A comparative spectroscopic study of two non-haem iron proteins lacking labile sulphide from Desulphovibrio gigas.

The ultraviolet visible, and near infrared spectrum of a two-iron protein from Desulphovibrio gigas, a new type of non-haem iron protein lacking labile sulphide, is compared with that of D. gigas rubredoxin. The charge transfer band maxima of rubredoxin at 495 and 565 nm are less separated in the new protein implying a higher symmetry of the two iron centres. The existence of a spin-spin interaction between the two iron centres in the new protein is suggested by the magnetic susceptibility measurements of the oxidized and reduced states of both proteins, which gives a smaller value per iron centre for the new protein. The oxidized form of the two iron-protein has a complex EPR spectrum with signals at g values of 8.97, 7.72, 5.73, 4.94, and 1.84. An EPR titration gives a value of --35 +/- 15 mV for the two signals at g values of 7.72 and 5.73. Rubredoxin has the characteristic spectrum of rubredoxins with two signals at g values of 9.4 and 4.27.     

Spectroscopic studies of cobalt-substituted ferredoxin fromClostridium pasteurianum

Summary Ferredoxin fromClostridium pasteurianum substituted with two Co atoms did not give any cobalt EPR signal at 8 K as isolated, but upon reduction with sodium dithionite, a broad signal appeared withg values that indicate highspin (S=3/2) Co(II). These signals were distinct from Co(II)-dithiothreitol signals, and disappeared upon reoxidation with air. Under anaerobic incubation of apoferredoxin with Co(II), a green derivative showed a visible spectrum typical of tetrahedral Co(Il)-thiolate coordination, which shifted dramatically upon exposure to air. The¹H-NMR spectrum of the aerobically isolated protein is reported at 300 MHz; magnetic susceptibility measurements were indicative of a diamagnetic species. These spectroscopic studies indicate that Co(II)-substituted ferredoxin is oxidized to low-spin Co(III)-ferredoxin in the presence of sulfide and oxygen. The diamagnetic Co(III) state could reversibly be reduced to highspin Co(II) by sodium dithionite.     

Evidence for polynuclear aggregates of ferric daunomycin. A M?ssbauer, EPR, X-ray absorption spectroscopy and magnetic susceptibility study.

The interaction of the antitumor agent daunomycin (DN) with ferric iron has been analysed by M?ssbauer spectroscopy, EPR, extended X-ray absorption fine structure (EXAFS), and magnetic susceptibility measurements. In contrast to literature data, at millimolar iron and anthracycline concentrations no solitary Fe(DN)3 complexes are formed in appreciable amounts. The M?ssbauer spectroscopic analysis revealed severe dependencies on temperature, on the preparation procedure, the time allowed for equilibration, and on the metal/ligand ratio. The M?ssbauer spectra exhibit two components: a broad magnetic sextet and a quadrupole doublet at an Fe/DN molar ratio of 1:3 and exclusively a doublet at a molar ratio of 1:20, indicating an equilibrium of these two spectral components. The EPR spectra are dominated by a signal at g(eff) = 2. Double integration of the EPR signals enabled the determination of their spin density and a correlation between EPR and M?ssbauer spectra. The M?ssbauer sextet species is EPR invisible and corresponds to magnetically ordered polynuclear aggregates with high magnetic anisotropy. EXAFS and susceptibility measurements provide additional evidence for the formation of polynuclear aggregates of ferric daunomycin. The quadrupole doublet species in the M?ssbauer spectra correlates with the g = 2 signal in EPR. This species is also related to a magnetically ordered system, exhibiting, however, superparamagnetic behavior due to less magnetic anisotropy. Since daunomycin forms dimers in aqueous solution at millimolar concentrations, we conclude that the cooperative phenomena observed in EPR and M?ssbauer spectra are a consequence of its stacking effects.     

1H NMR studies of porcine uteroferrin. Magnetic interactions and active site structure

Pink (reduced) uteroferrin exhibits well resolved paramagnetic NMR spectra with resonances ranging from 90 ppm downfield to 70 ppm upfield. The intensities of these signals depend on the degree of reduction and correlate well with the intensity of the EPR signals with gave = 1.74. Analyses of chemical shifts and the temperature dependence of the paramagnetically shifted resonances indicate that the Fe(III)-Fe(II) cluster in the reduced protein exhibits weak antiferromagnetic exchange coupling (-J approximately equal to 10 cm-1), in agreement with the estimate derived from the temperature dependence of the EPR signal intensity. Purple (oxidized) uteroferrin, on the other hand, exhibits no discernible paramagnetically shifted resonances, reflecting either strong antiferromagnetic coupling or an unfavorable electron spin-lattice relaxation time. Evans susceptibility comparisons between pink and purple uteroferrin show that the Fe(III)-Fe(III) cluster in the oxidized protein is more strongly coupled (-J greater than 40 cm-1). This value concurs with low temperature magnetic susceptibility measurements on both the porcine and splenic purple acid phosphatases. The isotropically shifted protons of tyrosine coordinated to the cluster are assigned by comparison with synthetic complexes. Tyrosine, earlier implicated as a ligand by resonance Raman spectroscopy, appears to coordinate only to the ferric site in pink uteroferrin. This is consistent with the relatively invariant extinction coefficients of uteroferrin in its oxidized and reduced forms and the ease of reduction of the nonchromophoric iron compared to its chromophoric partner. Other possible ligands to the cluster include histidine, suggested by the presence of downfield-shifted solvent-exchangeable resonances with appropriate isotropic shifts.     

Magnetic studies of Chromatium flavocytochrome C552. A mechanism for heme-flavin interaction.

Electron paramagnetic resonance and magnetic susceptibility studies of Chromatium flavocytochrome C552 and its diheme flavin-free subunit at temperatures below 45 degrees K are reported. The results show that in the intact protein and the subunit the two low-spin (S = 1/2) heme irons are distinguishable, giving rise to separate EPR signals. In the intact protein only, one of the heme irons exists in two different low spin environments in the pH range 5.5 to 10.5, while the other remains in a constant environment. Factors influencing the variable heme iron environment also influence flavin reactivity, indicating the existence of a mechanism for heme-flavin interaction.     

A magnetic study of acidic ferric hemoglobin

In this article, we have extensively studied and discussed the magnetic properties of acidic ferric hemoglobin and its isolated chains. The magnetic susceptibility, EPR and optical spectra of those samples were measured in the temperature region below 77 degrees K. By the magnetic susceptibility measurements, it could be made clear that at an acidic pH value, both ferric hemoglobin and its isolated chains were constituted of a mixture of two spin states (high-spin state S = 5/2 and low-spin state S = 1/2) and the ratio of this mixture varied in each protein sample, but was independent of the temperature change below 77 degrees K. The co-existence of these two components could be ascertained by the observation of EPR spectra at liquid hydrogen temperature. Acidic ferric hemoglobin and its isolated chains exhibited the two components of EPR spectra which corresponded to their magnetic susceptibility, and it was found that the relaxation time of the low-spin state was longer than that of the high-spin state. The low-spin component of EPR spectra was almost undetectable at liquid nitrogen temperature. The three principal g values of this low-spin were gz = 2.80, gy = 2.20, and gx = 1.70. At alkaline pH values these low-spin components and the high-spin component of EPR spectra were displaced by the different low-spin spectra which corresponded to the ferric hemoglobin-hydroxide complex. It seems that the magnetic properties of the high-spin component are the same as the acidic ferric myoglobin, and the fine structure of the iron ion also seems to be same. Optical spectroscopy also gave similar magnetic properties which corresponded to the magnetic measurements.     

Mechanistic features and structure of the nitrogenase alpha-Gln195 MoFe protein

EPR signals observed under CO and C(2)H(2) during nitrogenase turnover were investigated for the alpha-Gln(195) MoFe protein, an altered form for which the alpha-His(195) residue has been substituted by glutamine. Under CO, samples show S = 1/2 hi- and lo-CO EPR signals identical to those recognized for the wild-type protein, whereas the S = 3/2 signals generated under high CO/high flux conditions differ. Previous work has revealed that the EPR spectrum generated under C(2)H(2) exhibits a signal (S(EPR1)) originating from the FeMo-cofactor having two or more bound C(2)H(2) adducts and a second signal (S(EPR2)) arising from a radical species [S?rlie, M., Christiansen, J., Dean, D. R., and Hales, B. J. (1999) J. Am. Chem. Soc. 121, 9457-9458]. Pressure-dependent studies show that the intensity of these signals has a sigmoidal dependency at low pressures and maximized at 0.1 atm C(2)H(2) with a subsequent decrease in steady-state intensity at higher pressures. Analogous signals are not recognized for the wild-type MoFe protein. Analysis of the principal g-factors of S(EPR2) suggests that it either represents an unusual metal cluster or is a carboxylate centered radical possibly originating from homocitrate. Both S(EPR1) and S(EPR2) exhibit similar relaxation properties that are atypical for S = 1/2 signals originating from Fe-S clusters or radicals and indicate a coupled relaxation pathway. The alpha-Gln(195) MoFe protein also exhibits these signals when incubated under turnover conditions in the presence of C(2)H(4). Under these conditions, additional inflections in the g 4-6 region assigned to ground-state transitions of an S = 3/2 spin system are also recognized and assigned to turnover states of the MoFe protein without C(2)H(4) bound. The structure of alpha-Gln(195) was crystallographically determined and found to be virtually identical to that of the wild-type MoFe protein except for replacement of an NuH-S hydrogen bond interaction between FeMo-cofactor and the imidazole side chain of alpha-His(195) by an analogous interaction involving Gln.     

Magnetic susceptibility of laccases and ceruloplasmin.

1. Recent magnetic susceptibility measurements on laccase (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) from the lacquer tree Rhus vernicifera showed a deviation from Curie behaviour above 50 K, which was taken as evidence for an antiferromagnetically coupled Cu(II)-Cu(II) pair in the oxidized enzyme. The magnetic susceptibility of this protein has been reinvestigated. Further measurements on laccase from the fungus Polyporus versicolor and human ceruloplasmin (iron(II):oxygen oxidoreductase, EC 1.16.3.1) are presented. 2. The magnetic susceptibility of fungal laccase and lacquer tree laccase can be accounted for by the EPR detectable copper ions in the temperature range 40--300 K. 3. If an antiferromagnetically coupled Cu(II)-Cu(II) pair exists in the laccases, then the coupling, expressed as --J, should be at least of the order of 300 cm-1, as deduced from the Curie dependence of the susceptibility and the sensitivity in our measurements. 4. If an analogy with the laccases is assumed for the EPR invisible copper in ceruloplasmin then a limiting value of the coupling may be deduced also in this case, with --J at least of the order of 200 cm-1.     

Origin of the transient electron paramagnetic resonance signals in DNA photolyase

DNA photolyase repairs pyrimidine dimer lesions in DNA through light-induced electron donation to the dimer. During isolation of the enzyme, the flavin cofactor necessary for catalytic activity becomes one-electron-oxidized to a semiquinone radical. In the absence of external reducing agents, the flavin can be cycled through the semiquinone radical to the fully reduced state with light-induced electron transfer from a nearby tryptophan residue. This cycle provides a convenient means of studying the process of electron transfer within the protein by using transient EPR. By studying the excitation wavelength dependence of the time-resolved EPR signals we observe, we show that the spin-polarized EPR signal reported earlier from this laboratory as being initiated by semiquinone photochemistry actually originates from the fully oxidized form of the flavin cofactor. Exciting the semiquinone form of the flavin produces two transient EPR signals: a fast signal that is limited by the time response of the instrument and a slower signal with a lifetime of approximately 6 ms. The fast component appears to correlate with a dismutation reaction occurring with the flavin. The longer lifetime process occurs on a time scale that agrees with transient absorption data published earlier; the magnetic field dependence of the amplitude of this kinetic component is consistent with redox chemistry that involves electron transfer between flavin and tryptophan. We also report a new procedure for the rapid isolation of DNA photolyase.     

On the novel H2-activating iron-sulfur center of the "Fe-only" hydrogenases

The two hydrogenases (I and II) of the anaerobic N2-fixing bacterium Clostridium pasteurianum (Cp) and the hydrogenases of the anaerobes Megasphaera elsdenii (Me) and Desulfovibrio vulgaris (strain Hildenborough, Dv), contain iron-sulfur clusters but not nickel. They are the most active hydrogenases known. All four enzymes in their reduced states give rise to EPR signals typical of [4Fe-4S]1+ clusters but exhibit novel EPR signals in their oxidized states. For example, Cp hydrogenase I exhibits a sharp rhombic EPR signal when oxidized under mild conditions but the enzyme is inactivated by over-oxidation and then exhibits an axial EPR signal. A similar axial signal is observed from mildly oxidized hydrogenase I after treatment with CO. EPR, M?ssbauer and ENDOR spectroscopy indicate that the EPR signals from the oxidized enzyme and its CO derivative arise from a novel spin-coupled Fe center. Low temperature magnetic circular dichroism (MCD) studies reveal that an EPR-silent Fe-S cluster with S greater than 1/2 is also present in oxidized hydrogenase I. From a study of all spectroscopic properties of Cp, Dv, and Me hydrogenases, it is concluded that the H2-activating site of all four is a novel Fe-S cluster with S greater than 0 and integer, which in the oxidized state is exchange-coupled to a S = 1/2 species. The data are most consistent with the S = 1/2 species being a low spin Fe(III) center. The H2-activating site is susceptible to oxidative rearrangements to yield both active and inactive states of the enzyme. We discuss the possible implications of these finding to methods of enzyme oxidation and purification procedures currently used for hydrogenases.     

Luminescent/paramagnetic probes for detecting order in biological assemblies: transformation of luminescent probes into pi-radicals by photochemical reduction.

The spectroscopic methods of fluorescence polarization and electron paramagnetic resonance (EPR) are used to study order and orientation of extrinsically labeled protein elements of ordered biological systems. These methods generate complementary information about the order of the system, but a consistent quantitative interpretation of the related data is complicated because the signals arise from different donors. We introduce a new method that allows us to detect both signals from the same donor. Unsubstituted xanthene dyes (eosin, erythrosin, and fluorescein) were irradiated by laser light at their absorption maximum in the presence of different reducing agents. Due to photochemical reduction, the quinoidal structure of the xanthene ring is transformed into a semiquinone, and a pi-radical is formed having a characteristic EPR signal of an unpaired electron spin with proton hyperfine interactions. A strong EPR signal is observed from the dye in solution or when specifically attached to a protein following irradiation in the presence of dithiothreitol or cysteine. We applied this technique to the study of skeletal muscle fibers. The fluorescent dye (iodoacetamido)fluorescein was covalently attached to the reactive thiol of the myosin molecule in muscle fibers. Fluorescence polarization and EPR spectroscopy were performed on the labeled fibers in rigor. Both signals indicate a highly ordered system characteristic of cross-bridges bound to actin. Our use of the same signal donor for fluorescence and EPR studies of probe order is a promising new technique for the study of order in protein elements of biological assemblies.     

Comparison of redox and EPR properties of the molybdenum iron proteins of Clostridium pasteurianum and Azotobacter vinelandii nitrogenases

Both heterologous crosses of the Clostridium pasteurianum and Azotobacter vinelandii nitrogenase components are completely inactive, although the reasons for this incompatibility are not known. We have compared a number of properties of the MoFe proteins from these organisms (Cp1 and Av1, respectively) in an attempt to find differences that could explain this lack of functional activity. Optical and CD spectroscopic titrations are similar for both Av1 and Cp1, but EPR titrations are significantly different, suggesting different chemical reactivity patterns and/or magnetic interaction behavior. Similarly, reduction measurements on the six-electron-oxidized state of Cp1 and Av1 at controlled potentials indicate a difference in both the relative reduction sequence of the redox centers and the numerical values for their measured midpoint potentials. EPR measurements as a function of temperature also demonstrate that the relaxation behavior of the S = 3/2 MoFe centers associated with the proteins differ markedly. The Cp1 EPR signal only begins to undergo broadening above 65 K, whereas the Av1 signal is severely broadened above 25 K. These variations in the EPR properties for the two proteins are not likely to be due to differences in the stoichiometry and/or geometry of the MoFe cluster units themselves since similar EPR studies of the isolated cofactors showed them to be essentially identical. Thus, the different EPR behavior of the two proteins seems to arise either from protein constraints imposed on identical cofactors, and/or from magnetic interactions due to neighboring metal clusters.     

Improvements in technical assessment and protocol for EPR evaluation of magnetic fields effects on a radical pair reaction

The effects of either static or pulsed magnetic fields on the reaction rate of Fremy's salt-ascorbic acid were studied directly by EPR spectroscopy. Radical pair mechanism (RPM) accounts for the magnetic field effects, but the expected amounts are so small that they need to be observed with particular care with EPR technique. The method is based on the resolution of a pair of EPR signals by the addition of a stationary field gradient, where the signals are coming from the exposed and control capillary sample. To this purpose, a suitable device for the gradient generation was used. Others improvements were the strictly keeping of the same boundary temperature condition in the capillary pairs, obtained by a refrigerating system controlled by a thermocouple, and the use of a pair of Helmholtz coils to generate an external high homogeneous magnetic field. By this experimental set up, we found that the magnetic field induce the decrease of the studied radical reaction rate. This EPR approach is a significant alternative to the spectrophotometric one. Moreover, it offers the advantage to detect both the radicals and/or intermediates involved in the reaction.     

Complexes of copper(II) dipeptides with hexacyanoferrate(III). Magnetic and spectroscopic properties

The interaction between hexacyanoferrate(III) and two copper(II) dipeptide complexes, such as Cu(II)- glycylhistidine and Cu(II)-glycylphenylalanine, has been investigated by electronic and EPR spectroscopy and by magnetic susceptibility measurements. In both cases the magnetic susceptibility values sum to those corresponding to the patent complexes. However, the electronic relaxation time of the copper(II) ion in the mixed complexes is modified so much that the copper(II) EPR signal disappears suggesting the existence of a specific metal—metal interaction probably through a cyanide bridge. This hypothesis is also supported by the appearance of an hypsochromic shift of the Cu(II) electronic band after addition of hexacyanoferrate(III).     

Electron paramagnetic resonance studies of Pseudomonas cytochrome c peroxidase

The EPR spectrum at 15 K of Pseudomonas cytochrome c peroxidase, which contains two hemes per molecule, is in the totally ferric form characteristic of low-spin heme giving two sets of g-values with gz 3.26 and 2.94. These values indicate an imidazole-nitrogen : heme-iron : methionine-sulfur and an imidazole-nitrogen : heme-iron : imidazole-nitrogen hemochrome structure, respectively. The spectrum is essentially identical at pH 6.0 and 4.6 and shows only a very small amount of high-spin heme iron (g 5--6) also at 77 K. Interaction between the two hemes is shown to exist by experiments in which one heme is reduced. This induces a change of the EPR signal of the other (to gz 2.83, gy 2.35 and gx 1.54), indicative of the removal of a histidine proton from that heme, which is axially coordinated to two histidine residues. If hydrogen peroxide is added to the partially reduced protein, its EPR signal is replaced by still other signals (gz 3.5 and 3.15). Only a very small free radical peak could be observed consistent with earlier mechanistic proposals. Contrary to the EPR spectra recorded at low temperature, the optical absorption spectra of both totally oxidized and partially reduced enzyme reveal the presence of high-spin heme at room temperature. It seems that a transition of one of the heme c moieties from an essentially high-spin to a low-spin form takes place on cooling the enzyme from 298 to 15 K.     

Electron paramagnetic resonance and magnetic circular dichroism studies of a hexa-heme nitrite reductase from Wolinella succinogenes

The nature of the heme centers in the hexa-heme dissimilatory nitrite reductase from the bacterium Wolinella succinogenes has been investigated with EPR and magnetic circular dichroism spectroscopy. The EPR spectrum of the ferric enzyme is complex showing, in addition to magnetically isolated low-spin ferric hemes with g values of 2.93, 2.3 and 1.48, two sets of signals at g = 10.3, 3.7 and 4.8, 3.21, which we assign to two pairs of exchange coupled hemes. The MCD spectra show that the isolated hemes are bis-histidine coordinated and that there is one high-spin ferric heme. The exchange coupling is lost on treatment with SDS.     

Spectroscopic evidence for changes in the redox state of the nitrogenase P-cluster during turnover

Biological nitrogen fixation catalyzed by nitrogenase requires the participation of two component proteins called the Fe protein and the MoFe protein. Each alphabeta catalytic unit of the MoFe protein contains an [8Fe-7S] cluster and a [7Fe-9S-Mo-homocitrate] cluster, respectively designated the P-cluster and FeMo-cofactor. FeMo-cofactor is known to provide the site of substrate reduction whereas the P-cluster has been suggested to function in nitrogenase catalysis by providing an intermediate electron-transfer site. In the present work, evidence is presented for redox changes of the P-cluster during the nitrogenase catalytic cycle from examination of an altered MoFe protein that has the beta-subunit serine-188 residue substituted by cysteine. This residue was targeted for substitution because it provides a reversible redox-dependent ligand to one of the P-cluster Fe atoms. The altered beta-188(Cys) MoFe protein was found to reduce protons, acetylene, and nitrogen at rates approximately 30% of that supported by the wild-type MoFe protein. In the dithionite-reduced state, the beta-188(Cys) MoFe protein exhibited unusual electron paramagnetic resonance (EPR) signals arising from a mixed spin state system (S = 5/2, 1/2) that integrated to 0.6 spin/alphabeta-unit. These EPR signals were assigned to the P-cluster because they were also present in an apo-form of the beta-188(Cys) MoFe protein that does not contain FeMo-cofactor. Mediated voltammetry was used to show that the intensity of the EPR signals was maximal near -475 mV at pH 8.0 and that the P-cluster could be reversibly oxidized or reduced with concomitant loss in intensity of the EPR signals. A midpoint potential (Em) of -390 mV was approximated for the oxidized/resting state couple at pH 8.0, which was observed to be pH dependent. Finally, the EPR signals exhibited by the beta-188(Cys) MoFe protein greatly diminished in intensity under nitrogenase turnover conditions and reappeared to the original intensity when the MoFe protein returned to the resting state.     

Aldehyde oxidoreductase activity in Desulfovibrio alaskensis NCIMB 13491 EPR assignment of the proximal [2Fe-2S] cluster to the Mo site.

A novel molybdenum iron-sulfur-containing aldehyde oxidoreductase (AOR) belonging to the xanthine oxidase family was isolated and characterized from the sulfate-reducing bacterium Desulfovibrio alaskensis NCIMB 13491, a strain isolated from a soured oil reservoir in Purdu Bay, Alaska. D. alaskensis AOR is closely related to other AORs isolated from the Desulfovibrio genus. The protein is a 97-kDa homodimer, with 0.6 +/- 0.1 Mo, 3.6 +/- 0.1 Fe and 0.9 +/- 0.1 pterin cytosine dinucleotides per monomer. The enzyme catalyses the oxidation of aldehydes to their carboxylic acid form, following simple Michaelis-Menten kinetics, with the following parameters (for benzaldehyde): K(app/m)= 6.65 microM; V app = 13.12 microM.min(-1); k(app/cat) = 0.96 s(-1). Three different EPR signals were recorded upon long reduction of the protein with excess dithionite: an almost axial signal split by hyperfine interaction with one proton associated with Mo(V) species and two rhombic signals with EPR parameters and relaxation behavior typical of [2Fe-2S] clusters termed Fe/S I and Fe/S II, respectively. EPR results reveal the existence of magnetic interactions between Mo(V) and one of the Fe/S clusters, as well as between the two Fe/S clusters. Redox titration monitored by EPR yielded midpoint redox potentials of -275 and -325 mV for the Fe/S I and Fe/S II, respectively. The redox potential gap between the two clusters is large enough to obtain differentiated populations of these paramagnetic centers. This fact, together with the observed interactions among paramagnetic centers, was used to assign the EPR-distinguishable Fe/S I and Fe/S II to those seen in the reported crystal structures of homologous enzymes.     

Spectral resolution of the split EPR signals induced by illumination at 5 K from the S1, S3, and S0 states in photosystem II

S-State-dependent split EPR signals that are induced by illumination at cryogenic temperatures (5 K) have been measured in spinach photosystem II without interference from the Y(D)* radical in the g approximately 2 region. This allows us to present the first decay-associated spectra for the split signals, which originate from the CaMn4 cluster in magnetic interaction with a nearby radical, presumably Y(Z)*. The three split EPR signals that were investigated, "Split S1", "Split S3", and Split S0", all exhibit spectral features at g approximately 2.0 together with surrounding characteristic peaks and troughs. From microwave relaxation studies we can reach conclusions about which parts of the complex spectra belong together. Our analysis strongly indicates that the wings and the middle part of the split spectrum are parts of the same signal, since their decay kinetics in the dark at 5 K and microwave relaxation behavior are indistinguishable. In addition, our decay-associated spectra indicate that the g approximately 2.0 part of the "Split S1" EPR spectrum contains a contribution from magnetically uncoupled Y(Z)* as judged from the g value and 22 G line width of the EPR signal. The g value, 2.0033-2.0040, suggests that the oxidation of Y(Z) at 5 K results in a partially protonated radical. Irrespective of the S state, a small amount of a carotenoid or chlorophyll radical was formed by the illumination. However, this had relaxation and decay characteristics that clearly distinguish this radical from the split signal spectra. In this paper, we present the "clean" spectra from the low-temperature illumination-induced split EPR signals from higher plants, which will provide the basis for further simulation studies.     

Evidence for participation of iron in lipoxygenase reaction from optical and electron spin resonance studies.

Optical and EPR studies indicate that the iron present in lipoxygenase participates in catalysis. Addition of linoleic acid hydroperoxide to lipoxygenase 1 causes an increase in abosrbance over the range of 350 to 650 nm which is reversed when linoleic acid hydroperoxide is destroyed upon the addition of linoleic acid under anaerobic conditions. Lipoxygenase 1 alone exhibits no EPR signal but upon addition of linoleic acid hydroperoxide or linoleic acid several signals appear. Addition of linoleic acid hydroperoxide results in an EPR signal at g approximately equal to 6 accompanied by a small but relatively sharp signal at g approximately equal to 2. Under anaerobic conditions the latter is replaced by a broad anisotropic signal around g approximately equal to 2. The appearance of the EPR signal at g approximately equal to 6 coincides with the change in the optical spectrum of the enzyme. When linoleic acid is added under anaerobic conditions a broad anisotropic EPR signal around g approximately equal to 2 is observed. Thus it appears that lipoxygenase can exist in two forms: (a) a resting form with a very weak absorbance in the visible range of the light spectrum and no EPR signal and (b) an active form (after addition of linoleic acid hydroperoxide) with an increased optical absorbance and EPR signal at g approximately equal to 6. This observation may be related to the earlier discovery that the lipoxygenase reaction occurs with a lag which can be overcome by addition of product hydroperoxide. The EPR experiments indicate that lipoxygenase in the active form contains high spin ferric ion. Although EPR signals in the g approximately equal to 6 region are frequently observed with heme proteins, the only nonheme protein, other than lipoxygenase, reported to show an EPR signal in this region is the phenolytic dioxygenase, protocatechuate 3,4-dioxygenase (Peisach, J., Fujisawa, H., Blumberg, W. E., and Hayaishi, O. (1972) Fed. Proc. 31, 448).