The EPR spectrum of tyrosine Z* and its decay kinetics in O2-evolving photosystem II preparations

The O2-evolving complex of photosystem II, Mn 4Ca, cycles through five oxidation states, S0,..., S4, during its catalytic function, which involves the gradual abstraction of four electrons and four protons from two bound water molecules. The direct oxidant of the complex is the tyrosine neutral radical, YZ(*), which is transiently produced by the highly oxidizing power of the photoexcited chlorophyll species P680. EPR characterization of YZ(*) has been limited, until recently, to inhibited (non-oxygen-evolving) preparations. A number of relatively recent papers have demonstrated the trapping of YZ(*) in O2-evolving preparations at liquid helium temperatures as an intermediate of the S0 to S1, S1 to S2, and S2 to S3 transitions. The respective EPR spectra are broadened and split at g approximately 2 by the magnetic interaction with the Mn cluster, but this interaction collapses at temperatures higher than about 100K [Zahariou et al. (2007) Biochemistry 46, 14335 -14341]. We have conducted a study of the Tyr Z(*) transient in the temperature range 77-240 K by employing rapid or slow EPR scans. The results reveal for the first time high-resolution X-band spectra of Tyr Z(*) in the functional system and at temperatures close to the onset of the S-state transitions. We have simulated the S 2Y Z(*) spectrum using the simulation algorithm of Svistunenko and Cooper [(2004) Biophys. J. 87, 582 -595]. The small g(x) = 2.00689 value inferred from the analysis suggests either a H-bonding of Tyr Z (*) (presumably with His190) that is stronger than what has been assumed from studies of Tyr D(*) or Tyr Z(*) in Mn-depleted preparations or a more electropositive environment around Tyr Z(*). The study has also yielded for the first time direct information on the temperature variation of the YZ(*)/QA(-) recombination reaction in the various S states. The reaction follows biphasic kinetics with the slow phase dominating at low temperatures and the fast phase dominating at high temperatures. It is tentatively proposed that the slow phase represents the action of the YZ(*)/YZ(-) redox couple while the fast phase represents that of the YZ(*)/YZH couple; it is inferred that Tyr Z at elevated temperatures is protonated at rest. It is also proposed that YZ(*)/YZH is the couple that oxidizes the Mn cluster during the S1-S2 and S2-S3 transitions. A simple mechanism ensuring a rapid (concerted) protonation of Tyr Z upon oxidation of the Mn cluster is discussed, and also, a structure-based molecular model suggesting the participation of His190 into two hydrogen bonds is proposed.     

Cyclodecapeptides to mimic the radical site of tyrosyl-containing proteins.

Tyrosyl radicals are involved in many biologically important processes. The development of model compounds to mimic radical enzyme active sites, such as galactose oxidase (GO), has widely contributed to an enhanced understanding of their spectral properties, structural attributes and even reactivity. An emerging approach towards the synthesis of such active site mimetics is the use of peptidic ligands. The potential of cyclodecapeptides to bear phenoxyl radicals has been evaluated through three compounds. LH(4) (2+) is a cyclodecapetide containing two histidine residues (mimicking His(496) and His(581) of GO) and two tyrosine residues (mimicking Tyr(495) and the Tyr(272)* radical of GO). L(tBu)H(4) (2+) and L(OMe)H(4) (2+) incorporate 2,4,6-protected phenols in place of each tyrosine in LH(4) (2+). The deprotonation constants of each peptide determined by potentiometric titrations showed that there are some interactions between the acido-basic residues. Cyclic voltammetric studies revealed that only the peptides incorporating 2,4,6-protected phenolates exhibit reversible redox couples and are thus precursors of radicals stable enough to persist in solution. These studies also showed L(OMe2-) to possess the lower oxidation potential, indicating that this peptide, in its radical form, is the most stabilized. The electrochemically generated radical species have been characterized by EPR spectroscopy.     

Identification of hydrogen bonds to the Rieske cluster through the weakly coupled nitrogens detected by electron spin echo envelope modulation spectroscopy

The interaction of the reduced[2Fe-2S] cluster of isolated Rieske fragment from the bc1 complex of Rhodobacter sphaeroides with nitrogens (14N and 15N) from the local protein environment has been studied by X- and S-band pulsed EPR spectroscopy. The two-dimensional electron spin echo envelope modulation spectra of uniformly 15N-labeled protein show two well resolved cross-peaks with weak couplings of approximately 0.3-0.4 and 1.1 MHz in addition to couplings in the range of 6-8 MHz from two coordinating Ndelta of histidine ligands. The quadrupole coupling constants for weakly coupled nitrogens determined from S-band electron spin echo envelope modulation spectra identify them as Nepsilon of histidine ligands and peptide nitrogen (Np), respectively. Analysis of the line intensities in orientation-selected S-band spectra indicated that Np is the backbone N-atom of Leu-132 residue. The hyperfine couplings from Nepsilon and Np demonstrate the predominantly isotropic character resulting from the transfer of unpaired spin density onto the 2s orbitals of the nitrogens. Spectra also show that other peptide nitrogens in the protein environment must carry a 5-10 times smaller amount of spin density than the Np of Leu-132 residue. The appearance of the excess unpaired spin density on the Np of Leu-132 residue indicates its involvement in hydrogen bond formation with the bridging sulfur of the Rieske cluster. The configuration of the hydrogen bond therefore provides a preferred path for spin density transfer. Observation of similar splittings in the 15N spectra of other Rieske-type proteins and [2Fe-2S] ferredoxins suggests that a hydrogen bond between the bridging sulfur and peptide nitrogen is a common structural feature of [2Fe-2S] clusters.     

Repair of amino acid radicals of apolipoprotein B100 of low-density lipoproteins by flavonoids. A pulse radiolysis study with quercetin and rutin

Selective oxidative damage to apolipoprotein B in LDL can be effected radiolytically by (*)Br(2)(-) radicals. Twenty-seven Trp residues constitute major primary sites of oxidation, but two-thirds of oxidized Trps ((*)Trp) that are formed are repaired by intramolecular electron transfer from Tyr residues with formation of phenoxyl radicals (TyrO(*)). Analysis of (*)Trp and TyrO(*) transient absorbance changes suggests that other apolipoprotein B residues, probably Cys, are oxidized. LDL-bound quercetin can efficiently repair this damage. Absorption studies show that a LDL particle has the capacity to bind approximately 10 quercetin molecules through interaction with apolipoprotein B. The repair occurs by intramolecular electron transfer characterized by a rate constant of 2 x 10(3) s(-)(1). In contrast, rutin, a related flavonoid which does not bind to LDL, cannot repair oxidized apolipoprotein B. Urate is a hydrophilic plasma antioxidant which displays synergistic antioxidant properties with flavonoids. Urate radicals produced by (*)Br(2)(-) can also be repaired by LDL-bound quercetin. This repair occurs with a reaction rate constant of 6.8 x 10(7) M(-)(1) s(-)(1). Comparison with previous studies conducted with human serum albumin-bound quercetin suggests that quercetin analogues tailored to be carried preferentially by lipoproteins might be more powerful plasma antioxidants than natural quercetin carried by serum albumin.     

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.     

Peroxidase-catalyzed oxidation of eugenol: formation of a cytotoxic metabolite(s)

The oxidation of eugenol (4-allyl-2-methoxyphenol) by horseradish peroxidase was studied. Following the initiation of the reaction with hydrogen peroxide, eugenol was oxidized via a one-electron pathway to a phenoxyl radical which subsequently formed a transient, yellow-colored intermediate which was identified as a quinone methide. The eugenol phenoxyl radical was detected using fast-flow electron spin resonance. The radicals and/or quinone methide further reacted to form an insoluble complex polymeric material. The stoichiometry of the disappearance of eugenol versus hydrogen peroxide was approximately 2:1. The addition of glutathione or ascorbate prevented the appearance of the quinone methide and also prevented the disappearance of the parent compound. In the presence of glutathione, a thiyl radical was detected, and increases in oxygen consumption and in the formation of oxidized glutathione were also observed. These results suggested that glutathione reacted with the eugenol phenoxyl radical and reduced it back to the parent compound. Glutathione also reacted directly with the quinone methide resulting in the formation of a eugenol-glutathione conjugate(s). Using 3H-labeled eugenol, extensive covalent binding to protein was observed. Finally, the oxidation products of eugenol/peroxidase were observed to be highly cytotoxic using isolated rat hepatocytes as target cells.     

Oxidative dimerization of proteins: role of tyrosine accessibility

PURPOSE: To investigate the importance of two possible mechanisms of tyrosine oxidation on the yield of protein dimerization. The model chosen is hen and turkey egg-white lysozymes, which differ by seven amino acids, among which one tyrosine is in the 3 position. MATERIALS AND METHODS: Aqueous solutions of proteins were oxidized by OH(*) or N(*)(3) free radicals produced by gamma or pulse irradiation in an atmosphere of N(2)O. Protein dimers were quantified by SDS-PAGE and reverse-phase HPLC. Dityrosines were identified by absorption and fluorescence. RESULTS: Using N(*)(3) free radicals, the initial yields of dimerization are equal to (8.6 +/- 0.7) x 10(-9) mol J(-1) for both proteins. Using OH(*) free radicals, they become equal to (1.23 +/- 0.1) x 10(-8) and (4.42 +/- 0.1) x 10(-8) mol J(-1) for hen and turkey egg-white lysozymes, respectively (gamma radiolysis). DISCUSSION. N(*)(3) radicals react primarily with tryptophan residues only. Tyrosine gets oxidized by intramolecular long-range electron migration, whereas OH(*) may react directly with tyrosines. We propose a low participation of Tyr3 in turkey protein in the intramolecular process, because Tyr3 is far from all tryptophans. On the other hand, Tyr3 is very accessible to solvent and in a flexible area; thus collisions with OH(*) could easily be followed by intermolecular dimerization.     

Combinatorial approaches to functional models for galactose oxidase

The copper enzyme galactose oxidase (GOase, EC 1.1.3.9) catalyses the oxidation of D-galactose and other primary alcohols in air to the corresponding aldehydes and hydrogen peroxide. The current mechanistic hypothesis for this two-electron redox reaction involves a Cu(I)/Cu(II) couple and the reversible oxidation of a ligating phenolate (tyrosine residue of the Tyr272-Cys228 conjugate) to a phenoxyl radical. Our approaches to functional models for galactose oxidase comprise both the use of low-molecular-weight copper complexes of a Schiff-base and sulfonamide ligands, and the synthesis/screening of combinatorial libraries. With regard to the latter, we have synthesized (by the IRORI-directed synthesis approach) peptide libraries carrying either His or the redox-active amino acids Tyr, mod-Cys (a model for the Tyr272-Cys228 conjugate) or TOAC (a TEMPO-derived alpha-amino acid) at four variable positions. After incubation with copper ions, the catalytically active library members were identified by specially designed screening methods.     

Hydrogen bonds between nitrogen donors and the semiquinone in the Qi-site of the bc1 complex

The ubisemiquinone stabilized at the Qi-site of the bc1 complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of 14N and 15N have been studied by X-band (approximately 9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14N involved in H-bond formation and to assign it unambiguously to the Nepsilon of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and Ndelta in other quinone-processing sites. The experiments with 15N showed that the Nepsilon of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the Nepsilon of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38+/-0.13A. The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.     

Mutational analysis of cytochrome b at the ubiquinol oxidation site of yeast complex III

The cytochrome bc1 complex is a dimeric enzyme of the inner mitochondrial membrane that links electron transfer from ubiquinol to cytochrome c by a protonmotive Q cycle mechanism in which ubiquinol is oxidized at one center in the enzyme, referred to as center P, and ubiquinone is rereduced at a second center, referred to as center N. To better understand the mechanism of ubiquinol oxidation, we have examined catalytic activities and pre-steady-state reduction kinetics of yeast cytochrome bc1 complexes with mutations in cytochrome b that we expected would affect oxidation of ubiquinol. We mutated two residues thought to be involved in proton conduction linked to ubiquinol oxidation, Tyr132 and Glu272, and two residues proposed to be involved in docking ubiquinol into the center P pocket, Phe129 and Tyr279. Substitution of Phe129 by lysine or arginine yielded a respiration-deficient phenotype and lipid-dependent catalytic activity. Increased bypass reactions were detectable for both variants, with F129K showing the more severe effects. Substitution with lysine leads to a disturbed coordination of a b heme as deduced from changes in the midpoint potential and the EPR signature. Removal of the aromatic side chain in position Tyr279 lowers the catalytic activity accompanied by a low level of bypass reactions. Pre-steady-state kinetics of the enzymes modified at Glu272 and Tyr132 confirmed the importance of their functional groups for electron transfer. Altered center N kinetics and activation of ubiquinol oxidation by binding of cytochrome c in the Y132F and E272D enzymes indicate long range effects of these mutations.     

Probing the free radicals formed in the metmyoglobin-hydrogen peroxide reaction

The reaction between metmyoglobin and hydrogen peroxide results in the two-electron reduction of H2O2 by the protein, with concomitant formation of a ferryl-oxo heme and a protein-centered free radical. Sperm whale metmyoglobin, which contains three tyrosine residues (Tyr-103, Tyr-146, and Tyr-151) and two tryptophan residues (Trp-7 and Trp-14), forms a tryptophanyl radical at residue 14 that reacts with O2 to form a peroxyl radical and also forms distinct tyrosyl radicals at Tyr-103 and Tyr-151. Horse metmyoglobin, which lacks Tyr-151 of the sperm whale protein, forms an oxygen-reactive tryptophanyl radical and also a phenoxyl radical at Tyr-103. Human metmyoglobin, in addition to the tyrosine and tryptophan radicals formed on horse metmyoglobin, also forms a Cys-110-centered thiyl radical that can also form a peroxyl radical. The tryptophanyl radicals react both with molecular oxygen and with the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) traps the Tyr-103 radicals and the Cys-110 thiyl radical of human myoglobin, and 2-methyl-2-nitrosopropane (MNP) traps all of the tyrosyl radicals. When excess H2O2 is used, DBNBS traps only a tyrosyl radical on horse myoglobin, but the detection of peroxyl radicals and the loss of tryptophan fluorescence support tryptophan oxidation under those conditions. Kinetic analysis of the formation of the various free radicals suggests that tryptophanyl radical and tyrosyl radical formation are independent events, and that formation of the Cys-110 thiyl radical on human myoglobin occurs via oxidation of the thiol group by the Tyr-103 phenoxyl radical. Peptide mapping studies of the radical adducts and direct EPR studies at low temperature and room temperature support the conclusions of the EPR spin trapping studies.     

Comparative study of tyrosine radicals in hemoglobin and myoglobins treated with hydrogen peroxide

The reactions of hydrogen peroxide with human methemoglobin, sperm whale metmyoglobin, and horse heart metmyoglobin were studied by electron paramagnetic resonance (EPR) spectroscopy at 10 K and room temperature. The singlet EPR signal, one of the three signals seen in these systems at 10 K, is characterized by a poorly resolved, but still detectable, hyperfine structure that can be used to assign it to a tyrosyl radical. The singlet is detectable as a quintet at room temperature in methemoglobin with identical spectral features to those of the well characterized tyrosyl radical in photosystem II. Hyperfine splitting constants found for Tyr radicals were used to find the rotation angle of the phenoxyl group. Analysis of these angles in the crystal structures suggests that the radical resides on Tyr151 in sperm whale myoglobin, Tyr133 in soybean leghemoglobin, and either alphaTyr42, betaTyr35, or betaTyr130 in hemoglobin. In the sperm whale metmyoglobin Tyr103Phe mutant, there is no detectable tyrosyl radical present. Yet the rotation angle of Tyr103 (134 degrees) is too large to account for the observed EPR spectrum in the wild type. Tyr103 is the closest to the heme. We suggest that Tyr103 is the initial site of the radical, which then rapidly migrates to Tyr151.     

Pathways of dopamine oxidation mediated by nitric oxide

This study was aimed at establishing the interaction between dopamine and nitric oxide and elucidating the mechanistic aspects inherent in this interaction. At high (*) NO concentrations (microM range), dopamine underwent nitrosation with subsequent nitration. Nitrosation is proposed to occur via a nucleophilic attack to N(2)O(3) by dopamine. At low (*) NO concentrations (microM range), dopaminochrome was formed. EPR spin stabilization studies showed the occurrence of two o-semiquinone intermediates during dopaminochrome formation. Heats of formation obtained by AM1 semiempirical calculations supported the formation of the two o-semiquinone species. Hydroxyl radicals were detected by spin trapping EPR, and experiments performed with superoxide dismutase and catalase suggested that peroxynitrite was the source of HO(*). A mechanism is presented that considers the several factors influencing these reactions.     

Hydroxyl radical generation by photosystem II

The photogeneration of hydroxyl radicals (OH(*)) in photosystem II (PSII) membranes was studied using EPR spin-trapping spectroscopy. Two kinetically distinguishable phases in the formation of the spin trap-hydroxyl (POBN-OH) adduct EPR signal were observed: the first phase (t(1/2) = 7.5 min) and the second phase (t(1/2) = 30 min). The generation of OH(*) was found to be suppressed in the absence of the Mn-complex, but it was restored after readdition of an artificial electron donor (DPC). Hydroxyl radical generation was also lost in the absence of oxygen, whereas it was stimulated when the oxygen concentration was increased. The production of OH(*) during the first kinetic phase was sensitive to the presence of SOD, whereas catalase and EDTA diminished the production of OH(*) during the second kinetic phase. The POBN-OH adduct EPR signal during the first phase exhibits a similar pH-dependence as the ability to oxidize the non-heme iron, as monitored by the Fe(3+) (g = 8) EPR signal: both EPR signals gradually decreased as the pH value was lowered below pH 6.5 and were absent at pH 5. Sodium formate decreases the production of OH(*) in intact and Mn-deleted PSII membranes. Upon illumination of PSII membranes, both superoxide, as measured by EPR signal from the spin trap-superoxide (EMPO-OOH) adduct, and H(2)O(2), measured colormetrically, were generated. These results indicated that OH(*) is produced on the electron acceptor side of PSII by two different routes, (1) O(2)(*)(-), which is generated by oxygen reduction on the acceptor side of PSII, interacts with a PSII metal center, probably the non-heme iron, to form an iron-peroxide species that is further reduced to OH(*) by an electron from PSII, presumably via Q(A)(-), and (2) O(2)(*)(-) dismutates to form free H(2)O(2) that is then reduced to OH(*) via the Fenton reaction in the presence of metal ions, the most likely being Mn(2+) and Fe(2+) released from photodamaged PSII. The two different routes of OH(*) generation are discussed in the context of photoinhibition.     

Low-barrier hydrogen bond plays key role in active photosystem II--a new model for photosynthetic water oxidation

The function and mechanism of Tyr(Z) in active photosystem II (PSII) is one of the long-standing issues in the study of photosynthetic water oxidation. Based on recent investigations on active PSII and theoretical studies, a new model is proposed, in which D1-His190 acts as a bridge, to form a low-barrier hydrogen bond (LBHB) with Tyr(Z), and a coordination bond to Mn or Ca ion of the Mn-cluster. Accordingly, this new model differs from previous proposals concerning the mechanism of Tyr(Z) function in two aspects. First, the LBHB plays a key role to decrease the activation energy for Tyr(Z) oxidation and Tyr(Z)(.) reduction during photosynthetic water oxidation. Upon the oxidation of Tyr(Z), the hydrogen bond between Tyr(Z) and His190 changes from a LBHB to a weak hydrogen bond, and vice versa upon Tyr(Z)(.) reduction. In both stages, the electron transfer and proton transfer are coupled. Second, the positive charge formed after Tyr(Z) oxidation may play an important role for water oxidation. It can be delocalized on the Mn-cluster, thus helps to accelerate the proton release from substrate water on Mn-cluster. This model is well reconciled with observations of the S-state dependence of Tyr(Z) oxidation and Tyr(Z)(.) reduction, proton release, isotopic effect and recent EPR experiments. Moreover, the difference between Tyr(Z) and Tyr(D) in active PSII can also be readily rationalized. The His190 binding to the Mn-cluster predicted in this model is contradictious to the recent structure data, however, it has been aware that the crystal structure of the Mn-cluster and its environment are significantly modified by X-ray due to radiation damage and are different from that in active PSII. It is suggested that the His190 may be protonated during the radiation damage, which leads to the loss of its binding to Mn-cluster and the strong hydrogen bond with Tyr(Z). This type of change arising from radiation damage has been confirmed in other enzyme systems.     

Kinetic resolution of a tryptophan-radical intermediate in the reaction cycle of Paracoccus denitrificans cytochrome c oxidase

The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa(3) reacting with O(2) between 83 micros and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of Cu(A), heme a, heme a(3), and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). Cu(B) and heme a(3) were EPR silent during all stages of the reaction. Cu(A) and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of Cu(A) is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 micros and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a(3) oxoferryl species P(R) (maxima at 430 nm and 606 nm) was completed in approximately 130 micros, similar to the first oxidation phase of Cu(A) and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from P(R) and decays to a hitherto undetected intermediate named F(W)(*). F(W)(*) harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272(*)). The Trp-272(*) populates to 4-5% due to its relatively low rate of formation (t((1/2)) = 1.2 ms) and rapid rate of breakdown (t((1/2)) = 60 micros), which represents electron transfer from Cu(A)/heme a to Trp-272(*). The formation of the Trp-272(*) constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a(3) oxoferryl species or to Cu (2+)(B). The potential role of Trp-272 in proton pumping is discussed.     

Mitochondrial aconitase is a source of hydroxyl radical. An electron spin resonance investigation

Mitochondrial aconitase (m-aconitase) contains a [4Fe-4S](2+) cluster in its active site that catalyzes the stereospecific dehydration-rehydration of citrate to isocitrate in the Krebs cycle. It has been proposed that the [4Fe-4S](2+) aconitase is oxidized by superoxide, generating the inactive [3Fe-4S](1+) aconitase. In this reaction, the likely products are iron(II) and hydrogen peroxide. Consequently, the inactivation of m-aconitase by superoxide may increase the formation of hydroxyl radical ((*)OH) through the Fenton reaction in mitochondria. In this work, evidence for the generation of (*)OH from the reaction of m-aconitase with superoxide is provided using ESR spin trapping experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide and alpha-phenyl-N-tert-butylnitrone. Formation of free ( small middle dot)OH was verified with the (*)OH scavenger Me(2)SO, which forms methyl radical upon reacting with (*)OH. The addition of Me(2)SO to incubation mixtures containing m-aconitase and xanthine/xanthine oxidase yielded methyl radical, which was detected by ESR spin trapping. Methyl radical formation was further confirmed using [(13)C]Me(2)SO. Parallel low temperature ESR experiments demonstrated that the generation of the [3Fe-4S](1+) cluster increased with increasing additions of superoxide to m-aconitase. This reaction was reversible, as >90% of the initial aconitase activity was recovered upon treatment with glutathione and iron(II). This mechanism presents a scenario in which (*)OH may be continuously generated in the mitochondria.     

Purification and characterization of the epoxidase catalyzing the formation of fosfomycin from Pseudomonas syringae

The final step in the biosynthesis of fosfomycin in Streptomyces wedmorensis is catalyzed by ( S)-2-hydroxypropylphosphonic acid (HPP) epoxidase ( Sw-HppE). A homologous enzyme from Pseudomonas syringae whose encoding gene ( orf3) shares a relatively low degree of sequence homology with the corresponding Sw-HppE gene has recently been isolated. This purified P. syringae protein was determined to catalyze the epoxidation of ( S)-HPP to fosfomycin and the oxidation of ( R)-HPP to 2-oxopropylphosphonic acid under the same conditions as Sw-HppE. Therefore, this protein is indeed a true HPP epoxidase and is termed Ps-HppE. Like Sw-HppE, Ps-HppE was determined to be post-translationally modified by the hydroxylation of a putative active site tyrosine (Tyr95). Analysis of the Fe(II) center by EPR spectroscopy using NO as a spin probe and molecular oxygen surrogate reveals that Ps-HppE's metal center is similar, but not identical, to that of Sw-HppE. The identity of the rate-determining step for the ( S)-HPP and ( R)-HPP reactions was determined by measuring primary deuterium kinetic effects, and the outcome of these results was correlated with density functional theory calculations. Interestingly, the reaction using the nonphysiological substrate ( R)-HPP was 1.9 times faster than that with ( S)-HPP for both Ps-HppE and Sw-HppE. This is likely due to the difference in bond dissociation energy of the abstracted hydrogen atom for each respective reaction. Thus, despite the low level of amino acid sequence identity, Ps-HppE is a close mimic of Sw-HppE, representing a second example of a non-heme iron-dependent enzyme capable of catalyzing dehydrogenation of a secondary alcohol to form a new C-O bond.     

Tyrosine 167: the origin of the radical species observed in the reaction of cytochrome c oxidase with hydrogen peroxide in Paracoccus denitrificans

Determination of the three-dimensional structure of cytochrome c oxidase, the terminal enzyme of the respiratory chain, from Paracoccus denitrificans offers the possibility of site-directed mutagenesis studies to investigate the relationship between the structure and the catalytic function of the enzyme. The mechanism of electron-coupled proton transfer is still, however, poorly understood. The P(M) intermediate of the catalytic cycle is an oxoferryl state the generation of which requires one additional electron, which cannot be provided by the two metal centers. It is suggested that the missing electron is donated to this binuclear site by a tyrosine residue that forms a radical species, which can then be detected in both the P(M) and F(*) intermediates of the catalytic cycle. One possibility to produce P(M) and F(*) intermediates artificially in cytochrome c oxidase is the addition of hydrogen peroxide to the fully oxidized enzyme. Using electron paramagnetic resonance (EPR) spectroscopy, we assign a radical species detected in this reaction to a tyrosine residue. To address the question, which tyrosine residue is the origin of the radical species, several tyrosine variants of subunit I are investigated. These variants are characterized by their turnover rates, as well as using EPR and optical spectroscopy. From these experiments, it is concluded that the origin of the radical species appearing in P(M) and F(*) intermediates produced with hydrogen peroxide is tyrosine 167. The significance of this finding for the catalytic function of the enzyme is discussed.     

Carbon-13 and nitrogen-15 nuclear-magnetic-resonance investigation on Desulfovibrio vulgaris flavodoxin

Desulfovibrio vulgaris apoflavodoxin has been reconstituted with 15N and 13C-enriched riboflavin 5'-phosphate. For the first time all carbon atoms of the isoalloxazine ring of the protein-bound prosthetic group have been investigated. The reconstituted protein was studied in the oxidized and in the two-electron-reduced state. The results are interpreted in terms of specific interactions between the apoprotein and the prosthetic group, and the chemical structure of protein-bound FMN. In the oxidized state weak hydrogen bonds exist between the apoprotein and the N(5), N(3) and O(4 alpha) atoms of FMN. The N(1) and O(2 alpha) atoms of FMN form strong hydrogen bonds. The isoalloxazine ring of FMN is strongly polarized and the N(10) atom shows an increased sp2 hybridisation compared to that of free FMN in aqueous solution. The N(3)-H group is not accessible to bulk solvent, as deduced from the coupling constant of the N(3)-H group. In the reduced state the hydrogen bond pattern is similar to that in the oxidized state and in addition a strong hydrogen bond is observed between the N(5)-H group of FMN and the apoprotein. The reduced prosthetic group possesses a coplanar structure and is ionized. The N(3)-H and N(5)-H groups are not accessible to solvent water. Two-electron reduction of the protein leads to a large electron density increase in the benzene subnucleus of bound FMN compared to that in free FMN. The results are discussed in relation to the published crystallographic data on the protein.