.E. Wynne C.J. Searle I.R. Carroll B.J. Hamilton S. de Jersey J. Arch. Biochem. Biophys. 1999; 370: 183-189Crossref PubMed Scopus (141) Google Scholar). three enzymes the iron content is of the metal the red kidney bean and purple acid phosphatases, it be shown that the specific activity with the content of the (4Beck J.L. McConaghie L.A. Summors A.C. Arnold W.N. de Jersey J. Zerner B. Biochim. Biophys. Acta. 1986; 869: 61-68Crossref Scopus (139) Google Scholar, 8Schenk G. Ge Y. Carrington L.E. Wynne C.J. Searle I.R. Carroll B.J. Hamilton S. de Jersey J. Arch. Biochem. Biophys. 1999; 370: 183-189Crossref PubMed Scopus (141) Google Scholar). of manganese, which are present in of the enzyme, are by mm a in specific A similar with of the sweet potato enzyme characterized in our does not manganese, that the Fe-Mn binuclear in sweet potato purple acid phosphatase is for the catalytic To this the and were with the specific of sweet potato purple acid phosphatase at 4 °C in a of with a in specific when enzyme containing 1.0 g-atom of iron and g-atom of and a specific activity of was for one at 4 the content to and the specific activity to units/mg. The content incubation with Mn(II) the content to and the specific activity to the content not We the of the metal ion and red kidney bean purple acid phosphatases, a has been for the of enzyme (5Beck J.L. McArthur M.J. de Jersey J. Zerner B. Inorg. Chim. Acta. 1988; 153: 39-44Crossref Scopus (49) Google Scholar, J.L. Keough D.T. de Jersey J. Zerner B. Biochim. Biophys. Acta. PubMed Scopus Google Scholar). the same does not the metal ions from sweet potato purple acid phosphatase, short (10–60 s) with the sodium dithionite the of purple acid phosphatase samples with content but iron content ± the of present in the samples was the The specific activity of these was to be to the this is in a of content specific activity for and enzyme the specific activity with the in content in the range between and g-atom manganese/subunit = The specific activity for the enzyme with a full of iron and is to a number for p-nitrophenyl phosphate of of the enzyme in the presence of was to enzymatic activity to a to its of the enzyme or to the enzyme. the that zinc, present as binuclear centers in sweet potato enzyme may be for the catalytic The content of ions from one to and is likely by the of the which in content from to iron content and g-atom of no in specific activity (8Schenk G. Ge Y. Carrington L.E. Wynne C.J. Searle I.R. Carroll B.J. Hamilton S. de Jersey J. Arch. Biochem. Biophys. 1999; 370: 183-189Crossref PubMed Scopus (141) Google Scholar). reduction of purified enzyme g-atom of iron, g-atom of manganese, g-atom of zinc, and a specific activity of 675 and subsequent an in specific activity which with the content but not with the The iron content was to be the of binuclear centers present in the of sweet potato purple acid phosphatase are not for the catalytic to centers are present in sweet potato purple acid phosphatase, the enzyme to reduction with β-mercaptoethanol that of the activity of the oxidized pig enzyme. the specific activity of the sweet potato enzyme by only of this enzyme with peroxide that oxidized and the pig enzyme J.L. Keough D.T. de Jersey J. Zerner B. Biochim. Biophys. Acta. PubMed Scopus Google Scholar) no on the specific activity of the sweet potato enzyme. the EPR spectrum of the sweet potato purple acid phosphatase only very resonances to a binuclear center were that at best only a of sweet potato purple acid phosphatase contains and of the metal centers. the indicate that the catalytically active form of sweet potato purple acid phosphatase contains binuclear Fe-Mn in sweet potato purple acid phosphatase is to be likely in the Fe(III) of the of the visible absorption spectra the high of sequence of the sweet potato and red kidney bean (4Beck J.L. McConaghie L.A. Summors A.C. Arnold W.N. de Jersey J. Zerner B. Biochim. Biophys. Acta. 1986; 869: 61-68Crossref Scopus (139) Google Scholar) enzymes, which that the at 560 nm in the sweet potato enzyme from a Fe(III) The properties of this Fe-Mn center were using magnetic susceptibility and EPR Multifield saturation magnetization of the used by and (10Day E.P. David S.S. Peterson J. Dunham W.R. Bonvoisin J.J. Sands R.H. Que L.J. J. Biol. Chem. 1988; 263: 15561-15567Abstract Full Text PDF PubMed Google Scholar, 11Day E.P. Methods Enzymol. 1993; 227: 437-463Crossref PubMed Scopus (28) Google Scholar) on the pig purple acid phosphatase was employed to the magnetic susceptibility of the enzyme at magnetic and over a temperature range of 2 to 200 K The magnetization values over this temperature range are very and characteristic of an spin The of the 2 an S = 5/2 spin Hamiltonian good fits and a concentration of ± mm S = 5/2 centers. concentration to only of the concentration of Fe(III) and Mn(II) centers present in the excess of iron over manganese, present as Fe(III)-Zn(II) centers be to comprise the S = 5/2 excess ± mm) than ± We from on characterized S = 5/2 Fe(III) samples B. J. Inorg. Biochem. Google Scholar) that the between S = 5/2 concentration from metal ion and magnetization fits is at Fitting that a combination of S = and S = centers fits but with g and the concentrations of Fe(III) were less than that of the iron were present in centers. a antiferromagnetically coupled center purple acid with = = the susceptibility = be to a at K and this is not the susceptibility from a protein in which the metal ions at the binuclear active site are strongly antiferromagnetically coupled 140 an S = A temperature EPR spectrum of sweet potato purple acid phosphatase of resonances from high spin Fe(III) g = and Cu(II) The of that be to either or a binuclear metal center is with the susceptibility a strongly antiferromagnetically coupled center. Computer simulation of the resonances from high spin Fe(III) were performed using a spin Hamiltonian = g and a of and E/D The of E/D was characterized with using in more with more be A good was obtained with one at and a of and at with a of The are shown in and The presence of these the of with E/D = and E/D = only a very with E/D = is in the simulation to for the g = the spectrum be by the of the to E/D = it is more likely that is a present with a of of the concentration of high spin Fe(III) centers to the EPR spectrum shown in is and to experiments on high spin Fe(III) centers an of the determined from the spectrum Methods Enzymol. 1993; 227: Scopus Google as this affect the of the resonances in the spectrum. the be determined from the simulation of the spectrum as the parameter to cm−1) is than the microwave the of and E to the be and E are The best to the spin concentration is to the EPR spectra from the red kidney bean and sweet potato enzymes, which that the of sweet potato purple acid phosphatase containing high spin Fe(III) centers and is very less than is in with the from magnetic susceptibility A comparison of the spin Hamiltonian for the sweet potato enzyme with those for red kidney bean purple acid phosphatase E/D = that the Fe(III) site is more in the sweet potato enzyme and from either the or centers. the enzymatic and spectroscopic data indicate the presence of a strongly antiferromagnetically coupled binuclear Fe(III)-Mn(II) center in sweet potato purple acid phosphatase, which is for the biological containing or bridging in Fe(III)-Mn(II) antiferromagnetism with 20 C.J. A. J. S. J. Chem. Soc. Scholar, C. M.P. Que L.J. J. Am. Chem. Soc. Scopus Google R. S. K. Inorg. Chem. PubMed Scopus Google Scholar). B. K. S. and data on a of the μ-oxo are likely to be present than a μ-hydroxo bridge, which is in the Fe(III)-Zn(II) and the centers of the and red kidney bean purple acid phosphatase S. P. J. R. Krebs B. Inorg. Chim. Acta. Google Scholar). are known in Fe(III) and in but to in Mn(II) The acid which are known to to the metal ions in the binuclear centers in pig and red kidney bean purple acid phosphatases, are in the sweet potato enzyme (8Schenk G. Ge Y. Carrington L.E. Wynne C.J. Searle I.R. Carroll B.J. Hamilton S. de Jersey J. Arch. Biochem. Biophys. 1999; 370: 183-189Crossref PubMed Scopus (141) Google Scholar). We have recently reported the of and data on the enzyme G. Carrington L.E. Hamilton de Jersey J. Biol. 1999; PubMed Scopus Google Scholar). a of its it is that these acid to the metal and the Fe(III)-Mn(II) at is than in the μ-hydroxo enzymes from pig Hamilton S. de Jersey J. J.L. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) and red kidney bean T. R. Krebs B. J. Biol. PubMed Scopus Google Scholar). is with the presence of a μ-oxo bridge than a μ-hydroxo bridge in sweet potato purple acid of with Fe(III)-Mn(II) centers and of the of the sweet potato enzyme with and are in in this to the in the active site that may to the of the The presence of a μ-oxo bridge in the sweet potato enzyme described may be of for studies on binuclear characterized in metal ion are and a to be present in which the is by an Chem. PubMed Scopus Google Scholar). The identity of the The of red kidney bean purple acid phosphatase that a is a likely T. R. Krebs B. J. Biol. PubMed Scopus Google Scholar). metal studies on purple acid phosphatase an in which a of the metal ion as a to a in the which the phosphate of the substrate B.A. J. Am. Chem. Soc. 1999; Scopus Google Scholar). of in pig and purple acid phosphatases Que J. Am. Chem. Soc. 1999; Scopus Google Scholar, B.A. 1999; PubMed Scopus Google Scholar) has to the that in the the phosphate of the substrate and a ion bridge the metal ions in the active It is that the of this bridging is for an on the phosphate the is the metal ion substrate may this for a on the of the substrate Que J. Am. Chem. Soc. 1999; Scopus Google Scholar). the bridge in sweet potato purple acid phosphatase is to be a it be out at present that a from the bridging the in this enzyme. The in metal ion content and bridging in purple acid phosphatases purified from red kidney and sweet or in different may affect the reduction and substrate leading to different biological in The of three cDNAs encoding different isoforms of purple acid phosphatase in sweet potato may the for of the in metal content and EPR spectra by different in on the enzyme. The enzyme by and S. B. Biochem. Biophys. Res. Commun. 1987; PubMed Scopus Google Scholar) was reported to contain of (λmax = nm, εmax = The enzyme showed no evidence for a = signal characteristic of the antiferromagnetically coupled center of the purple acid phosphatases, but the a signal in the = The S. B. Biochem. Biophys. Res. Commun. 1987; PubMed Scopus Google Scholar) that the enzyme may contain iron centers is now in of the sequence between the three sweet potato isoforms and the red kidney bean purple acid phosphatase (7Durmus A. Eicken C. Sift B.H. Kratel A. Kappl R. Hüttermann J. Krebs B. Eur. J. Biochem. 1999; 260: 709-716Crossref PubMed Scopus (84) Google Scholar, 8Schenk G. Ge Y. Carrington L.E. Wynne C.J. Searle I.R. Carroll B.J. Hamilton S. de Jersey J. Arch. Biochem. Biophys. 1999; 370: 183-189Crossref PubMed Scopus (141) Google Scholar). al. (7Durmus A. Eicken C. Sift B.H. Kratel A. Kappl R. Hüttermann J. Krebs B. Eur. J. Biochem. 1999; 260: 709-716Crossref PubMed Scopus (84) Google Scholar), a similar to that of and S. B. Biochem. Biophys. Res. Commun. 1987; PubMed Scopus Google Scholar), showed that enzyme contains an Fe(III)-Zn(II) center with spectral properties similar to the red kidney bean enzyme. the of the resonances to high spin Fe(III) in enzyme (pH are very similar to those in the present a and A. Eicken C. Sift B.H. Kratel A. Kappl R. Hüttermann J. Krebs B. Eur. J. Biochem. 1999; 260: 709-716Crossref PubMed Scopus (84) Google as from the sequence that the have similar iron The sweet potato purple acid phosphatase by al. Y. S. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) may be different from the Fe-Mn and the enzyme. Metal ion analysis of this enzyme indicated the presence of one and iron The enzyme exhibited a of nm and εmax = on a mass of 55 values that are different from those of the and Fe-Mn enzyme was but in strong acid 100 resonances g of were The that the enzyme contained a to a It is that the Mn(II) by EPR as a of of to Mn(II) and the of the metal center of the purple acid phosphatase purified by the Y. Biochim. Biophys. Acta. PubMed Scopus Google Scholar, Y. S. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) it is that it may an by the which and al. have and but not the metal center is a binuclear the reported metal ion appear to be strongly antiferromagnetically coupled Fe(III)-Mn(II) or of which be EPR-silent and a Mn(II) signal with