Spectroscopic studies on the interaction of phosphate with uteroferrin

The effect of phosphate on the binuclear iron center of pink (reduced) uteroferrin was examined by magnetic resonance and optical spectroscopy. The purple (oxidized) protein, which contains 1 mol of tightly bound phosphate per mol of enzyme at isolation, does not give rise to a 31P NMR signal. Phosphate binding to phosphate-stripped pink uteroferrin is indistinguishable from that in the native purple phosphoprotein. As measured by EPR and optical spectroscopy, the rate of reaction between phosphate and pink uteroferrin is pH-dependent, decreasing as the pH increases. Phosphate is capable of binding to the reduced protein between pH 3 and 7.8, resulting in formation of the purple uteroferrin-phosphate complex. Evans susceptibility measurements at pH 4.9 indicate that the EPR silent species with a maximum absorption at 535 nm, generated upon phosphate addition to pink uteroferrin, is diamagnetic. Moreover, phosphate causes disappearance of the hyperfine-shifted resonances in the 1H NMR spectra of the reduced protein. We therefore have not been able to identify the paramagnetic "purple reduced enzyme-phosphate complex" reported by Pyrz et al. (Pyrz, J. W., Sage, J. T., Debrunner, P. G., and Que, Jr., L. (1986) J. Biol Chem. 261, 11015-11020) using Mossbauer spectroscopy and dithionite-reduced 57Fe-reconstituted uteroferrin. Our present data with native unmodified enzyme are in accord with our earlier results (Antanaitis, B. C., and Aisen, P. (1985) J. Biol. Chem. 260, 751-756) and with the results of Burman et al. (Burman, S., Davis, J. C., Weber, M. J., and Averill, B. A. (1986) Biochem. Biophys. Res. Commun. 136, 490-497) on bovine spleen phosphatase, suggesting that phosphate binding to reduced protein rapidly induces oxidation of the binuclear iron center.     

Effects of perturbants on the pink (reduced) active form of uteroferrin. Phosphate-induced anaerobic oxidation

The binuclear iron cluster of uteroferrin in its reduced and enzymatically active pink form is sensitive to a variety or perturbants. Orthophosphate, in the presence or absence of oxygen, rapidly shifts the absorption maximum of pink uteroferrin from 510 to 545 nm, concurrently abolishing the protein's g'av = 1.74 EPR signal. Apparently, therefore, dioxygen is not required for phosphate-induced oxidation of the pink protein's ferrous iron. Pyrophosphate and arsenate produce changes which differ only in degree from those induced by phosphate, suggesting that all of these structurally similar competitive inhibitors bind to a common site. Molybdate, an inhibitor even more potent than phosphate, quantitatively converts the rhombic EPR signal of pink uteroferrin into an axial signal that remains invariant to subsequent additions of phosphate. Thus, there can be inhibition without oxidation, as further evidenced by the complex EPR spectrum of undiminished intensity produced by sulfate. Fluoride, too, induces an axial component in the EPR signal of pink uteroferrin, but at high concentration abolishes the signal entirely. Vanadate also drives the protein to its oxidized, EPR-silent state, serving as an electron acceptor itself to yield the characteristic g' = 2 signal of the vanadyl (VO2+) cation. Remarkably, however, the protein remains pink, demonstrating a dissociation between color and oxidation state. Guanidinium, in contrast, causes a sizeable red shift in the pink protein's absorption maximum without loss of EPR signal intensity, showing dissociation of color and oxidation state in a complementary way.     

The binding of molybdate to uteroferrin. Hyperfine interactions of the binuclear center with 95Mo, 1H, and 2H

Uteroferrin, an acid phosphatase with a spin-coupled and redox-active binuclear iron center, is paramagnetic in its pink, enzymatically active, mixed-valence (S = 1/2) state. Phosphate, a product and inhibitor of the enzymatic activity of uteroferrin, converts the pink, EPR-active form of the protein to a purple, EPR-silent species. In contrast, molybdate, a tetrahedral oxyanion analog of phosphate, transforms the EPR spectrum of uteroferrin from a rhombic to an axial form. With both electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance (ENDOR) spectroscopies, we observe a hyperfine interaction of [95Mo]molybdate with the S = 1/2, Fe(II)-Fe(III) center of the protein. A pair of 95Mo resonances centered at the 95Mo Larmor frequency at the applied magnetic field and separated by a hyperfine coupling constant of 1.2 MHz is evident. Therefore, a single monomeric species of molybdate is close to, and likely a ligand of, the binuclear cluster. 1H ENDOR studies on uteroferrin reveal at least six sets of lines mirrored about the 1H Larmor frequency. Two pairs of these lines become reduced in intensity when the protein is exchanged against D2O. Moreover, ESEEM and 2H ENDOR spectra display resonances at the 2H Larmor frequency. Therefore, the metal-binding region of the protein is accessible to solvent. Additional deuterium lines observable by ESEEM spectroscopy provide evidence for a population of strongly coupled, readily exchangeable protons associated with the binuclear center. The measured hyperfine coupling constants for these deuterons are orientation-dependent with splittings of nearly 4 MHz at g3 = 1.59 and less than 1 MHz at g1 = 1.94. In the presence of molybdate, ESEEM spectra of D2O-exchanged samples reveal a resonance at the 2H Larmor frequency, with no evidence of spectral components due to strongly coupled deuterons. 1H ENDOR studies of the uteroferrin-molybdate complex show at least seven pairs of lines, mirrored about the 1H Larmor frequency, of which one pair becomes attenuated in amplitude upon deuteration. The active site thus remains accessible to solvent in the presence of molybdate.     

Purple acid phosphatase from bovine spleen. Interactions at the active site in relation to the reaction mechanism.

Oxidation of the reduced (pink) phosphate-free bovine spleen acid phosphatase with 1.5 mol H2O2 or sodium peroxodisulfate/mol, in the presence of Mes or Bistris pH 5, leads to a species with an absorption maximum at 558 nm. Addition of acetate or oxidation in the presence of acetate buffer engenders a species with a maximum at 550 nm. Addition of phosphate to both species shifts the maximum immediately to 540 nm; this is the species also found after preparation from the spleen. The assumption that these species represent strongly bidentate-binding hydroxo, acetato and phosphato complexes of the Fe(III)-Fe(III) system is supported by replacement reactions with other ligating oxoanions followed by their typical spectral shifts. These oxoanion complexes cannot be dissociated by gel filtration; this is possible only after reduction to the Fe(II)-Fe(III) system. The oxidized species without EPR signals below g values of 2 still reveals 5% activity which cannot be reduced to zero even in the presence of higher concentrations of peroxodisulfate. The pH optimum of the reaction with alpha-naphthyl phosphate shifts from 5.9 to 5.3 in the oxidized species. The apparent pK values around 4.5 as derived from the pH dependence of activity, of the EPR spectra, and the spectral shifts of the phosphate-saturated reduced and oxidized species are assigned to an aquo/hydroxo equilibrium at the Fe(III) or an equilibrium, where the phosphato ligand is replaced by a hydroxo ligand. A reaction mechanism is proposed in which a hydroxo ligand at the chromophoric Fe(III) attacks the phosphoric acid ester group only when that is monoprotonated and pre-oriented by electrostatic interaction with the nonchromophoric metal ion. Binding and inhibition studies with the oxoanions indicate that they compete with the catalytically active hydroxo group of the reduced and oxidized enzyme with nearly the same inhibition constants. Catalysis is not affected by the oxoanions which replace the additional mu-hydroxo ligand in the 558-nm-absorbing Fe(III)-Fe(III) species. In contrast to hemerythrin and ribonucleotide reductase, a binuclear iron center is proposed for the purple acid phosphatase, which is bridged by a carboxylato and two aquo/hydroxo groups, but without a mu-oxo bridge.     

1H NMR studies of a two-iron: two-sulfur ferredoxin from halobacterium of the dead sea

Ferredoxin isolated from Halobacterium of the Dead Sea (HFd) was found to be stable and retain its conformation in 4–0.5 M salt solutions. Reconstitution of the denatured protein to the oxidized form in ²H₂O indicated that the resonances shifted to the 8–10 ppm region, which include 18 protons, are nonexchangeable -NH protons. The C₂H and C₄H resonances of His-119 were assigned in both oxidized and reduced HFd. pH titration curves of these resonances yielded a pK_a for this His of 6.57 ± 0.1 and 6.65 ± 0.1 in oxidized and reduced HFd, respectively. pH titration curves, T₁ relaxation times, and the temperature dependence of the chemical shift were obtained for resonances between 6 and 10 ppm of oxidized HFd. In oxidized HFd a paramagnetically shifted resonance was observed at 15 ppm with 1 H intensity, and an anti-Curie temperature dependence. In reduced HFd eight resonances each with 1 H intensity were shifted downfield by 10–50 ppm and one resonance with 1 H intensity was shifted upfield to ?6.8 ppm. Four of these resonances exhibited an anti-Curie temperature dependence, two exhibited a moderate Curie dependence, and three were temperature independent.     

Hydrogen-1 nuclear magnetic resonance of the nitrogenase iron protein (Cp2) from Clostridium pasteurianum

Proton NMR spectra (250 MHz) of the nitrogenase iron protein from Clostridium pasteurianum (Cp2) were found to display 9 or 10 paramagnetically shifted resonances in the 15-50 ppm range. The most shifted resonances belonged to two approximately equal subsets having temperature dependences of opposite sign. The latter occurrence is consistent with the interaction of the corresponding protons with an antiferromagnetically coupled metal center. The number of proton resonances of Cp2, their positions, and their temperature dependences were similar to those observed in spectra of (4Fe-4S)+ ferredoxins, particularly those of the latter that contain a single tetranuclear cluster, such as the ferredoxin from Bacillus stearothermophilus. The effects of several adenine nucleotides on the paramagnetically shifted proton resonances of Cp2 have been investigated. Whereas MgAMP had no effect at all, MgADP and MgATP were found to induce different modifications, which in both cases involved approximately half only of the shifted proton resonances. These data suggest that nucleotide binding affects mainly one part of the iron-sulfur cluster. A remarkable feature of the spectra of Cp2 in the presence of MgATP is the grouping of the shifted proton resonances in sets of two or four having identical chemical shifts and temperature dependences. A nearly perfect 2-fold symmetry is thus suggested for the arrangement of the cysteine protons around the active site. These observations lend support to the proposal that the (4Fe-4S) cluster is held symmetrically between the two identical subunits and are consistent with the existence of two MgATP binding sites on nitrogenase iron proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H NMR and NOE studies of the purple acid phosphatases from porcine uterus and bovine spleen.

The diiron active sites of the purple acid phosphatases from porcine uterus (also called uteroferrin, Uf) and bovine spleen (BSPAP) and their complexes with tungstate are compared by 1H NMR and NOE techniques. The paramagnetically shifted features of the 1H NMR spectrum of reduced BSPAP are similar to those of reduced Uf, while the spectra of the tungstate complexes are almost identical. These observations suggest that the two active sites are quite similar, in agreement with the greater than 90% sequence homology found in the two enzymes. Nuclear Overhauser effect (NOE) experiments on the His N-H resonances show that the Fe(III)-His residue is N epsilon-coordinated, while the Fe(II)-His is H delta-coordinated in both enzymes. On the basis of the above NMR and NOE results, our previously proposed model for the dinuclear iron active site of Uf [Scarrow, R. C., Pyrz, J. W., & Que, L., Jr. (1990) J. Am. Chem. Soc. 112, 657-665] is corroborated, refined, and found to represent the diiron center of BSPAP as well.     

Reduced forms of the iron-containing small subunit of ribonucleotide reductase from Escherichia coli

The B2 subunit of ribonucleotide reductase from Escherichia coli contains a stable tyrosyl free radical and an antiferromagnetically coupled dimeric iron center with high-spin ferric ions. The tyrosyl radical is an oxidized form of tyrosine-122. This study shows that the B2 protein has a fully reduced state, denoted reduced B2, characterized by a normal nonradical tyrosine-122 residue and a dimeric ferrous iron center. Reduced B2 can be formed either from active B2 by a three-electron reduction in the presence of suitable mediators or from apoB2 by addition of two equimolar amounts of ferrous ions in the absence of oxygen. The oxidized tyrosyl radical and the ferric iron center can be generated from reduced B2 by the admission of air. The tyrosyl radical can be selectively reduced by one-electron reduction in the presence of a suitable mediator, yielding metB2, a form that seems identical with the form resulting from treatment of active B2 with hydroxyurea. 1H NMR was used to characterize the paramagnetically shifted resonances associated with the reduced iron center. Prominent resonances were observed around 45 ppm (nonexchangeable with solvent) and 57 ppm (exchangeable with solvent) at 37 degrees C. From the temperature dependence of the chemical shifts of these resonances it was concluded that the ferrous ions in reduced B2 are only weakly, if at all, antiferromagnetically coupled. By comparison with data on the similar iron center of deoxyhemerythrin it is suggested that the 57 ppm resonance should be assigned to protons in histidine ligands of the iron center.     

1H NMR spectra of vertebrate [2Fe-2S] ferredoxins. Hyperfine resonances suggest different electron delocalization patterns from plant ferredoxins

We report the observation of paramagnetically shifted (hyperfine) proton resonances from vertebrate mitochondrial [2Fe-2S] ferredoxins. The hyperfine signals of human, bovine, and chick [2Fe-2S] ferredoxins are described and compared with those of Anabaena 7120 vegetative ferredoxin, a plant-type [2Fe-2S] ferredoxin studied previously [Skjeldal, L., Westler, W. M., & Markley, J. L. (1990) Arch. Biochem. Biophys. 278, 482-485]. The hyperfine resonances of the three vertebrate ferredoxins were very similar to one another both in the oxidized state and in the reduced state, and slow (on the NMR scale) electron self-exchange was observed in partially reduced samples. For the oxidized vertebrate ferredoxins, hyperfine signals were observed downfield of the diamagnetic envelope from +13 to +50 ppm, and the general pattern of peaks and their anti-Curie temperature dependence are similar to those observed for the oxidized plant-type ferredoxins. For the reduced vertebrate ferredoxins, hyperfine signals were observed both upfield (-2 to -18 ppm) and downfield (+15 to +45 ppm), and all were found to exhibit Curie-type temperature dependence. This pattern and temperature dependence are distinctly different from those found with reduced plant-type ferredoxins which have signal centered around +120 ppm with Curie-type temperature dependence, assigned to cysteines which interact with Fe(III), and signals centered around +20 ppm with anti-Curie temperature dependence, assigned to cysteines which interact with Fe(II) [Dugad, L. B., La Mar, G. N., Banci, L., & Bertini, I. (1990) Biochemistry 29, 2263-2271].(ABSTRACT TRUNCATED AT 250 WORDS)     

Paramagnetically shifted resonances in 1H NMR spectra of ribonucleotide reductase from Escherichia coli

The 400-MHz 1H NMR spectra of the subunit B2 of ribonucleotide reductase from Escherichia coli show paramagnetically shifted resonances at 24 ppm (exchangeable protons) and at 19 ppm (nonexchangeable protons). The protein contains an antiferromagnetically coupled dimeric iron center and a tyrosyl free radical. The paramagnetically shifted resonances must be due to the iron center, since they remain essentially unchanged in protein B2 with and without free radical. In analogy with recently published results for hemerythrin from Phascolopsis gouldii, which has a similar iron center, the 24-ppm resonance is suggested to arise from histidine ligands to the iron ions.     

Magnetization of the oxidized and reduced three-iron cluster of Desulfovibrio gigas ferredoxin II

The saturation magnetizations of the three iron cluster of ferredoxin II of Desulfovibrio gigas in both the oxidized and reduced states have been studied at fixed magnetic fields up to 4.5 tesla over the temperature range from 1.8 to 200 K. The low field (0.3 tesla) susceptibility of oxidized ferredoxin II obeys the Curie law over this entire temperature range. This establishes -2Jox greater than 200 cm-1 as the lower limit for the antiferromagnetic exchange coupling of oxidized ferredoxin II. The saturation magnetizations of reduced ferredoxin II at several fixed fields yield a nested family of curves which can be fit with spin S = 2 and D = -2.7(4) cm-1 (with E/D assigned the value 0.23 as determined by M?ssbauer and EPR spectra). The low field susceptibility of reduced ferredoxin II also obeys the Curie law from approximately 4 up to 200 K. This establishes -2Jred greater than 40 cm-1 as the lower limit for the antiferromagnetic coupling of reduced ferredoxin II.     

Absence of iron transfer from uteroferrin to transferrin

Transfer of iron from native porcine uteroferrin to apotransferrin was investigated using EPR spectroscopy. Purple (oxidized) or pink (reduced) forms of uteroferrin were incubated with porcine or human apotransferrin under conditions of temperature (37 degrees C) and pH (6.8) approximating those found in the allantoic fluid of the pregnant sow. Studies were also performed in the presence of mediators such as ascorbate, citrate, and ATP in concentrations previously claimed to be effective in promoting large-scale transfer of iron (Buhi, W. C., Ducsay, C. A., Bazer, F. W., and Roberts, R. M. (1982) J. Biol. Chem. 257, 1712-1723). Our experiments indicate that even in the presence of mediators, less than 20% of the iron in uteroferrin is transferred to apotransferrin at the end of 24 h and such transfer may be accompanied by denaturation of uteroferrin. We therefore conclude that the direct transfer of iron to apotransferrin is unlikely to be a physiological role of uteroferrin.     

Magnetization and electron paramagnetic resonance studies of reduced uteroferrin and its "EPR-silent" phosphate complex

The exchange coupling of reduced uteroferrin has been measured (19.8(5) cm-1 S1.S2) using recently developed techniques for studying metalloprotein magnetization. A spin Hamiltonian describing the coupled binuclear Fe(II).Fe(III) center has been used to fit the low and high field magnetization data, the EPR g values, and the highly anisotropic effective hyperfine tensor of the ferric site. The exchange coupling of the phosphate complex of reduced uteroferrin has also been measured (6.0(5) cm-1 S1.S2) using the same techniques. The smaller exchange coupling of the phosphate complex is comparable with the zero field splittings of the iron sites. This results in increased sensitivity of the system g values (found by calculation from the spin Hamiltonian) to variations of the zero field splitting parameters arising from heterogeneities in the protein microenvironment. Consequently, there is a very significant (9-fold) increase in the "effective g strain" of the system compared to the situation in the absence of phosphate. This, together with the larger g anisotropy (g = (1.06, 1.51, 2.27)), gives rise to an EPR signal for the phosphate complex of reduced uteroferrin which is extremely broad and difficult to detect but which has now been identified for the first time.     

M?ssbauer, EPR, and magnetization studies of the Azotobacter vinelandii Fe protein. Evidence for a [4Fe-4S]1+ cluster with spin S = 3/2

We have studied the Fe protein (Av2) of the Azotobacter vinelandii nitrogenase system with M?ssbauer and EPR spectroscopies and magnetic susceptometry. In the oxidized state the protein exhibits M?ssbauer spectra typical of diamagnetic [4Fe-4S]2+ clusters. Addition of Mg.ATP or Mg.ADP causes a pronounced decline in the quadrupole splitting of the M?ssbauer spectra of the oxidized protein. Our studies show that reduced Av2 in the native state is heterogeneous. Approximately half of the molecules contain a [4Fe-4S]1+ cluster with electronic spin S = 1/2 and half contain a [4Fe-4S]1+ cluster with spin S = 3/2. The former yields the characteristic g = 1.94 EPR signal whereas the latter exhibits signals around g = 5. The magnetization of reduced Av2 is dominated by the spin S = 3/2 form of its [4Fe-4S]1+ clusters. These results explain a long standing puzzle, namely why the integrated spin intensity of the g = 1.94 EPR signal is substantially less than 1 spin/4 Fe atoms. In 50% ethylene glycol, 90% of the clusters are in the spin S = 1/2 form whereas, in 0.4 M urea, 85% are in the S = 3/2 form. In 0.4 M urea, the EPR spectrum of reduced Av2 exhibits well defined resonances at g = 5.8 and 5.15, which we assign to the S = 3/2 system. The EPR and M?ssbauer studies yield a zero-field splitting of 2D approximately equal to -5 cm-1 for this S = 3/2 state.     

19F NMR studies on 8-fluoroflavins and 8-fluoro flavoproteins

The 19F NMR spectra of the oxidized and reduced forms of 8-fluororiboflavin, 8-fluoro-FAD, and the 8-fluoroflavin-reconstituted flavoproteins flavodoxin, riboflavin binding protein, D-amino acid oxidase, p-hydroxybenzoate hydroxylase, Old Yellow Enzyme, anthranilate hydroxylase, general acyl-CoA dehydrogenase, glucose oxidase, and L-lactate oxidase were measured. For the proteins studied the oxidized resonances appeared over a 10.1-ppm range, while the reduced resonances were spread over 10.3 ppm. Reduction caused an upfield shift of about 27 ppm for the free 8-fluoroflavins and most of the 8-fluoro flavoproteins. The notable exception was 8-fluoro-FMN flavodoxin, which was shifted 37.6 ppm, indicating an unusually high electron density in the benzene ring. Ligand binding to the oxidized 8-fluoro flavoproteins caused either upfield or downfield shifts of 1.5-5 ppm, depending on the protein/ligand combination. The 8-fluoro-FAD anthranilate hydroxylase resonance was shifted downfield and split into two peaks in the presence of anthranilate. The 8-fluoro-FMN Old Yellow Enzyme resonance was shifted upfield upon complexation with charge-transfer-forming, para-substituted phenolates. The upfield shift increased from less than 1 to 5 ppm as the electron-donating capacity of the phenolate increased. Complexation of native Old Yellow Enzyme with 2,4-difluorophenol caused the fluorine resonances of the ligand to shift and split into two pairs of signals. Each pair of signals was associated with a different isozyme of Old Yellow Enzyme.     

31 P nuclear magnetic resonance studies of the interaction of pyridine nucleotide coenzymes with dehydrogenases.

31P nuclear magnetic resonance spectra of the pyrophosphate group in NAD+ and NADH were recorded in the presence of beef heart lactate dehydrogenase and rabbit muscle glyceraldehyde-3-phosphate dehydrogenase. At high lactate dehydrogenase concentrations (60 mg/ml), two NADH resonances are observed: a slowly exchanging peak which is shifted to 1.9 ppm downfield (relative to free NADH) and a rapidly exchanging peak with a downfield shift of 0.5-0.6 ppm. At lover concentrations (15 mg/ml) only the rapidly exchanging peak is observed thus indicating that the peak observed at-1.9 ppm is due to coenzyme bound to an aggregated enzyme species. With NAD+, rapid exchange and downfield shifts are observed at both enzyme and concentrations, with shifts of about 1.5 ppm and 0.6 ppm at 60 and 15 mg/ml, respectively. In the presence of glyceraldehydephosphate dehydrogenase, the results are independent of enzyme concentration, and slow exchange and upfield shifts of 0.4-0.6 ppm occur with each coenzyme. These data indicate that the environment of the pyrophosphate group of oxidized and reduced coenzyme is the same for a given dehydrogenase, but is different in one enzyme from the other. The resonances observed with glyceraldehydephosphate dehydrogenase are broader than those observed with lactate dehydrogenase. This is indicative of either shorter relaxation times with the former enzyme, or the presence of multiple, unresolved resonances.     

Detection and characterization of hyperfine-shifted resonances in the proton nuclear magnetic resonance spectrum of Anabaena 7120 ferredoxin at high magnetic fields

This paper presents previously unobserved signals in the 1H NMR spectra of oxidized and reduced [2Fe-2S]-ferredoxin from Anabaena 7120 detected at 400, 500, and 600 MHz. The signals shifted to low field exhibited longitudinal relaxation (T1) values in the range of 100-400 microseconds and line widths in the range of 1-10 kHz (at 400 MHz), and the chemical shifts of all signals showed strong temperature dependence. Although the line widths were smaller at lower magnetic fields, the resolution was better at higher magnetic fields. In the oxidized state, a broad signal was detected at 37 ppm, which corresponds to at least 6 protons, and whose chemical shift exhibits positive temperature dependence. This signal also was found in oxidized ferredoxin reconstituted in 2H2O, which excludes the signal as arising from solvent-exchangeable amide protons. In the reduced state, four signals detected between 90 and 140 ppm exhibited negative temperature dependence. These consisted of two pairs of signals, each pair having one component with half the linewidth of the other. On the basis of their chemical shifts, linewidths, longitudinal relaxation properties, and temperature dependence we assigned these resonances to four of the beta hydrogens of the ligated cysteines. Two solvent-exchangeable hyperfine-shifted signals were found in the reduced state; these are located upfield of the diamagnetic region. The low-field hyperfine resonances of half-reduced ferredoxin in the presence of sodium dithionite showed a self electron transfer exchange rate that was slow on the NMR scale as observed earlier (Chan, T., and Markley, J. L. (1983) Biochemistry 22, 5982-5987), but the exchange rate was accelerated in the presence of methyl viologen.     

Detection of a g' = 1.74 EPR signal in bovine spleen purple acid phosphatase

When purified with hydroxylapatite, bovine spleen purple acid phosphatase, bearing two iron atoms/molecule, is EPR-silent. In contrast, enzyme purified without hydroxylapatite exhibits the distinctive g' = 1.74 EPR signal characteristic of porcine uteroferrin, with an intensity accounting for about 10% of the total iron. The intensity of the signal is increased 8-fold by the addition of ferrous iron. This treatment, while shifting the visible absorption maximum of the protein from 550 to 525 nm, does not significantly alter the intensity of its visible absorption. Loss of the g' = 1.74 EPR signal upon addition of phosphate to EPR-active preparations and the detection of virtually stoichiometric amounts of phosphate in the protein as isolated suggest that phosphate-binding may abolish the g' = 1.75 EPR signal. Such binding may bring the two iron atoms of the enzyme into juxtaposition, causing loss of EPR signal intensity either through spin-lattice relaxation broadening or antiferromagnetic exchange coupling, perhaps involving phosphate or other ligands intercalated between the two paramagnetic iron atoms.     

Interaction of porcine uterine fluid purple acid phosphatase with vanadate and vanadyl cation.

Uteroferrin, the purple acid phosphatase from porcine uterine fluid, is noncompetitively inhibited by vanadate in a time-dependent manner under both aerobic and anaerobic conditions. This time-dependent inhibition is observed only with the diiron enzyme and is absent when the FeZn enzyme is used. The observations are attributed to the sequential formation of two uteroferrin-vanadium complexes. The first complex forms rapidly and reversibly, while the second complex forms slowly and results in the production of catalytically inactive oxidized uteroferrin and V(IV), which is observed by EPR. The redox reaction can be reversed by treatment of the oxidized enzyme first with (V(IV)) and then EDTA to generate a catalytically active uteroferrin. Multiple inhibition kinetics suggests that vanadate is mutually exclusive with molybdate, tungstate, and vanadyl cation. The binding site for each of these anions is distinct from the site to which the competitive inhibitors phosphate and arsenate bind. The time-dependent inhibition by vanadate of uteroferrin containing the diiron core represents a new type of mechanism by which vanadium can interact with proteins and gives additional insight into the binding of anions to uteroferrin.     

Sequential binding of cobalt(II) to metallo-beta-lactamase CcrA

In an effort to probe Co(II) binding to metallo-beta-lactamase CcrA, EPR, EXAFS, and (1)H NMR studies were conducted on CcrA containing 1 equiv (1-Co(II)-CcrA) and 2 equiv (Co(II)Co(II)-CcrA) of Co(II). The EPR spectra of 1-Co(II)-CcrA and Co(II)Co(II)-CcrA are distinct and indicate 5/6-coordinate Co(II) ions. The EPR spectra also reveal the absence of significant spin-exchange coupling between the Co(II) ions in Co(II)Co(II)-CcrA. EXAFS spectra of 1-Co(II)-CcrA suggest 5/6-coordinate Co(II) with two or more histidine ligands. EXAFS spectra of Co(II)Co(II)-CcrA also indicate 5/6 ligands at a similar average distance to 1-Co(II)-CcrA, including an average of about two histidines per Co(II). (1)H NMR spectra for 1-Co(II)-CcrA revealed seven paramagnetically shifted resonances, three of which were solvent-exchangeable, while the NMR spectra for Co(II)Co(II)-CcrA showed at least 16 shifted resonances, including an additional solvent-exchangeable resonance and a resonance at 208 ppm. The data indicate sequential binding of Co(II) to CcrA and that the first Co(II) binds to the consensus Zn(1) site in the enzyme.