Broad EPR Signal Research Articles

Reaction of ferrous cytochrome c peroxidase with dioxygen: site-directed mutagenesis provides evidence for rapid reduction of dioxygen by intramolecular electron transfer from the compound I radical site.

The reaction of dioxygen with the ferrous forms of the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was studied by photolysis of the respective ferrous-CO complexes in the presence of dioxygen. Reaction of ferrous CCP(MI) with dioxygen transiently formed a FeII-O2 complex (bimolecular rate constant = (3.8 +/- 0.3) x 10(4) M-1 s-1 at pH 6.0; 23 degrees C) that reacted further (first-order rate constant = 4 +/- 1 s-1) to form a product with an absorption spectrum and an EPR radical signal at g = 2.00 that were identical to those of compound I formed by the reaction of CCP(MI)III with peroxide. Thus, the product of the reaction of CCP(MI)II with dioxygen retained three of the four oxidizing equivalents of dioxygen. Gel electrophoresis of the CCP(MI)II + dioxygen reaction products showed that covalent dimeric and trimeric forms of CCP(MI) were produced by the reaction of CCP(MI)II with dioxygen. Photolysis of the CCP(MI)II-CO complex in the presence of ferrous cytochrome c prevented the appearance of the cross-linked forms and resulted in the oxidation of 3 mol of cytochrome c/mol of CCP(MI)II-CO added. The results provide evidence that reaction of CCP(MI)II with dioxygen causes transient oxidation of the enzyme by 1 equiv above the normal compound I oxidation state. Mutations that eliminate the broad EPR signal at g = 2.00 characteristic of the compound I radical also prevented the rapid oxidation of the ferrous enzyme by dioxygen.(ABSTRACT TRUNCATED AT 250 WORDS)

INVESTIGATION OF THE SAMARIUM ROLE IN THE PHYSICAL PROPERTIES OF Sm0.5Re0.5Ba2Cu3O7−δ COMPOUNDS IN THE ORTHORHOMBIC AND TETRAGONAL STRUCTURES

Perovskites of type Sm 0.5 Re 0.5 Ba 2 Cu 3 O 7−δ (Re=Y, La, Nd, Eu, Gd, Dy, Ho, Er, Tm, Yb, and Lu) have been investigated by XRD, magnetic and EPR measurements in both orthorhombic and tetragonal phases. In the former case the usual superconducting phase transition in the range 90 to 95 K has been detected. In all the compounds an EPR spectrum of Cu 2+ ions has been observed. In the tetragonal phase, the EPR spectra of trivalent rare earth ions have been recorded for some members of the series. The observation of a broad EPR signal, tentatively attributed to superexchange interaction over the oxygen bridges < O 2), has also been recorded. Furthermore, the intensity of low-field microwave absorption has been found to depend strongly on the nature of the substituted rare earth ion at the sites of Y ions of the YBa 2 Cu 3 O 7−δ type compounds.

EPR characterization of the cytochrome b- c1 complex from Rhodobacter sphaeroides

EPR characteristics of cytochrome c 1, cytochromes b-565 and b-562, the iron-sulfur cluster, and an antimycin-sensitive ubisemiquinone radical of purified cytochrome b- c 1 complex of Rhodobacter sphaeroides have been studied. The EPR specra of cytochrome c 1 shows a signal at g = 3.36 flanked with shoulders. The oxidized form of cytochrome b-562 shows a broad EPR signal at g = 3.49, while oxidized cytochrome b-565 shows a signal at g = 3.76, similar to those of two b cytochromes in the mitochondrial complex. The distribution of cytochromes b-565 and b-562 in the isolated complex is 44 and 56%, respectively. Antimycin and 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone (DBMIB) have little effect on the g = 3.76 signal, but they cause a slight downfield and upfield shifts of the g = 3.49 signal, respectively. 5-Undecyl-6-hydroxyl-4,7-dioxobenzothiazole (UHDBT) shifts the g = 3.49 signal downfield to g = 3.56 and sharpens the g = 3.76 signal slightly. Myxothiazol causes an upfield shift of both g = 3.49 and g = 3.76 signals. EPR characteristics of the reduced iron-sulfur cluster in bacterial cytochrome b- c 1 complex are: g x = 1.8 with a small shoulder at g = 1.76, g y = 1.89 and g z = 2.02, similar to those observed with the mitochondrial enzyme. The g x = 1.8 signal decreased and the shoulder increased concurrently as the redox potential decreased, indicating that the environment of the iron-sulfur cluster is sensitive to the redox state of the complex. UHDBT sharpens the g z and and shifts it downfield from g = 2.02 to 2.03, and shifts g x upfield from g = 1.80 to 1.78. UHDBT also causes an upfield shift of g y but to a much lesser extent compared to the other two signals. Addition of DBMIB causes a downfield shift of the g y from 1.89 to 1.94 and broadens the g x signal with an upfield to g = 1.75. Myxothiazol and antimycin show little effect on the g y and g z signals, but they broaden and shift the g x signal upfield to g = 1.74. However, the myxothiazol effect is partially reversed by UHDBT. An antimycin-sensitive ubisemiquinone radical was detected in the cytochrome b- c 1 complex. At pH 8.4, the antimycin-sensitive ubisemiquinone radical has a maximal concentration of 0.66 mol per mol complex at 100 mV. The concentration of antimycin sensitive Q-radical correlates well with the enzymatic activity of the complex. The antimycin-sensitive ubisemiquinone radical exhibits a pH dependency of approx. 60 mV / pH unit, between pH 8.0 and 9.0, suggesting the possible involvement of a dissociable group, with a pK a between 8.0 and 9.0, in Q-binding.

Redox Intermediates of Desulfovibrio gigas [NiFe] Hydrogenase Generated Under Hydrogen: MÖssbauer and epr characterization of the metal centers

The hydrogenase (EC 1.2.2.1) of Desulfovibrio gigas is a complex enzyme containing one nickel center, one [3Fe-4S] and two [4Fe-4S] clusters. Redox intermediates of this enzyme were generated under hydrogen (the natural substrate) using a redox-titration technique and were studied by EPR and Mossbauer spectroscopy. In the oxidized states, the two [4Fe-4S]2+ clusters exhibit a broad quadrupole doublet with parameters (apparent delta EQ = 1.10 mm/s and delta = 0.35 mm/s) typical for this type of cluster. Upon reduction, the two [4Fe-4S]1+ clusters are spectroscopically distinguishable, allowing the determination of their midpoint redox potentials. The cluster with higher midpoint potential (-290 +/- 20 mV) was labeled Fe-S center I and the other with lower potential (-340 +/- 20 mV), Fe-S center II. Both reduced clusters show atypical magnetic hyperfine coupling constants, suggesting structural differences from the clusters of bacterial ferredoxins. Also, an unusually broad EPR signal, labeled Fe-S signal B', extending from approximately 150 to approximately 450 mT was observed concomitantly with the reduction of the [4Fe-4S] clusters. The following two EPR signals observed at the weak-field region were tentatively attributed to the reduced [3Fe-4S] cluster: (i) a signal with crossover point at g approximately 12, labeled the g = 12 signal, and (ii) a broad signal at the very weak-field region (approximately 3 mT), labeled the Fe-S signal B. The midpoint redox potential associated with the appearance of the g = 12 signal was determined to be -70 +/- 10 mV. At potentials below -250 mV, the g = 12 signal began to decrease in intensity, and simultaneously, the Fe-S signal B appeared. The transformation of the g = 12 signal into the Fe-S signal B was found to parallel the reduction of the two [4Fe-4S] clusters indicating that the [3Fe-4S]o cluster is sensitive to the redox state of the [4Fe-4S] clusters. Detailed redox profiles for the previously reported Ni-signal C and the g = 2.21 signal were obtained in this study, and evidence was found to indicate that these two signals represent two different oxidation states of the enzyme. Finally, the mechanistic implications of our results are discussed.

EPR and Mössbauer 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.

Correlation of Zero Field Splittings and Site Distortions V. Mn2+ in Cs2Zn3S4

Abstract In platelets of Cs2Zn3S4 with about 4% of the Zn substituted by Mn two nonequivalent centers of isolated Mn2+ were observed in addition to a broad EPR signal near g = 2 which is assigned to clusters of interconnected MnS4 units. The fine structure and hyperfine structure parameters for the single-ion centers (all in units of 1CT 4 cm -1) are b0 2 = -318.3 ± 3; b2 2 = -210.3 ± 2; Ay = -62.7; Az = -63.6 ± 0.8; b0 2 = -890 ± 18; b2 2 = -743 ± 36; Ay = -60.6; Az = - 61.4 ± 0.8; for centers I and II, resp. Center II arises from Zn sites of C 2 site symmetry while for center I assignment to either one of two sites with D2 site symmetry is possible. The larger hyperfine splitting constants as well as the superposition analysis favor the larger sites which are not occupied by Zn, but are partly occupied in the analogous Mn (and Co) compounds. Superposition analysis yields the same value of + 0.12 ± 0.02 cm-1 for the intrinsic zero field splitting parameter b̄2 of the bridging MnS4 units in both sites. gz is significantly higher than the free ion value indicating a higher degree of covalency than in Cds and CdGa2S4.

Zero-field splitting of Fe3+ in horseradish peroxidase and of Fe4+ in horseradish peroxidase compound I from electron spin relaxation data

From the temperature dependence of the Orbach relaxation rate of the paramagnetic center in horseradish peroxidase (HRP), we deduce an excited-state energy of 40.9 +/- 1.1 K. Similar studies on the broad EPR signal of HRP compound I indicate a much weaker Orbach relaxation process involving an excited state at 36.8 +/- 2.5 K. The strength of the Orbach process in HRP-I is weaker than one would normally estimate by 2-4 orders of magnitude. This fact lends support to the model of HRP-I involving a spin 1/2 free radical coupled to a spin 1 Fe4+ heme iron via a weak exchange interaction. Such a system should exhibit an Orbach relaxation process involving delta E, the excited state of the Fe4+ ion, but reduced in strength by (Jyy/delta E)2, where Jyy is related to the strength of the exchange interaction between the two spin systems.

The detection of two electron paramagnetic resonance radical signals associated with chloroperoxidase compound I.

The electron paramagnetic resonance spectra of chloroperoxidase Compound I and native enzyme are compared. Upon the formation of Compound I, the g = 2.62, 2.26, and 1.82 signals associated with native enzyme disappear and are replaced by two new EPR signals, a sharp signal at g = 2.008 and a broad signal at g = 1.73. The g = 2.008 signal accounts for only 2% of the theoretical spins while the broad signal at g = 1.73 accounts for 60 to 70% of the theoretical spins in Compound I. The g = 1.73 broad signal is reminiscent of the broad EPR signal associated with horseradish peroxidase Compound I. however, the chloroperoxidase Compound I signal has a significantly different g value. The results suggest that the g = 1.73 signal represents a porphyrin pi cation radical which has a stronger coupling to the heme ferryl iron than is the case with horseradish peroxidase Compound I.

Nature of the radical formed during electrolytic reduction of 4-(2′-thienyl)quinazoline in dimethylformamide

EPR studies and HMO calculations are carried out in order to understand the nature of the electrolytically generated radical of 4(2′-thienyl)quinazoline (4-TQ) in dimethylformamide (DMF). Single broad EPR signal obtained is explained on the basis of theoretical hyperfine splitting constants. By correlating the experimental half-wave potentials and the colour of the radical with HMO energies, it is concluded that 4-TQ protonated at N1-position is getting reduced to give the neutral radical.

An EPR study of the lineshape of copper in cytochrome c oxidase

The EPR spectrum of copper in cytochrome c oxidase (EC 1.9.3.1) has been studied between 5 and 220°K, and the spectral parameters have been determined for both forms of EPR-detectable copper by computer simulation methods. Numerical methods have been developed to separate the spectra of intrinsic copper and inactive copper. Evidence is presented to show that inactive copper is probably formed by denaturation. The EPR parameters for intrinsic copper were determined as g x - 1.99, g y = 2.03, g z = 2.185, ∥ A x(Cu) ∥ = 0.0020cm −1, ∥ A y(Cu) ∥ = 0.0025cm −1, ∥ A z(Cu) ∥ = 0.0030cm −1. The principal values of the g tensor and the small value of ∥ A z(Cu) ∥ are interpreted in terms of mixing of 3d, 4s, and 4p metal orbitals. A flattened-tetrahedral stereochemistry about Cu 2+ with an additional rhombic distortion is in best agreement with all of the data. The peak-to-peak linewidth is found to be orientation dependent, and is described by a tensor with principal values ΔH x = 45G, ΔH y = 65G, ΔH z = 85G. A weak dipolar interaction with a low-spin ferric species about 7 Å distant is proposed to account for this anisotropy. The proposed site and stereochemistry for the copper ion is consistent with the electron transport function of the enzyme. Broad EPR signals with a very short spin-lattice relaxation time have been observed near g = 14and g = 3 at 5°K in oxidized cytochrome oxidase but not in the reduced or denatured enzyme. The possibility that these are due to the “EPR-undetectable” iron and copper is raised.

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).

Carotenoid chromophore length and protection against photosensitization.

Abstract— Carotenoid pigments were extracted and purified from wild‐type and mutants 7 and 93a of Sarcina lutea, and tested for their ability to quench 1O2. The wild‐type pigment (P‐438, 9 conjugated double bonds) is as active in quenching 1O2 as is β‐carotene. On the other hand, the pigment P‐422 (8 conjugated double bonds) from mutant 7 is 2 or 3 times less efficient, whereas phytofluene and phytoene from S. lutea are 100 and 1000 times less efficient, respectively, than is β‐carotene at quenching 1O2. It was also found that the broad EPR signal, induced by light in benzene solutions of chlorophyll a and hydroquinone, and related to chlorophyll oxidation, is efficiently quenched by P‐438 and to a much smaller extent also by Sarcina phytoene.