Photoaccumulation of the PsaB phyllosemiquinone in photosystem I of Chlamydomonas reinhardtii

Photoaccumulation of membrane preparations of Chlamydomonas reinhardtii at pH 8 and 220 K reduces the primary and secondary electron acceptors in the Photosystem I (PSI) reaction centre, and produces a maximum of two spins per P700(z.rad;+). Proton electron nuclear double resonance (ENDOR) spectra demonstrate that the phyllosemiquinone produced is that attributed to the PsaA branch of electron transfer. Photoaccumulation at pH 10 and 220 K produces a maximum of four spins per P700(z.rad;+), and proton ENDOR spectra indicate that a second phyllosemiquinone is being photoaccumulated, with markedly different proton hyperfine couplings (hfcs). This phyllosemiquinone is unaffected by mutation of PsaAW693, confirming that it does not arise from the PsaA branch of electron transfer, and we therefore attribute it to the PsaB phyllosemiquinone.     

Photoaccumulation of the PsaB phyllosemiquinone in Photosystem I of Chlamydomonas reinhardtii

Photoaccumulation of membrane preparations of Chlamydomonas reinhardtii at pH 8 and 220 K reduces the primary and secondary electron acceptors in the Photosystem I (PSI) reaction centre, and produces a maximum of two spins per P700⁺. Proton electron nuclear double resonance (ENDOR) spectra demonstrate that the phyllosemiquinone produced is that attributed to the PsaA branch of electron transfer. Photoaccumulation at pH 10 and 220 K produces a maximum of four spins per P700⁺, and proton ENDOR spectra indicate that a second phyllosemiquinone is being photoaccumulated, with markedly different proton hyperfine couplings (hfcs). This phyllosemiquinone is unaffected by mutation of PsaAW693, confirming that it does not arise from the PsaA branch of electron transfer, and we therefore attribute it to the PsaB phyllosemiquinone.     

Bidirectional electron transfer in photosystem I: electron transfer on the PsaA side is not essential for phototrophic growth in Chlamydomonas

We have used pulsed electron paramagnetic resonance (EPR) measurements of the electron spin polarised (ESP) signals arising from the geminate radical pair P700(z.rad;+)/A(1)(z.rad;-) to detect electron transfer on both the PsaA and PsaB branches of redox cofactors in the photosystem I (PSI) reaction centre of Chlamydomonas reinhardtii. We have also used electron nuclear double resonance (ENDOR) spectroscopy to monitor the electronic structure of the bound phyllosemiquinones on both the PsaA and PsaB polypeptides. Both these spectroscopic assays have been used to analyse the effects of site-directed mutations to the axial ligands of the primary chlorophyll electron acceptor(s) A(0) and the conserved tryptophan in the PsaB phylloquinone (A(1)) binding pocket. Substitution of histidine for the axial ligand methionine on the PsaA branch (PsaA-M684H) blocks electron transfer to the PsaA-branch phylloquinone, and blocks photoaccumulation of the PsaA-branch phyllosemiquinone. However, this does not prevent photoautotrophic growth, indicating that electron transfer via the PsaB branch must take place and is alone sufficient to support growth. The corresponding substitution on the PsaB branch (PsaB-M664H) blocks kinetic electron transfer to the PsaB phylloquinone at 100 K, but does not block the photoaccumulation of the phyllosemiquinone. This transformant is unable to grow photoautotrophically although PsaA-branch electron transfer to and from the phyllosemiquinone is functional, indicating that the B branch of electron transfer may be essential for photoautotrophic growth. Mutation of the conserved tryptophan PsaB-W673 to leucine affects the electronic structure of the PsaB phyllosemiquinone, and also prevents photoautotrophic growth.     

Density functional calculated spin densities and hyperfine couplings for hydrogen bonded 1,4-naphthosemiquinone and phyllosemiquinone anion radicals: a model for the A1 free radical formed in photosystem I

The B3LYP hybrid density functional method is used to calculate spin densities and hyperfine couplings for the 1,4-naphthosemiquinone anion radical and a model of the phyllosemiquinone anion radical. The effect of hydrogen bonding on the spin density distribution is shown to lead to a redistribution of pi spin density from the semiquinone carbonyl oxygens to the carbonyl carbon atoms. The effect of in plane and out of plane hydrogen bonding is examined. Out of plane hydrogen bonding is shown to give rise to a significant delocalisation of spin density on to the hydrogen bond donor heavy atom. Excellent agreement is observed between calculated and experimental hyperfine couplings. Comparison of calculated hyperfine couplings with experimental determinations for the A1 phyllosemiquinone anion radical present in Photosystem I (PS I) of higher plant photosynthesis indicates that the in vivo radical may have a hydrogen bond to the O4 atom only as opposed to hydrogen bonds to each oxygen atom in alcohol solvents. The hydrogen bonding situation appears to be the reverse of that observed for QA in the bacterial type II reaction centres where the strong hydrogen bond occurs to the quinone O1 oxygen atom. For different types of reaction centre the presence or absence of the non-heme Fe(II) atom may well determine which type of hydrogen bonding situation prevails at the primary quinone site which in turn may influence the direction of subsequent electron transfer.     

Electron spin echo envelope modulation spectroscopy in photosystem I.

The applications of electron spin echo envelope modulation (ESEEM) spectroscopy to study paramagnetic centers in photosystem I (PSI) are reviewed with special attention to the novel spectroscopic techniques applied and the structural information obtained. We briefly summarize the physical principles and experimental techniques of ESEEM, the spectral shapes and the methods for their analysis. In PSI, ESEEM spectroscopy has been used to the study of the cation radical form of the primary electron donor chlorophyll species, P(700)(+), and the phyllosemiquinone anion radical, A(1)(-), that acts as a low-potential electron carrier. For P(700)(+), ESEEM has contributed to a debate concerning whether the cation is localized on a one or two chlorophyll molecules. This debate is treated in detail and relevant data from other methods, particularly electron nuclear double resonance (ENDOR), are also discussed. It is concluded that the ESEEM and ENDOR data can be explained in terms of five distinct nitrogen couplings, four from the tetrapyrrole ring and a fifth from an axial ligand. Thus the ENDOR and ESEEM data can be fully accounted for based on the spin density being localized on a single chlorophyll molecule. This does not eliminate the possibility that some of the unpaired spin is shared with the other chlorophyll of P(700)(+); so far, however, no unambiguous evidence has been obtained from these electron paramagnetic resonance methods. The ESEEM of the phyllosemiquinone radical A(1)(-) provided the first evidence for a tryptophan molecule pi-stacked over the semiquinone and for a weaker interaction from an additional nitrogen nucleus. Recent site-directed mutagenesis studies verified the presence of the tryptophan close to A(1), while the recent crystal structure showed that the tryptophan was indeed pi-stacked and that a weak potential H-bond from an amide backbone to one of the (semi)quinone carbonyls is probably the origin of the to the second nitrogen coupling seen in the ESEEM. ESEEM has already played an important role in the structural characterization on PSI and since it specifically probes the radical forms of the chromophores and their protein environment, the information obtained is complimentary to the crystallography. ESEEM then will continue to provide structural information that is often unavailable using other methods.     

Recruitment of a foreign quinone into the A(1) site of photosystem I. II. Structural and functional characterization of phylloquinone biosynthetic pathway mutants by electron paramagnetic resonance and electron-nuclear double resonance spectroscopy

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance studies of the photosystem (PS) I quinone acceptor, A(1), in phylloquinone biosynthetic pathway mutants are described. Room temperature continuous wave EPR measurements at X-band of whole cells of menA and menB interruption mutants show a transient reduction and oxidation of an organic radical with a g-value and anisotropy characteristic of a quinone. In PS I complexes, the continuous wave EPR spectrum of the photoaccumulated Q(-) radical, measured at Q-band, and the electron spin-polarized transient EPR spectra of the radical pair P700(+) Q(-), measured at X-, Q-, and W-bands, show three prominent features: (i) Q(-) has a larger g-anisotropy than native phylloquinone, (ii) Q(-) does not display the prominent methyl hyperfine couplings attributed to the 2-methyl group of phylloquinone, and (iii) the orientation of Q(-) in the A(1) site as derived from the spin polarization is that of native phylloquinone in the wild type. Electron spin echo modulation experiments on P700(+) Q(-) show that the dipolar coupling in the radical pair is the same as in native PS I, i.e. the distance between P700(+) and Q(-) (25.3 +/- 0.3 A) is the same as between P700(+) and A(1)(-) in the wild type. Pulsed electron-nuclear double resonance studies show two sets of resolved spectral features with nearly axially symmetric hyperfine couplings. They are tentatively assigned to the two methyl groups of the recruited plastoquinone-9, and their difference indicates a strong inequivalence among the two groups when in the A(1) site. These results show that Q (i) functions in accepting an electron from A(0)(-) and in passing the electron forward to the iron-sulfur clusters, (ii) occupies the A(1) site with an orientation similar to that of phylloquinone in the wild type, and (iii) has spectroscopic properties consistent with its identity as plastoquinone-9.     

EPR, ENDOR, and TRIPLE resonance spectroscopy on the neutral flavin radical in Escherichia coli DNA photolyase

Ultraviolet radiation promotes the formation of a cyclobutane ring between adjacent pyrimidine residues on the same DNA strand to form a pyrimidine dimer. Such dimers may be restored to their monomeric forms through the action of a light-absorbing enzyme named DNA photolyase. The redox-active cofactor involved in the light-induced electron transfer reactions of DNA repair and enzyme photoactivation is a noncovalently bound FAD. In this paper, the FAD cofactor of Escherichia coli DNA photolyase was characterized as the neutral flavin semiquinone by EPR spectroscopy at 9.68 and 94.5 GHz. From the high-frequency/high-field EPR spectrum, the principal values of the axially symmetric g-matrix of FADH(*) were extracted. Both EPR spectra show an emerging hyperfine splitting of 0.85 mT that could be assigned to the isotropic hyperfine coupling constant (hfc) of the proton at N(5). To obtain more information about the electron spin density distribution ENDOR and TRIPLE resonance spectroscopies were applied. All major proton hfc's could be measured and unambiguously assigned to molecular positions at the isoalloxazin moiety of FAD. The isotropic hfc's of the protons at C(8alpha) and C(6) are among the smallest values reported for protein-bound neutral flavin semiquinones so far, suggesting a highly restricted delocalization of the unpaired electron spin on the isoalloxazin moiety. Two further hfc's have been detected and assigned to the inequivalent protons at C(1'). Some conclusions about the geometrical arrangement of the ribityl side chain with respect to the isoalloxazin ring could be drawn: Assuming tetrahedral angles at C(1') the dihedral angle between the C(1')-C(2') bond and the 2p(z)() orbital at N(10) has been estimated to be 170.4 degrees +/- 1 degrees.     

Correlating EPR and X-ray structural analysis of arsenite-inhibited forms of aldehyde oxidoreductase

Two arsenite-inhibited forms of each of the aldehyde oxidoreductases from Desulfovibrio gigas and Desulfovibrio desulfuricans have been studied by X-ray crystallography and electron paramagnetic resonance (EPR) spectroscopy. The molybdenum site of these enzymes shows a distorted square-pyramidal geometry in which two ligands, a hydroxyl/water molecule (the catalytic labile site) and a sulfido ligand, have been shown to be essential for catalysis. Arsenite addition to active as-prepared enzyme or to a reduced desulfo form yields two different species called A and B, respectively, which show different Mo(V) EPR signals. Both EPR signals show strong hyperfine and quadrupolar couplings with an arsenic nucleus, which suggests that arsenic interacts with molybdenum through an equatorial ligand. X-ray data of single crystals prepared from EPR-active samples show in both inhibited forms that the arsenic atom interacts with the molybdenum ion through an oxygen atom at the catalytic labile site and that the sulfido ligand is no longer present. EPR and X-ray data indicate that the main difference between both species is an equatorial ligand to molybdenum which was determined to be an oxo ligand in species A and a hydroxyl/water ligand in species B. The conclusion that the sulfido ligand is not essential to determine the EPR properties in both Mo–As complexes is achieved through EPR measurements on a substantial number of randomly oriented chemically reduced crystals immediately followed by X-ray studies on one of those crystals. EPR saturation studies show that the electron transfer pathway, which is essential for catalysis, is not modified upon inhibition. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

EPR/ENDOR characterization of the physical and electronic structure of the OEC Mn cluster

Electron paramagnetic resonance (EPR) spectroscopy has often played a crucial role in characterizing the various cofactors and processes of photosynthesis, and photosystem II and its oxygen evolving chemistry is no exception. Until recently, the application of EPR spectroscopy to the characterization of the oxygen evolving complex (OEC) has been limited to the S2-state of the Kok cycle. However, in the past few years, continuous wave-EPR signals have been obtained for both the S0- and S1-state as well as for the S2 (radical)(Z)-state of a number of inhibited systems. Furthermore, the pulsed EPR technique of electron spin echo electron nuclear double resonance spectroscopy has been used to directly probe the 55Mn nuclei of the manganese cluster. In this review, we discuss how the EPR data obtained from each of these states of the OEC Kok cycle are being used to provide insight into the physical and electronic structure of the manganese cluster and its interaction with the key tyrosine, Y(Z).     

Further characterisation of the FAD and Fe2S2 redox centres of component C, the NADH:acceptor reductase of the soluble methane monooxygenase of Methylococcus capsulatus (Bath)

The absorbance contributions of the FAD and Fe2S2 redox centres of component C of the soluble methane monooxygenase complex have been resolved, using mersalyl to destroy the Fe2S2 centre. The Fe2S2 seems to be very similar to that of spinach ferredoxin, by its absorbance and electron paramagnetic resonance (EPR) spectra, and the FAD semiquinone is a neutral semiquinone. Spectrophotometry near room temperature and EPR spectroscopy near liquid-helium temperature allow the three redox couples of component C to be ordered. Component C can exist in Oe-1 (oxidised), 1e-1 (semiquinone), 2e-1 (mostly semiquinone and reduced Fe2S2), and 3e-1 forms (dihydroquinone and reduced Fe2S2), under equilibrium conditions. The ability of component C to support odd-electron forms is consistent with its proposed role as a 2e-1/1e-1 transformase, splitting electron pairs from NADH for passage to component A in one-electron steps. (The FAD appears to interact with NADH, and transfers single electrons to the Fe2S2, for donation to component A at a constant redox potential.) The mid-point potentials of component C were found using redox dyes and EPR spectroscopy and were: FAD/FAD., Em = -150 mV; Fe2S2/Fe2.S2,Em = -220 mV; FAD./FAD..,Em = -260 mV. the presence of NADH did not alter these mid-point potentials. These mid-point potentials are consistent with the role of component C as the NADH:component A reductase, passing electrons from NADH (Em = -320 mV) onto component A (Em = +150 mV and Em = -150 mV). The reducing power from NADH appears to be required by component A to activate one atom of oxygen, to insert into methane, and the reducing equivalents derived from NADH end up with the other oxygen atom, as water.     

Evidence for an EPR-detectable semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase A (NarGHI) from Escherichia coli

Nitrate reductase A (NRA, NarGHI) is expressed in Escherichia coli by growing the bacterium in anaerobic conditions in the presence of nitrate. This enzyme reduces nitrate to nitrite and uses menaquinol (or ubiquinol) as the electron donor. The location of quinones in the enzyme, their number, and their role in the electron transfer mechanism are still controversial. In this work, we have investigated the spectroscopic and thermodynamic properties of a semiquinone (SQ) in membrane samples of overexpressed E. coli nitrate reductase poised in appropriate redox conditions. This semiquinone is highly stabilized with respect to free semiquinone. The g-values determined from the numerical simulation of its Q-band (35 GHz) EPR spectrum are equal to 2.0061, 2.0051, 2.0023. The midpoint potential of the Q/QH(2) couple is about -100 mV, and the SQ stability constant is about 100 at pH 7.5. The semiquinone EPR signal disappears completely upon addition of the quinol binding site inhibitor 2-n-nonyl-4-hydroxyquinoline N-oxide (NQNO). A semiquinone radical could also be stabilized in preparations where only the NarI membrane subunit is overexpressed in the absence of the NarGH catalytic dimer. Its thermodynamic and spectroscopic properties show only slight variations with those of the wild-type enzyme. The X-band continuous wave (cw) electron nuclear double resonance (ENDOR) spectra of the radicals display similar proton hyperfine coupling patterns in NarGHI and in NarI, showing that they arise from the same semiquinone species bound to a single site located in the NarI membrane subunit. These results are discussed with regard to the location and the potential function of quinones in the enzyme.     

Studies by electron paramagnetic resonance spectroscopy of xanthine oxidase enriched with molybdenum-95 and with molybdenum-97

Investigations have been carried out on the nature of the species from the enzyme xanthine oxidase that give rise to two molybdenum (V) electron paramagnetic resonance (EPR) signals. Isotopic enrichment with 95Mo, 97Mo, 33S, and 17O was employed. Computer simulations of the EPR spectra recorded at 9- and 35-GHz microwave frequencies were used to evaluate the various hyperfine couplings and angular relations between the principal axes of g and A, as well as the nuclear electric quadrupole interaction for 97Mo. The results support the presence of an oxo ligand in the Rapid and of both an oxo and a sulfido ligand in the Very Rapid signal-giving species.     

Reaction of bovine cytochrome c oxidase with hydrogen peroxide produces a tryptophan cation radical and a porphyrin cation radical

Oxidized bovine cytochrome c oxidase reacts with hydrogen peroxide to generate two electron paramagnetic resonance (EPR) free radical signals (Fabian, M., and Palmer, G. (1995) Biochemistry 34, 13802-13810). These radicals are associated with the binuclear center and give rise to two overlapped EPR signals, one signal being narrower in line width (DeltaHptp = 12 G) than the other (DeltaHptp = 45 G). We have used electron nuclear double resonance (ENDOR) spectrometry to identify the two different chemical species giving rise to these two EPR signals. Comparison of the ENDOR spectrum associated with the narrow signal with that of compound I of horseradish peroxidase (formed by reaction of that enzyme with hydrogen peroxide) demonstrates that the two species are virtually identical. The chemical species giving rise to the narrow signal is therefore identified as an exchange-coupled porphyrin cation radical similar to that formed in horseradish peroxidase compound I. Comparison of the ENDOR spectrum of compound ES (formed by the reaction of hydrogen peroxide with cytochrome c peroxidase) with that of the broad signal indicates that the chemical species giving rise to the broad EPR signal in cytochrome c oxidase is probably an exchange coupled tryptophan cation radical. This is substantiated using H(2)O/D(2)O solvent exchange experiments where the ENDOR difference spectrum of the broad EPR signal of cytochrome c oxidase shows a feature consistent with hyperfine coupling to the exchangeable N(1) proton of a tryptophan cation radical.     

Semiquinone and ascorbyl radicals in the gut fluids of caterpillars measured with EPR spectrometry

The biological activity of phenolic compounds ingested by caterpillars is commonly believed to result from their oxidation, although the products of oxidation have been well-characterized in only a few cases. The initial oxidation products of phenols (semiquinone or phenoxyl radicals) can be measured with electron paramagnetic resonance (EPR) spectrometry. In this study semiquinone radicals formed from tannic acid and gallic acid in the gut fluids of two species of caterpillars were measured. In Orgyia leucostigma, in which ingested phenols are not oxidized, semiquinone radicals were absent or at very low intensities. By contrast, in Malacosoma disstria, in which ingested phenols are oxidized, high semiquinone radical intensities were measured. In the absence of detectable levels of semiquinone radicals, ascorbyl radicals were detected in the EPR spectra instead. High molar ratios of ascorbate to phenols in an artificial diet produced ascorbyl radicals in the midgut fluids of both species, while diets containing low molar ratios produced semiquinone radicals. Similar results were obtained in M. disstria fed the leaves of red oak or sugar maple. The results of this study provide further evidence that ascorbate is an essential antioxidant that prevents the oxidation of phenols in the gut fluids of caterpillars, and demonstrate that EPR spectrometry is a valuable method for determining the degree of oxidative activation of phenols ingested by herbivorous insects.     

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.     

Exposing the complex III Qo semiquinone radical

Complex III Qo site semiquinone has been assigned pivotal roles in productive energy-conversion and destructive superoxide generation. After a 30-year search, a genetic heme bH knockout arrests this transient semiquinone EPR radical, revealing the natural engineering balance pitting energy-conserving, short-circuit minimizing, split electron transfer and catalytic speed against damaging oxygen reduction.     

Binding of amines to the O2-evolving center of photosystem II

The binding of several primary amines to the O2-evolving center (OEC) of photosystem II (PSII) has been studied by using low-temperature electron paramagnetic resonance (EPR) spectroscopy of the S2 state. Spinach PSII membranes treated with NH4Cl at pH 7.5 produce a novel S2-state multiline EPR spectrum with a 67.5-G hyperfine line spacing when the S2 state is produced by illumination at 0 degrees C [Beck, W. F., de Paula, J. C., & Brudvig, G. W. (1986) J. Am. Chem. Soc. 108, 4018-4022]. The altered hyperfine line spacing and temperature dependence of the S2-state multiline EPR signal observed in the presence of NH4Cl are direct spectroscopic evidence for coordination of one or more NH3 molecules to the Mn site in the OEC. In contrast, the hyperfine line pattern and temperature dependence of the S2-state multiline EPR spectrum in the presence of tris(hydroxymethyl)aminomethane, 2-amino-2-ethyl-1,3-propanediol, or CH3NH2 at pH 7.5 were the same as those observed in untreated PSII membranes. We conclude that amines other than NH3 do not readily bind to the Mn site in the S2 state because of steric factors. Further, NH3 binds to an additional site on the OEC, not necessarily located on Mn, and alters the stability of the S2-state g = 4.1 EPR signal species. The effects on the intensities of the g = 4.1 and multiline EPR signals as the NH3 concentration was varied indicate that both EPR signals arise from the same paramagnetic site and that binding of NH3 to the OEC affects an equilibrium between two configurations exhibiting the different EPR signals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen permeation profile in lipid membranes: comparison with transmembrane polarity profile

Permeation of oxygen into membranes is relevant not only to physiological function, but also to depth determinations in membranes by site-directed spin labeling. Spin-lattice (T(1)) relaxation enhancements by air or molecular oxygen were determined for phosphatidylcholines spin labeled at positions (n = 4-14, 16) of the sn-2 chain in fluid membranes of dimyristoyl phosphatidylcholine, by using nonlinear continuous-wave electron paramagnetic resonance (EPR). Both progressive saturation and out-of-phase continuous-wave EPR measurements yield similar oxygen permeation profiles. With pure oxygen, the T(2)-relaxation enhancements determined from homogeneous linewidths of the linear EPR spectra are equal to the T(1)-relaxation enhancements determined by nonlinear EPR. This confirms that both relaxation enhancements occur by Heisenberg exchange, which requires direct contact between oxygen and spin label. Oxygen concentrates in the hydrophobic interior of phospholipid bilayer membranes with a sigmoidal permeation profile that is the inverse of the polarity profile established earlier for these spin-labeled lipids. The shape of the oxygen permeation profile in fluid lipid membranes is controlled partly by the penetration of water, via the transmembrane polarity profile. At the protein interface of the KcsA ion channel, the oxygen profile is more diffuse than that in fluid lipid bilayers.     

Histidinyl radical formation in the self-peroxidation reaction of bovine copper-zinc superoxide dismutase.

In the absence of suitable oxidizable substrates, the peroxidase reaction of copper-zinc superoxide dismutase (SOD) oxidizes SOD itself, ultimately resulting in its inactivation. A SOD-centered free radical adduct of 2-methyl-2-nitrosopropane (MNP) was detected upon incubation of SOD with the spin trap and a hydroperoxide (either H(2)O(2) or peracetic acid). Proteolysis by Pronase converted the anisotropic electron paramagnetic resonance (EPR) spectrum of MNP/(center dot)SOD to a nearly isotropic spectrum with resolved hyperfine couplings to several atoms with non-zero nuclear spin. Authentic histidinyl radical (from histidine + HO(center dot)) formed a MNP adduct with a very similar EPR spectrum to that of the Pronase-treated MNP/(center dot)SOD, suggesting that the latter was centered on a histidine residue. An additional hyperfine coupling was detected when histidine specifically (13)C-labeled at C-2 of the imidazole ring was used, providing evidence for trapping at that atom. All of the experimental spectra were convincingly simulated assuming hyperfine couplings to 2 nearly equivalent nitrogen atoms and 2 different protons, also consistent with trapping at C-2 of the imidazole ring. Free histidinyl radical consumed oxygen, implying peroxyl radical formation. MNP-inhibitable oxygen consumption was also observed when cuprous SOD but not cupric SOD was added to a H(2)O(2) solution. Formation of 2-oxohistidine, the stable product of the SOD-hydroperoxide reaction, required oxygen and was inhibited by MNP. These results support formation of a transient SOD-peroxyl radical.