Spectroscopic and kinetics studies of a high-salt-stabilized form of the purple acid phosphatase from bovine spleen.

Use of a revised purification procedure that maintains the enzyme in a high-salt environment has resulted in the isolation of a new form of the bovine spleen purple acid phosphatase. This enzyme cannot be distinguished from that previously described [Davis, J. C., Lin, S. S., & Averill, B. A. (1981) Biochemistry 20, 4062] by electrophoresis, isoelectric focusing, Western blot analysis, or N-terminal amino acid sequence and exhibits identical catalytic properties and EPR spectra in the reduced (pink) form. It does, however, possess a much more highly ordered structure as shown by CD spectra and exhibits markedly different reactivity upon oxidation and different visible spectra upon binding of inhibitory anions or changing pH. The properties of the new high-salt-stabilized form of the enzyme have permitted an extensive examination of the visible absorption spectra of complexes of the oxidized and reduced enzyme with inhibitory anions. It is found that these anions may be grouped into three classes on the basis of their effect on the visible absorption maximum and their sensitivity to pH: phosphate, arsenate, and AMP; tungstate and molybdate; and fluoride. This grouping is reinforced by a detailed examination of the steady-state kinetics of the enzyme in the presence of these inhibitors, which reveals that the first class exhibits mixed-type inhibition due to the presence of competitive and noncompetitive binding sites, while the second class exhibits simple non-competitive inhibition. Fluoride exhibits complex inhibition behavior characterized by curved Lineweaver-Burk plots; this behavior cannot be attributed to the presence of inhibitory aluminum fluoride complexes. Taken together, the spectral and kinetics data are consistent with a picture in which tetrahedral oxyanions bind in a noncompetitive fashion by bridging the two iron atoms in the dinuclear center, with the smaller anions also being able to bind in a competitive manner at a single iron atom.     

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.     

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.     

Determination of the exchange integral in binuclear iron-sulfur clusters in proteins of varying complexity.

The coupling constants J between the iron atoms in ferredoxin type iron-sulfur proteins containing binuclear clusters were evaluated by two parallel methods. The temperature dependence of the EPR linewidths and integrated abosrption intensities are both related to the energy of the first excited state. The values of J obtained were: center S-1 in succinate dehydrogenase, 90 cm-1; Rieske's iron-sulfur center, 65 cm-1; adrenodoxin, 270 cm-1. The behavior of iron-sulfur center N-1a in NADH:UQ reductase was also examined; its similarity to that of center S-1 indicates that center N-1a is also a binuclear iron-sulfur center, with J = 90 cm-1. Greater rhombic distortion present in the EPR spectrum of a binuclear cluster was associated with smaller values of J.     

The interaction of phosphate with uteroferrin. Characterization of a reduced uteroferrin-phosphate complex

The interaction of phosphate with reduced uteroferrin has been re-examined in light of disagreements on the oxidation state of the binuclear iron cluster (Keough, D. T., Beck, J. L., de Jersey, J., and Zerner, B. (1982) Biochem. Biophys. Res. Commun. 108, 1643-1648; Antanaitis, B. C., and Aisen, P. (1985) J. Biol. Chem. 260, 751-756). Our results based on Mossbauer observations and the kinetics of spectral change and activity loss show clearly that phosphate binds to reduced uteroferrin to form a reduced uteroferrin-phosphate complex. This complex exhibits a pair of quadrupole doublets at 119 K with parameters typical of a high spin ferric and a high spin ferrous center, respectively, but distinct from those of the native reduced enzyme. The reduced phosphate complex exhibits a pH-dependent visible absorption maximum ranging from 530 to 561 nm. In air, the reduced phosphate complex converts to the oxidized phosphate complex with a first order rate constant of 4 X 10(-3) min-1, as monitored by spectral changes and loss of enzyme activity.     

Angular dependences of perpendicular and parallel mode electron paramagnetic resonance of oxidized beef heart cytochrome c oxidase

Cytochrome c oxidase catalyzes the reduction of oxygen to water with a concomitant conservation of energy in the form of a transmembrane proton gradient. The enzyme has a catalytic site consisting of a binuclear center of a copper ion and a heme group. The spectroscopic parameters of this center are unusual. The origin of broad electron paramagnetic resonance (EPR) signals in the oxidized state at rather low resonant field, the so-called g' = 12 signal, has been a matter of debate for over 30 years. We have studied the angular dependence of this resonance in both parallel and perpendicular mode X-band EPR in oriented multilayers containing cytochrome c oxidase to resolve the assignment. The "slow" form and compounds formed by the addition of formate and fluoride to the oxidized enzyme display these resonances, which result from transitions between states of an integer-spin multiplet arising from magnetic exchange coupling between the five unpaired electrons of high spin Fe(III) heme a(3) and the single unpaired electron of Cu(B). The first successful simulation of similar signals observed in both perpendicular and parallel mode X-band EPR spectra in frozen aqueous solution of the fluoride compound of the closely related enzyme, quinol oxidase or cytochrome bo(3), has been reported recently (Oganesyan et al., 1998, J. Am. Chem. Soc. 120:4232-4233). This suggested that the exchange interaction between the two metal ions of the binuclear center is very weak (|J| approximately 1 cm(-1)), with the axial zero-field splitting (D approximately 5 cm(-1)) of the high-spin heme dominating the form of the ground state. We show that this model accounts well for the angular dependences of the X-band EPR spectra in both perpendicular and parallel modes of oriented multilayers of cytochrome c oxidase derivatives and that the experimental results are inconsistent with earlier schemes that use exchange coupling parameters of several hundred wavenumbers.     

Direct observation of multiple protonation states in recombinant human purple acid phosphatase

To date, most spectroscopic studies on mammalian purple acid phosphatases (PAPs) have been performed at a single pH, typically pH 5. The catalytic activity of these enzymes is, however, pH dependent, with optimal pH values of 5.5–6.2 (depending on the form). For example, the pH optimum of PAPs isolated as single polypeptides is around pH 5.5, which is substantially lower that of proteolytically cleaved PAPs (ca. pH 6.2). In addition, the catalytic activity of single polypeptide PAPs at their optimal pH values is four to fivefold lower than that of the proteolytically cleaved enzymes. In order to elucidate the chemical basis for the pH dependence of these enzymes, the spectroscopic properties of both the single polypeptide and proteolytically cleaved forms of recombinant human PAP (recHPAP) and their complexes with inhibitory anions have been examined over the pH range 4 to 8. The EPR spectra of both forms of recHPAP are pH dependent and show the presence of three species: an inactive low pH form (pH<pK _a,1), an active form (pK _a,1<pH<pK _a,2), and an inactive high pH form (pH>pK _a,2). The pK _a,1 values observed by EPR for the single polypeptide and proteolytically cleaved forms are similar to those previously observed in kinetics studies. The spectroscopic properties of the enzyme–phosphate complex (which should mimic the enzyme–substrate complex), the enzyme–fluoride complex, and the enzyme–fluoride–phosphate complex (which should mimic the ternary enzyme–substrate–hydroxide complex) were also examined. EPR spectra show that phosphate binds to the diiron center of the proteolytically cleaved form of the enzyme, but not to that of the single polypeptide form. EPR spectra also show that fluoride binds only to the low pH form of the enzymes, in which it presumably replaces a coordinated water molecule. The binding of fluoride and phosphate to form a ternary complex appears to be cooperative.Electronic Supplementary Material Supplementary material is available for this article at     

Evidence for a mu-oxo-bridged binuclear iron cluster in the hydroxylase component of methane monooxygenase. M?ssbauer and EPR studies

M?ssbauer and EPR studies of a highly active hydroxylase component of methane monooxygenase isolated from Methylosinus trichosporium OB3b are reported. The M?ssbauer spectra of the oxidized (as isolated) hydroxylase show iron in a diamagnetic cluster containing an even number of Fe3+ sites. The parameters are consistent with an antiferromagnetically coupled binuclear cluster similar to those of hemerythrin and purple acid phosphatases. Upon partial reduction of the hydroxylase, an S = 1/2 EPR spectrum with g values at 1.94, 1.86, and 1.75 (gav = 1.85) is observed. Such spectra are characteristic of oxo-bridged iron dimers in the mixed valent Fe(II).Fe(III) state. Further reduction leads to the appearance of a novel EPR resonance at g = 15. Comparison with an inorganic model compound for mu-oxo-bridged binuclear iron suggests that the g = 15 signal is characteristic of the doubly reduced state of the cluster in the protein. In this state, the M?ssbauer spectra exhibit two quadrupole doublets typical of high spin Fe2+, consistent with the Fe(II).Fe(II) form of the cluster. The spectral features of the iron center of the hydroxylase in three oxidation states are all similar to those reported for mu-oxo (or mu-hydroxo)-bridged binuclear iron clusters. Since no known monooxygenase contains such a cluster, a new oxygenase mechanism is suggested. Three different preparative methods yielded hydroxylases spanning a 9-fold range in specific activity, yet the same cluster concentration and spectral characteristics were observed. Thus, other parameters than those measured here have a major influence on the activity.     

Fluoride inhibition of bovine spleen purple acid phosphatase: characterization of a ternary enzyme-phosphate-fluoride complex as a model for the active enzyme-substrate-hydroxide complex.

Purple acid phosphatases (PAPs) employ a dinuclear Fe(3+)Fe(2+) or Fe(3+)Zn(2+) center to catalyze the hydrolysis of phosphate monoesters. The interaction of fluoride with bovine spleen purple acid phosphatase (BSPAP) has been studied using a combination of steady-state kinetics and spectroscopic methods. For FeZn-BSPAP, the nature of the inhibition changes from noncompetitive at pH 6.5 (K(i(comp)) approximately K(i(uncomp)) approximately 2 mM) to uncompetitive at pH 5.0 (K(i(uncomp)) = 0.2 mM). The inhibition constant for AlZn-BSPAP at pH 5.0 (K(i) = 3 microM) is approximately 50-70-fold lower than that observed for both FeZn-BSAP and GaZn-BSPAP, suggesting that fluoride binds to the trivalent metal. Fluoride binding to the enzyme-substrate complex was found to be remarkably slow; hence, the kinetics of fluoride binding were studied in some detail for FeZn-, AlZn-, and FeFe-BSPAP at pH 5.0 and for FeZn-BSPAP at pH 6.5. Since the enzyme kinetics studies indicated the formation of a ternary enzyme-substrate-fluoride complex, the binding of fluoride to FeZn-BSPAP was studied using optical and EPR spectroscopies, both in the presence and absence of phosphate. The characteristic optical and EPR spectra of FeZn-BSPAP. F and FeZn-BSPAP.PO(4).F are similar at pH 5.0 and pH 6.5, indicating the formation of similar fluoride complexes at both pHs. A structural model for the ternary enzyme-(substrate/phosphate)-fluoride complexes is proposed that can explain the results from both the spectroscopic and the enzyme kinetics experiments. In this model, fluoride binds to the trivalent metal replacing the water/hydroxide ligand that is essential for the hydrolysis reaction to take place, while phosphate or the phosphate ester coordinates to the divalent metal ion.     

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.     

Physical and chemical studies on bacterial superoxide dismutases. Purification and some anion binding properties of the iron-containing protein of Escherichia coli B.

Highly purified iron superoxide dismutase was obtained from Escherichia coli B using a modification of the procedure of Yost and Jridovich (Yost, F. J., Jr., and Fridovich, I. (1973) J. Biol. Chem. 248, 4905-4908). The protein contained 1.8 +/- 0.2 atoms of iron per 38,700 g of protein. We have found that cyanide does not bind to the Fe3+ ion of iron dismutase but fluoride and azide have moderately large binding constants. Optical and electron paramagnetic resonance (EPR) measurements suggested that 2 fluoride ions could associate with each iron atom with the first having an association constant of approximately 520 M-1 and the second with an estimated value of 24 M-1. Activity measurements yielded an inhibition constant for fluoride of 30 M-1. At room temperature only one azide binds to the Fe3+ (K = 760 M-1) and this does not interfere with superoxide dismutase activity. Upon freezing solutions of iron superoxide dismutase in the presence of excess azide their color changes from yellow to pink. Combined EPR and optical titrations with azide suggest the presence of two binding sites on Fe3+ with only the first being occupied at room temperature and the second binding azide only upon freezing the solution. The results suggest that each Fe3+ ion of this superoxide dismutase has two coordination positions available for interaction with solute molecules but only one is necessary for catalysis of the superoxide dismutation reaction. The EPR, optical, and circular dichroism spectra of the native protein and the various fluoride and azide complexes are presented.     

Activation of the binuclear metal center through formation of phosphotriesterase-inhibitor complexes

Phosphotriesterase (PTE) from Pseudomonas diminuta is a binuclear metalloenzyme that catalyzes the hydrolysis of organophosphate nerve agents at rates approaching the diffusion-controlled limit. The proposed catalytic mechanism postulates the interaction of the substrate with the metal center and subsequent nucleophilic attack by the bridging hydroxide. X-band EPR spectroscopy was utilized to monitor the active site of Mn/Mn-substituted PTE upon addition of two inhibitors, diisopropyl methyl phosphonate and triethyl phosphate, and the product of hydrolysis, diethyl phosphate. The effects of inhibitor and product binding on the magnetic properties of the metal center and the hydroxyl bridge were evaluated by measuring changes in the features of the EPR spectra. The EPR spectra support the proposal that the binding of substrate analogues to the binuclear metal center diminishes the population of hydroxide-bridged species. These results, in conjunction with previously published kinetic and crystallographic data, suggest that substrate binding via the phosphoryl oxygen at the beta-metal weakens the coordination of the hydroxide bridge to the beta-metal. The weakened coordination to the beta-metal ion increases the nucleophilic character of the hydroxide and is coupled to the increase in the electrophilic character of the substrate.     

Binding of isotopically labeled substrates, inhibitors, and cyanide by protocatechuate 3,4-dioxygenase

Binding of ligands to the active site Fe3+ of protocatechuate 3,4-dioxygenase is investigated using EPR-detected transferred hyperfine coupling from isotopically labeled substrates, inhibitors, and cyanide. Broadening is observed in EPR resonances from the anaerobic enzyme complex with homoprotocatechuate (3,4-dihydroxyphenylacetate), a slow substrate, enriched with 17O (I = 5/2) in either the 3-OH or the 4-OH group. This shows that this substrate binds directly to the Fe3+ and strongly suggests that an iron chelate can be formed. Cyanide is known to bind to the enzyme in at least two steps, forming first a high spin and then a low spin complex (Whittaker, J. W., and Lipscomb, J. D. (1984) J. Biol. Chem. 259, 4487-4495). Hyperfine broadening from [13C]cyanide (I = 1/2) is observed in the EPR spectra of both complexes, showing that cyanide is an Fe3+ ligand in each case. Cyanide binding is also at least biphasic in the presence of protocatechuate (PCA). The initial high spin enzyme-PCA-cyanide complex forms rapidly and exhibits a unique EPR spectrum. Broadening from PCA enriched with 17O in either the 3-OH or the 4-OH group is detected showing that PCA binds to the iron, probably as a chelate complex. In contrast, no broadening from [13C]cyanide is detected for this complex suggesting that cyanide binds at a site away from the Fe3+. Steady state kinetic measurements of cyanide inhibition of PCA turnover are consistent with two rapidly exchanging cyanide binding sites that inhibit PCA binding and which can be simultaneously occupied. Formation of the nearly irreversible, low spin enzyme-PCA-cyanide complex is competitively inhibited by PCA. Transient kinetics of the formation of this complex are second order in cyanide implying that two cyanides bind. Broadening in the EPR spectrum of this complex is detected from [13C]cyanide, but not from [17O]PCA, suggesting that PCA is displaced. This study provides the first direct evidence for chelation of the active site Fe3+ by substrates and for a small molecule binding site away from the iron in intradiol dioxygenases.     

Protonation of the binuclear metal center within the active site of phosphotriesterase

Phosphotriesterase (PTE) is a binuclear metalloenzyme that catalyzes the hydrolysis of organophosphates, including pesticides and chemical warfare agents, at rates approaching the diffusion controlled limit. The catalytic mechanism of this enzyme features a bridging solvent molecule that is proposed to initiate nucleophilic attack at the phosphorus center of the substrate. X-band EPR spectroscopy is utilized to investigate the active site of Mn/Mn-substituted PTE. Simulation of the dominant EPR spectrum from the coupled binuclear center of Mn/Mn-PTE requires slightly rhombic zero-field splitting parameters. Assuming that the signal arises from the S = 2 manifold, an exchange coupling constant of J = -2.7 +/- 0.2 cm(-)(1) (H(ex) = -2JS(1) x S(2)) is calculated. A kinetic pK(a) of 7.1 +/- 0.1 associated with loss in activity at low pH indicates that a protonation event is responsible for inhibition of catalysis. Analysis of changes in the EPR spectrum as a function of pH provides a pK(a) of 7.3 +/- 0.1 that is assigned as the protonation of the hydroxyl bridge. From the comparison of kinetic and spectral pK(a) values, it is concluded that the loss of catalytic activity at acidic pH results from the protonation of the hydroxide that bridges the binuclear metal center.     

EPR and M?ssbauer studies of protocatechuate 4,5-dioxygenase. Characterization of a new Fe2+ environment

Protocatechuate 4,5-dioxygenase from Pseudomonas testosteroni has been purified to homogeneity and crystallized. The iron containing, extradiol dioxygenase is shown to be composed of two subunit types (alpha, Mr = 17,700 and beta, Mr = 33,800) in a 1:1 ratio; such a composition has not been observed for other extradiol dioxygenases. The 4.2 K M?ssbauer spectrum of native protocatechuate 4,5-dioxygenase prepared from cells grown in 57Fe-enriched media consists of a doublet with quadrupole splitting, delta EQ = 2.22 mm/s, and isomer shift delta Fe = 1.28 mm/s, demonstrating a high spin Fe2+ site. These parameters, and the temperature dependence of delta EQ, are unique among enzymes but are strikingly similar to those reported for the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, suggesting very similar ligand environments. The Fe2+ of protocatechuate 4,5-dioxygenase can be oxidized, for instance by H2O2, to yield high spin Fe3+ with EPR g values around g = 6 (and g = 4.3). In the oxidized state, protocatechuate 4,5-dioxygenase is inactive; the iron, however, can be rereduced by ascorbate to yield active enzyme. Our data suggest that protocatechuate binds to Fe2+; the spectra indicate that the ligand binding is heterogenous. The M?ssbauer spectra observed here are fundamentally different from those reported earlier (Zabinski, R., Münck, E., Champion, P., and Wood, J. M. (1972) Biochemistry 11, 3212-3219). The spectra of the earlier (reconstituted) preparations, which had substantially lower specific activities, probably reflect adventitiously bound Fe3+. We discuss here how adventitiously bound iron can be identified and removed. The Fe2+ which is present in native protocatechuate 4,5-dioxygenase and its complexes with substrates and inhibitors reacts quantitatively with nitric oxide to produce a species with electronic spin S = 3/2. The EPR and M?ssbauer spectra of these complexes compare favorably with EDTA . Fe(II) . NO. We have studied the latter complex extensively and have analyzed the M?ssbauer spectra with an S = 3/2 spin Hamiltonian. EPR spectra show that protocatechuate 4,5-dioxygenase-NO complexes with substrates or inhibitors are heterogeneous and consist of several well defined subspecies. The data show that NO, and presumably also O2, has access to the active site Fe2+ in the enzyme-substrate complex. The use of EPR-detectable NO complexes as a rapid and sensitive tool for the study of the EPR silent active site iron of extradiol dioxygenases is discussed.     

Characterization of the cycle of iron-mediated electron transfer from Adriamycin to molecular oxygen

The anticancer drug adriamycin binds iron and these complexes cycle to reduce molecular oxygen (Zweier, J. L. (1984) J. Biol. Chem. 259, 6056-6058). Optical absorption, EPR, and M?ssbauer spectroscopic data are correlated with polarographic O2 consumption and chemical Fe2+ extraction measurements in order to characterize each step in this cycle. Fe3+ binds to adriamycin at physiologic pH forming a complex with an optical absorbance maximum at 600 nm. EPR signals at g = 4.2 and g = 2.01, and a doublet M?ssbauer spectrum with isomer shift delta = 0.57 mm/s and quadrupole splitting delta EQ = 0.74 mm/s are observed indicating that the Fe3+ bound to adriamycin is high spin S = 5/2. Under anaerobic conditions the absorbance maximum at 600 nm decreases with an exponential decay constant = 0.77 h-1, and the EPR and M?ssbauer spectra of Fe3+-adriamycin similarly decrease as the Fe3+ is reduced to EPR silent Fe2+. The Fe2+-adriamycin complex which is formed exhibits a M?ssbauer spectrum with delta = 1.18 mm/s and delta EQ = 1.82 mm/s indicative of high spin Fe2+. As the EPR spectra of Fe3+-adriamycin decrease on reduction of the Fe3+ to Fe2+ a signal of the oxidized adriamycin free radical appears at g = 2.004 with line width of 8 G. On exposure to O2 the absorption maximum at 600 nm, the Fe3+ EPR, and the Fe3+ M?ssbauer spectra all return. Polarographic measurements demonstrate that O2 is consumed and that H2O2 is formed. Addition of high affinity Fe2+ chelators block O2 consumption indicating that Fe2+ formation is essential for O2 reduction. This cycle of iron-mediated O2 reduction can explain the formation of the reactive reduced oxygen and adriamycin radicals which are thought to mediate the biological activity of adriamycin.     

The EPR spectrum for CuB in cytochrome c oxidase

Incubation of cytochrome c oxidase (CcO) in its resting state in saturated ammonium sulfate, at room temperature overnight, gave EPR signals characteristic of a single Cu(II) center. From the g// and A// values it is concluded that this is a square-planar type 2 copper center, and superhyperfine splitting shows the presence of three nearly equivalent 14N nuclei in the plane. It is suggested that this center, also formed by incubating the enzyme in 10% methanol followed by direct irradiation, must be the CuB center. This type 2 copper EPR spectrum is identical to the EPR spectrum of CuB reported for the isolated cytochrome bo3 complex from Escherichia coli; and to the EPR spectrum reported for the sulfobetaine 12 heat-treated cytochrome c oxidase complex. It is argued that a small perturbation in the system causes decoupling of the magnetic coupling of the heme a3-CuB binuclear center and the appearance of the type 2 EPR signal.     

The active centers of adenylylsulfate reductase from Desulfovibrio gigas. Characterization and spectroscopic studies

In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (approximately 400 kDa). It is an iron-sulfur, flavin-containing protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10-0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin-spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and II). M?ssbauer studies on 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential less than or equal to 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical M?ssbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin-type [4Fe-4S] cluster, suggesting a different structure and/or a distinct cluster-ligand environment.     

Conformational changes of spin-labeled native and modified phosphorylase B

The phosphorylase B labelled with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-iodacetamide (phosphorylase I) and with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-ethylmaleinimide (phosphorylase II) was studied. It was shown that label I is characterized by a greater mobility with respect to the protein as compared to label II. In spin-labelled preparations of phosphorylase B the 1,5--2,0 SH-groups of the enzyme monomer having no effect on the enzyme activity were modified. The effects of AMP, glucose-1-phosphate and glucose-6-phosphate on the EPR spectrum of phosphorylase I were studied. The greatest changes in the spectrum (especially in the high field line) were found to occur in the presence of glucose-6-phosphate. These changes are due to the increase in the degree of anisotropic spin rotation. The experimental and theoretical spectra allowing to determine the correlation time for the protein moiety (tau b = 160 ns) were shown to be similar. The local conformation changes were found to occur in the vicinity of one of the two label-bound SH-groups of phosphorylase I. The EPR spectra demonstrate the S-shaped dependence of mobility of phosphorylase I label on concentration of glucose-6-phosphate (0,1--10 mM). In the presence of AMP no S-shaped dependence is observed. Reduced NaBH4 phosphorylase I does not reveal the S-shaped dependence of the label mobility on concentration of glucose-6-phosphate. The degree of the label immobilization in the apo-phosphorylase I--pyridoxal-5-chloromethylphosphonate complex in the presence of glucose-6-phosphate and AMP is the same as in cholophosphorylase I; however, in contrast to the choloenzyme it does not depend on glucose-6-phosphate (0,1--10,0 mM). The changes in the mobility of the spin label of apophosphorylase I and its complex with the AMP analog--adenosine-5'-chloromethylphosphonate--during the choloenzyme reconstruction by pyridoxalphosphate are indicative of participation of AMP and the phosphate group of AMP in the formation of the enzyme active center.