Electron paramagnetic resonance investigation of photosynthetic reaction centers from Rhodobacter sphaeroides R-26 in which Fe2+ was replaced by Cu2+. Determination of hyperfine interactions and exchange and dipole-dipole interactions between Cu2+ and QA-.

We report electron paramagnetic resonance (EPR) experiments in frozen solutions of unreduced and reduced photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides R-26 in which Fe2+ has been chemically replaced by the isotope 65Cu2+. Samples in which the primary quinone acceptor QA is unreduced (Cu2+QA:RCs) give a powder EPR spectrum typical for Cu2+ having axial symmetry, corresponding to a d(x2 - y2) ground state orbital, with g values g parallel = 2.314 +/- 0.001 and g perpendicular = 2.060 +/- 0.003. The spectrum shows a hyperfine structure for the nuclear spin of copper (65I = 3/2) with A parallel = (-167 +/- 1) x 10(-4) cm-1 and /A perpendicular/ = (16 +/- 2) x 10(-4) cm-1, and hyperfine couplings with three nitrogen ligands. This has been verified in samples containing the naturally occurring 14N isotope (l = 1), and in samples where the nitrogen ligands to copper were replaced by the isotope 15N (l = 1/2). We introduce a model for the electronic structure at the position of the metal ion which reflects the recently determined three-dimensional structure of the RCs of Rb. sphaeroides (Allen, J. P., G. Feher, T. O. Yeates, H. Komiya, and D. C. Rees. 1987. Proc. Natl. Acad. Sci. USA. 84:5730: Allen, J. P., G. Feher, T. O. Yeates, H. Komiya, and D. C. Rees. 1988. Proc. Natl. Acad. Sci. USA, 85:8487) as well as our EPR results. In this model the copper ion is octahedrally coordinated to three nitrogens from histidine residues and to one carboxylate oxygen from a glutamic acid, forming a distorted square in the plane of the d(x2 = y2) ground state orbital. It is also bound to a nitrogen of another histidine and to the other carboxylate oxygen of the same glutamic acid residue, in a direction approximately normal to this plane. The EPR spectrum changes drastically when the quinone acceptor QA is chemically reduced (Cu2+QA-:RCs); the change is due to the exchange and dipole-dipole interactions between the Cu2+ and QA- spins. A model spin Hamiltonian proposed for this exchange coupled cooper-quinone spin dimer accounts well for the observed spectra. From a comparison of the EPR spectra of the Cu2+QA:RC and CU2+QA-:RC complexes we obtain the values /J0/ = (0.30 +/- 0.02) K for the isotropic exchange coupling, and /d/ = (0.010 +/- 0.002) K for the projection of the dipole-dipole interaction tensor on the symmetry axis of the copper spin. From the EPR experiments only the relative signs of J0 and d can be deduced; it was determined that they have the same sign. The magnitude of the exchange coupling calculated for Cu2+QA-:RC is similar to that observed for the Fe2+QA-:RC complex (J0 = -0.43K). The exchange coupling is discussed in terms of the superexchange paths connecting the Cu2+ ion and the quinone radical using the structural data for the RCs of Rb. sphaeroides. From the value of the dipole-dipole interaction, d, we determined R approximately 8.4 A for the weighted distance between the metal ion and the quinone in reduced RCs, which is to be compared with 10 A obtained from x-ray analysis of unreduced RCs. This points to a shortening of the Cu2+ -QA- distance upon reduction of the quinone, as has been proposed by Allen et al. (1988).     

The electronic structure of Fe2+ in reaction centers from Rhodopseudomonas sphaeroides. III. EPR measurements of the reduced acceptor complex.

Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.     

Magnetization of manganese superoxide dismutase from Thermus thermophilus.

The ground state magnetic properties of manganese superoxide dismutase from Thermus thermophilus in its native and reduced forms have been determined using saturation magnetization data. Parallel EPR measurements were used to verify that commonly encountered paramagnetic impurities were at low concentration relative to the metalloprotein. The native enzyme contains high spin Mn(III) (S = 2) with D = +2.44(5) cm-1 and E/D = 0. The reduced enzyme contains high spin Mn(II) (S = 5/2) with D = +0.50(5) cm-1 and E/D = 0.027. These results are in keeping with the suggestions of several previous groups of workers concerning the permissible oxidation and spin states of the manganese, but the zero field splitting parameters are unlike those of known manganese model compounds. In addition, the extinction coefficient for the visible region absorption maximum of the native enzyme and the corresponding difference extinction coefficient (native minus reduced) have been measured using saturation magnetization data to quantitate Mn(III) present. The result, epsilon 480 = 950(80) M-1 cm-1 (delta epsilon 480 = 740(60) M-1 cm-1) agrees with the previously reported value of epsilon 480 = 910 M-1 cm-1 found by total manganese determination (Sato, S. and Nakazawa, K. (1978) J. Biochem. 83, 1165-1171). The wide variation in the reported visible region extinction coefficients of manganese superoxide dismutases from different sources is discussed.     

Crossing of low-lying electronic levels of high-spin ferrous ion in deoxyhemoglobin and deoxymyoglobin.

The electronic structure of high-spin S = 2 ferrous ion in deoxy forms of hemoglobin and myoglobin is considered in terms of spin Hamiltonian formalism. Spin Hamiltonian parameters of the second order B0(2)(D), B2(2)(E) and, for the first time in the available literature, of fourth order B0(4), B2(4) and B4(4), are calculated for the rhombic symmetry case of Fe2+. The Hamiltonian matrix is diagonalized for several sets of Bq(k) parameters compatible with other experimental data. The low-lying Fe2+ levels exhibit crossings in a high magnetic field, applied along the z-axis perpendicular to the heme plane. The cross-over values of the magnetic field are determined to be Hc1 = 46 kOe and Hc2 = 168 kOe for D = 5.2, E = 0.6 cm-1 (close to the magnetic data of Nakano, N., Otsuka, J. and Tasaki, A. (1972) Biochim. Biophys. Acta 278, 355-371) and with B0(4) = 0.037, B2(4) = 0.005, B4(4) = 0.013 cm-1 and gz = 2.028. Experimental techniques for measurement of the crossing effects are discussed.     

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.     

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.     

Radical-pair energetics and decay mechanisms in reaction centers containing anthraquinones, naphthoquinones or benzoquinones in place of ubiquinone

In reaction centers from Rhodobacter sphaeroides (formerly called Rhodopseudomonas sphaeroides), light causes an electron-transfer reaction that forms the radical pair state (P+I-, or PF) from the initial excited singlet state (P) of a bacteriochlorophyll dimer (P). Subsequent electron transfer to a quinone (Q) produces the state P+Q-. Back electron transfer can regenerate P from P+Q-, giving rise to 'delayed' fluorescence that decays with approximately the same lifetime as P+Q-. The free-energy difference between P+Q- and P can be determined from the initial amplitude of the delayed fluorescence. In the present work, we extracted the native quinone (ubiquinone) from Rps. sphaeroides reaction centers, and replaced it by various anthraquinones, naphthoquinones, and benzoquinones. We found a rough correlation between the halfwave reduction potential (E1/2) of the quinone used for reconstitution (as measured polarographically in dimethylformamide) and the apparent free energy of the state P+Q- relatively to P. As the E1/2 of the quinone becomes more negative, the standard free-energy gap between P+Q- and P decreases. However, the correlation is quantitatively weak. Apparently, the effective midpoint potentials (Em) of the quinones in situ depend subtly on interactions with the protein environment in the reaction center. Using the value of the Em for ubiquinone determined in native reaction centers as a reference, and the standard free energies determined for P+Q- in reaction centers reconstituted with other quinones, the effective Em values of 12 different quinones in situ are estimated. In native reaction centers, or in reaction centers reconstituted with quinones that give a standard free-energy gap of more than about 0.8 eV between P+Q- and P*, charge recombination from P+Q- to the ground state (PQ) occurs almost exclusively by a temperature-insensitive mechanism, presumably electron tunneling. When reaction centers are reconstituted with quinones that give a free-energy gap between P+Q- and P* of less than 0.8 with quinones that give a free-energy gap between P+Q- and P* of less than 0.8 eV, part or all of the decay proceeds through a thermally accessible intermediate. There is a linear relationship between the log of the rate constant for the decay of P+Q- via the intermediate state and the standard free energy of P+Q-. The higher the free energy, the faster the decay. The kinetic and thermodynamic properties of the intermediate appear not to depend strongly on the quinone used for reconstitution, indicating that the intermediate is probably not simply an activated form of P+Q-.(ABSTRACT TRUNCATED AT 400 WORDS)     

The nature of the nitric oxide complexes of lipoxygenase.

The NO complex of lipoxygenase with EPR signals near g = 4.0 is an S = 3/2 system with D approximately 15 cm-1 similar to Fe2+-EDTA-NO. This may result from antiferromagnetic coupling of axial (D greater than E) high spin ferrous iron to NO. The other NO complex of lipoxygenase, with EPR signals below ge, may result from rhombic high spin ferrous iron coupled to NO with D greater than J. The quenching of both signals by a hydroperoxy derivative of linoleic acid probably represents replacement of NO by an oxygen ligand.     

Spectroscopic characterization of the novel iron-sulfur cluster in Pyrococcus furiosus ferredoxin

Pyrococcus furiosus ferredoxin is the only known example of a ferredoxin containing a single [4Fe-4S] cluster that has non-cysteinyl ligation of one iron atom, as evidenced by the replacement of a ligating cysteine residue by an aspartic acid residue in the amino acid sequence. The properties of the iron-sulfur cluster in both the aerobically and anaerobically isolated ferredoxin have been characterized by EPR, magnetic circular dichroism, and resonance Raman spectroscopies. The anaerobically isolated ferrodoxin contains a [4Fe-4S]+,2+ cluster with anomalous properties in both the oxidized and reduced states which are attributed to aspartate and/or hydroxide coordination of a specific iron atom. In the reduced form, the cluster exists with a spin mixture of S = 1/2 (20%) and S = 3/2 (80%) ground states. The dominant S = 3/2 form has a unique EPR spectrum that can be rationalized by an S = 3/2 spin Hamiltonian with E/D = 0.22 and D = +3.3 +/- 0.2 cm-1. The oxidized cluster has an S = 0 ground state, and the resonance Raman spectrum is characteristic of a [4Fe-4S]2+ cluster except for the unusually high frequency for the totally symmetric breathing mode of the [4Fe-4S] core, 342 cm-1. Comparison with Raman spectra of other [4Fe-4S]2+ centers suggests that this behavior is diagnostic of anomalous coordination of a specific iron atom. The iron-sulfur cluster is shown to undergo facile and quantitative [4Fe-4S] in equilibrium [3Fe-4S] interconversion, and the oxidized and reduced forms of the [3Fe-4S] cluster have S = 1/2 and S = 2 ground states, respectively. In both redox states the [3Fe-4S]0,+ cluster exhibits spectroscopic properties analogous to those of similar clusters in other bacterial ferredoxins, suggesting non-cysteinyl coordination for the iron atom that is removed by ferricyanide oxidation. Aerobic isolation induces partial degradation of the [4Fe-4S] cluster to yield [3Fe-4S] and possibly [2Fe-2S] centers. Evidence is presented to show that only the [4Fe-4S] form of this ferredoxin exists in vivo.     

Infrared evidence of cyanide binding to iron and copper sites in bovine heart cytochrome c oxidase. Implications regarding oxygen reduction

Cyanide binding to bovine heart cytochrome c oxidase at five redox levels has been investigated by use of infrared and visible-Soret spectra. A C-N stretch band permits identification of the metal ion to which the CN- is bound and the oxidation state of the metal. Non-intrinsic Cu, if present, is detected as a cyanide complex. Bands can be assigned to Cu+CN at 2093 cm-1, Cu2+CN at 2151 or 2165 cm-1, Fe3+CN at 2131 cm-1, and Fe2+CN at 2058 cm-1. Fe2+CN is found only when the enzyme is fully reduced whereas the reduced Cu+CN occurs in 2-, 3-, and 4-electron reduced species. A band for Fe3+CN is not found for the complex of fully oxidized enzyme but is for all partially reduced species. Cu2+CN occurs in both fully oxidized and 1-electron-reduced oxidase. CO displaces the CN- at Fe2+ to give a C-O band at 1963.5 cm-1 but does not displace the CN- at Cu+. Another metal site, noted by a band at 2042 cm-1, is accessible only in fully reduced enzyme and may represent Zn2+ or another Cu+. Binding of either CN- or CO may induce electron redistribution among metal centers. The extraordinary narrowness of ligand infrared bands indicates very little mobility of the components that line the O2 reduction site, a property of potential advantage for enzyme catalysis. The infrared evidence that CN- can bind to both Fe and Cu supports the possibility of an O2 reduction mechanism in which an intermediate with a mu-peroxo bridge between Fe and Cu is formed. On the other hand, the apparent independence of Fe and Cu ligand-binding sites makes a heme hydroperoxide (Fe-O-O-H) intermediate an attractive alternative to the formation an Fe-O-O-Cu linkage.     

EPR and M?ssbauer studies of nucleotide-bound nitrogenase iron protein from Azotobacter vinelandii

We have recently shown (Lindahl, P. A., Day, E. P., Kent, T. A., Orme-Johnson, W. H., and Münck, E. (1985) J. Biol. Chem. 260, 11160-11173) that the [4Fe-4S]1+ cluster of the native Fe protein can exist in two forms characterized by different cluster spin: an S = 1/2 state exhibiting a g = 1.94 type EPR signal and an S = 3/2 state yielding signals at g approximately 5.8 and 5.1. We have now extended our study of the Fe protein to include the MgATP- and MgADP-bound forms. The [4Fe-4S]1+ cluster of the nucleotide-bound Fe protein exists in a similar S = 1/2, S = 3/2 spin mixture. The S = 3/2 cluster exhibits a broad EPR signal at g approximately 4.8. In spectra of the MgATP-bound protein, we have also observed a g = 4.3 signal from an S = 5/2 state (D = 1 - 3 cm-1, E/D approximately 0.32). Various experiments strongly suggest that this signal does not originate from adventitiously bound Fe3+. The g = 4.3 signal may arise from approximately 2% of the [4Fe-4S]1+ clusters when MgATP is protein-bound. We have also discovered substoichiometric amounts of what appears to be ADP in some nominally nucleotide-free Fe protein samples. MgATP binds to Fe protein in the presence of perturbing solvents, resulting in EPR spectra similar to those of MgATP-bound samples in aqueous solutions; MgADP binding, on the other hand, results in signals more typical of the solvent state. Spectra of samples frozen during turnover of the nitrogenase system exhibit a mixture of spin states. Characterization of the Fe protein EPR signature described here should aid future mechanistic and nucleotide-binding studies.     

The involvement of iron and ubiquinone in electron transfer reactions mediated by reaction centers from photosynthetic bacteria.

Reaction centers from Rhodopseudomonas sphaeroides strain R-26 were prepared with varying Fe and ubiquinone (Q) contents. The photooxidation of P-870 to P-870+ was found to occur with the same quantum yield in Fe-depleted reaction centers as in control samples. The kinetics of electron transfer from the initial electron acceptor (I) to Q also were unchanged upon Fe removal. We conclude that Fe has no measurable role in the primary photochemical reaction. The extent of secondary reaction from the first quinone acceptor (QA) to the second quinone acceptor (QB) was monitored by the decay kinetics of P-870+ after excitation of reaction centers with single flashes in the absence of electron donors, and by the amount of P-870 photooxidation that occurred on the second flash in the presence of electron donors. In reaction centers with nearly one iron and between 1 and 2 ubiquinones per reaction center, the amount of secondary electron transfer is proportional to the ubiquinone content above one per reaction center. In reaction centers treated with LiClO4 and o-phenanthroline to remove Fe, the amount of secondary reaction is decreased and is proportional to Fe content. Fe seems to be required for the secondary reaction. In reaction centers depleted of Fe by treatment with SDS and EDTA, the correlation between Fe content and secondary activity is not as good as that found using LiClO4. This is probably due in part to a loss of primary photochemical activity in samples treated with SDS; but the correlation is still not perfect after correction for this effect. The nature of the back reaction between P-870+ and Q-B was investigated using stopped flow techniques. Reaction centers in the P-870+ Q-B state decay with a 1-s half-time in both the presence and absence of o-phenanthroline, an inhibitor of electron transfer between Q-B and QB. This indicates that the back reaction between P-870+ and Q-A is direct, rather than proceeding via thermal repopulation of Q-A. The P-870+ Q-B state is calculated to lie at least 100 mV in free energy below the P-870+ Q-A state.     

On the mechanism of photoinduced electron transfer in photosystem II reaction centers

The EPR spectrum of the triplet state of photosystem II reaction centers has been studied in the case of the singly reduced primary acceptor complex QAFe2+. It was demonstrated that the shape of the spectrum does not change much when the relaxation of the primary acceptor is accelerated and when magnetic interaction between the reduced quinone molecule QA and the non-heme iron Fe2+ is disrupted. This observation confirms the earlier conclusion that the anomalous shape of the EPR spectrum is due mainly to the anisotropy of the quatum yield of the triplet state. A scheme of primary events in photosystem II is discussed, which is consistent with the observed properties of the EPR spectrum of the triplet state.     

Evidence for siroheme-Fe4S4 interaction in spinach ferredoxin-sulfite reductase

Spinach ferredoxin-sulfite reductase (SiR) contains one siroheme and one Fe4S4 center per polypeptide subunit. The heme is entirely in the high-spin Fe3+ state in the oxidized enzyme. When SiR is photochemically reduced with ethylenediaminetetraacetate (EDTA)-deazaflavin, the free enzyme and its CN- and CO complexes show changes in absorption spectra associated with the heme even after the heme has been reduced from the Fe3+ to the Fe2+ state. With CO- or CN--SiR, these spectral changes are associated with the appearance of a classical "g = 1.94" type of EPR spectrum characteristic of reduced Fe4S4 centers. The line shapes and exact g values of the g = 1.94 EPR spectra vary with the nature of the ligand bound to the heme Fe. Photoreduction of free SiR results in production of a novel type of EPR signal, with g = 2.48, 2.34, and 2.08 in the fully reduced enzyme; this signal accounts for 0.6 spin per heme. (A small g = 1.94 type EPR signal, representing 0.2 spin per heme, is also found.) These data suggest the presence of a strong magnetic interaction between the siroheme and Fe4S4 centers in spinach SiR, this interaction giving rise to different EPR signals depending on the spin state of the heme Fe in the reduced enzyme.     

A study of the magnetic properties of haem a3 in cytochrome c oxidase by using magnetic-circular-dichroism spectroscopy.

M.c.d. (magnetic-circular-dichroism) spectroscopy was used to study the magnetization properties of the haem centres in cytochrome c oxidase with magnetic fields of between 0 and 5.3 T over the temperature range 1.5--200 K. The oxidized, oxidized cyanide and partially reduced cyanide forms of the enzyme were studied. In the oxidized state only cytochrome a3+ is detectable by m.c.d. spectroscopy, and its magnetization characteristics show it to be a low-spin ferric haem. In the partially reduced cyanide form of the enzyme cytochrome a is in the diamagnetic low-spin ferrous form, whereas cytochrome a3--CN is e.p.r.-detectable and gives an m.c.d.-magnetization curve typical of a low-spin ferric haem. In the oxidized cyanide form of the enzyme both cytochrome a and cytochrome a3--CN are detectable by m.c.d. spectroscopy, although only cytochrome a gives an e.p.r. signal. The magnetization characteristics of haem a3--CN show clearly that its ground state is an electronic doublet and that another state, probably a spin singlet, lies greater than 10 cm-1 above this. These features are well accounted for by an electronic state of spin S = 1 with a predominantly axial distortion, which leaves the doublet, Ms = +/- 1, as the ground state and the component Ms = 0 as the excited state. This state would not give an e.p.r. signal. Such an electronic state could arise either from a ferromagnetic coupling between haem a3+(3)-CN and the cupric ion, Cua3, or form a haem in the Fe(IV) state.     

Multi-frequency EPR and high-resolution M?ssbauer spectroscopy of a putative [6Fe-6S] prismane-cluster-containing protein from Desulfovibrio vulgaris (Hildenborough). Characterization of a supercluster and superspin model protein.

The putative [6Fe-6S] prismane cluster in the 6-Fe/S-containing protein from Desulfovibrio vulgaris, strain Hildenborough, has been enriched to 80% in 57Fe, and has been characterized in detail by S-, X-, P- and Q-band EPR spectroscopy, parallel-mode EPR spectroscopy and high-resolution 57Fe M?ssbauer spectroscopy. In EPR-monitored redox-equilibrium titrations, the cluster is found to be capable of three one-electron transitions with midpoint potentials at pH 7.5 of +285, +5 and -165 mV. As the fully reduced protein is assumed to carry the [6Fe-6S]3+ cluster, by spectroscopic analogy to prismane model compounds, four valency states are identified in the titration experiments: [6Fe-6S]3+, [6Fe-6S]4+, [6Fe-6S]5+, [6Fe-6S]6+. The fully oxidized 6+ state appears to be diamagnetic at low temperature. The prismane protein is aerobically isolated predominantly in the one-electron-reduced 5+ state. In this intermediate state, the cluster exists in two magnetic forms: 10% is low-spin S = 1/2; the remainder has an unusually high spin S = 9/2. The S = 1/2 EPR spectrum is significantly broadened by ligand (2.3 mT) and 57Fe (3.0 mT) hyperfine interaction, consistent with a delocalization of the unpaired electron over 6Fe and indicative of at least some nitrogen ligation. At 35 GHz, the g tensor is determined as 1.971, 1.951 and 1.898. EPR signals from the S = 9/2 multiplet have their maximal amplitude at a temperature of 12 K due to the axial zero-field splitting being negative, D approximately -0.86 cm-1. Effective g = 15.3, 5.75, 5.65 and 5.23 are observed, consistent with a rhombicity of [E/D] = 0.061. A second component has g = 9.7, 8.1 and 6.65 and [E/D] = 0.108. When the protein is reduced to the 4+ intermediate state, the cluster is silent in normal-mode EPR. An asymmetric feature with effective g approximately 16 is observed in parallel-mode EPR from an integer spin system with, presumably, S = 4. The fully reduced 3+ state consists of a mixture of two S = 1/2 ground state. The g tensor of the major component is 2.010, 1.825 and 1.32; the minor component has g = 1.941 and 1.79, with the third value undetermined. The sharp line at g = 2.010 exhibits significant convoluted hyperfine broadening from ligands (2.1 mT) and from 57Fe (4.6 mT). Zero-field high-temperature M?ssbauer spectra of the protein, isolated in the 5+ state, quantitatively account for the 0.8 fractional enrichment in 57Fe, as determined with inductively coupled plasma mass spectrometry.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for variable metal-radical spin coupling in oxoferrylporphyrin cation radical complexes

Oxoferrylporphyrin cation radical complexes were generated by m-chloroperoxybenzoic acid oxidation of the chloro and trifluoromethanesulfonato complexes of tetramesitylporphyrinatoiron(III) [(TMP)Fe] and the trifluoromethanesulfonato complex of tetra(2,6-dichlorophenyl)porphyrinatoiron(III) [TPP(2,6-Cl)Fe]. Coupling between ferryl iron (S = 1) and porphyrin radical (S' = 1/2) spin systems was investigated by M?ssbauer and EPR spectroscopy. The oxoferrylporphyrin cation radical systems generated from the TMP complexes show strong ferromagnetic coupling. Analysis of the magnetic M?ssbauer spectra, using a spin Hamiltonian explicitly including a coupling tensor J, suggests an exchange-coupling constant J greater than 80 cm-1. The EPR spectra show non-zero rhombicity, the origin of which is discussed in terms of contributions from the usual zero-field effects of iron and from iron-radical spin-dipolar interaction. A consistent estimate of zero-field splitting parameter D approximately + 6 cm-1 was obtained by EPR and M?ssbauer measurements. EPR and M?ssbauer parameters are shown to be slightly dependent on solvent, but not on the axial ligand in the starting (TMP)Fe complex. In contrast to the TMP complex, the oxoferrylporphyrin cation radical system generated from [TPP(2,6-Cl)FeOSO2CF3] exhibits M?ssbauer and EPR spectra consistent with weak iron-porphyrin radical coupling of magnitude of J approximately 1 cm-1.     

Nitrogenase. VIII. M?ssbauer and EPR spectroscopy. The MoFe protein component from Azotobacter vinelandii OP.

We have studied the molybdenum-iron protein (MoFe protein, also known as component I) from Azobacter vinelandi using M?ssbauer spectroscopy and electron paramagnetic resonance on samples enriched with 57Fe. These spectra can be interpreted in terms of two EPR active centers, each of which is reducible by one electron. A total of four different chemical environments of Fe can be discerned. One of them is a cluster of Fe atoms with a net electronic spin of 3/2, one of them is high-spin ferrous iron and the remaining two are iron in a reduced state (probably in clusters). The results are as follows: Chemical analysis yields 11.5 Fe atoms and 12.5 labile sulfur atoms per molybdenum atom; the molecule contains two Mo atoms per 300 000 daltons. The EPR spectrum of the MoFe protein exhibits g values at 4.32, 3.65 and 2.01, associated with the ground state doublet of a S = 3/2 spin system. The spin Hamiltonian H = D(S2/z minus 5/4 + lambda(S2/x minus S2/y)) + gbeta/o S-H fits the experimental data for go = 2.00 and lambda = 0.055. Quantitative analysis of the temperature dependence of the EPR spectrum yields D/k = 7.5 degrees K and 0.91 spins/molybdenum atom, which suggests that the MoFe protein has two EPR active centers. Quantitative evaluation of M?ssbauer spectra shows that approximately 8 iron atoms give rise to one quadrupole doublet; at lower temperatures magnetic spectra, associated with the groud electronic doublet, are observed; at least two magnetically inequivalent sites can be distinguished. Taken together the data suggest that each EPR center contains 4 iron atoms. The EPR and M?ssbauer data can only be reconciled if these iron atoms reside in a spin-coupled (S = 3/2) cluster. Under nitrogen fixing conditions the magnetic M?ssbauer spectra disappeared concurrently with the EPR signal and quadrupole doublets are obserced at all temperatures. The data suggest that each EPR active center is reduced by one electron. The M?ssbauer investigation reveals three other spectral components characteristic of iron nuclei in an environment of integer or zero electronic spin, i.e. they reside in complexes which are "EPR-silent". One of the components (3-4 iron atoms) has M?ssbauer parameters characteristic of the high-spin ferrous iron as in reduced ruberdoxin. However, measurements in strong fields indicate a diamagnetic environment. Another component, representing 9-11 iron atoms, seems to be diamagnetic also. It is suggested that these atoms are incorporated in spin-coupled clusters.     

Identification of the iron-sulfur center of spinach ferredoxin-nitrite reductase as a tetranuclear center, and preliminary EPR studies of mechanism.

EPR spectroscopic and chemical analyses of spinach nitrite reductase show that the enzyme contains one reducible iron-sulfur center, and one site for binding either cyanide or nitrite, per siroheme. The heme is nearly all in the high spin ferric state in the enzyme as isolated. The extinction coefficient of the enzyme has been revised to E386 = 7.6 X 10(4) cm-1 (M heme)-1. The iron-sulfur center is reduced with difficulty by agents such as reduced methyl viologen (equilibrated with 1 atm of H2 at pH 7.7 in the presence of hydrogenase) or dithionite. Complexation of the enzyme with CO (a known ligand for nitrite reductase heme) markedly increases the reducibility of the iron-sulfur center. New chemical analyses and reinterpretation of previous data show that the enzyme contains 6 mol of iron and 4 mol of acid-labile S2-/mol of siroheme. The EPR spectrum of reduced nitrite reductase in 80% dimethyl sulfoxide establishes clearly that the enzyme contains a tetranuclear iron-sulfur (Fe4S4) center. The ferriheme and Fe4S4 centers are reduced at similar rates (k = 3 to 4 s-1) by dithionite. The dithionite-reduced Fe4S4 center is rapidly (k = 100 s-1) reoxidized by nitrite. These results indicate a role for the Fe4S4 center in catalysis.     

The iron-sulfur cluster composition of Escherichia coli nitrate reductase

Nitrate reductase from Escherichia coli has been investigated by low-temperature magnetic circular dichroism and electron paramagnetic resonance (EPR) spectroscopies, as well as by Fe-S core extrusion, to determine the Fe-S cluster composition. The results indicate approximately one 3Fe and three or four [4Fe-4S]2+,1+ centers/molecule of isolated enzyme. The magnetic circular dichroism spectra and magnetization characteristics show the oxidized and reduced 3Fe and [4Fe-4S] centers to be electronically analogous to those in bacterial ferredoxins. The form and spin quantitation of the EPR spectra from [4Fe-4S]1+ centers in the reduced enzyme were found to vary with the conditions of reduction. For the fully reduced enzyme, the EPR spectrum accounted for between 2.9 and 3.5 spins/molecule, and comparison with partially reduced spectra indicates weak intercluster magnetic interactions between reduced paramagnetic centers. In common with other Fe-S proteins, the 3Fe center was not extruded intact under standard conditions. The results suggest that nitrate reductase is the first example of a metalloenzyme where enzymatic activity is associated with a form that contains an oxidized 3Fe center. However, experiments to determine whether or not the 3Fe center is present in vivo were inconclusive.