Autoreduction of ferryl myoglobin: discrimination among the three tyrosine and two tryptophan residues as electron donors

Ferric myoglobin undergoes a two-electron oxidation in its reaction with H(2)O(2). One oxidation equivalent is used to oxidize Fe(III) to the Fe(IV) ferryl species, while the second is associated with a protein radical but is rapidly dissipated. The ferryl species is then slowly reduced back to the ferric state by unknown mechanisms. To clarify this process, the formation and stability of the ferryl forms of the Tyr --> Phe and Trp --> Phe mutants of recombinant sperm whale myoglobin (SwMb) were investigated. Kinetic studies showed that all the mutants react normally with H(2)O(2) to give the ferryl species. However, the rapid phase of ferryl autoreduction typical of wild-type SwMb was absent in the triple Tyr --> Phe mutant and considerably reduced in the Y103F and Y151F mutants, strongly implicating these two residues as intramolecular electron donors. Replacement of Tyr146, Trp7, or Trp14 did not significantly alter the autoreduction, indicating that these residues do not contribute to ferryl reduction despite the fact that Tyr146 is closer to the iron than Tyr151 or Tyr103. Furthermore, analysis of the fast phase of autoreduction in the dimer versus recovered monomer of the Tyr --> Phe mutant K102Q/Y103F/Y146F indicates that the Tyr151-Tyr151 cross-link is a particularly effective electron donor. The presence of an additional, slow phase of reduction in the triple Tyr --> Phe mutant indicates that alternative but normally minor electron-transfer pathways exist in SwMb. These results demonstrate that internal electron transfer is governed as much by the tyrosine pK(a) and oxidation potential as by its distance from the electron accepting iron atom.     

H2O2-mediated cross-linking between lactoperoxidase and myoglobin: elucidation of protein-protein radical transfer reactions

The H(2)O(2)-dependent reaction of lactoperoxidase (LPO) with sperm whale myoglobin (SwMb) or horse myoglobin (HoMb) produces LPO-Mb cross-linked species, in addition to LPO and SwMb homodimers. The HoMb products are a LPO(HoMb) dimer and LPO(HoMb)(2) trimer. Dityrosine cross-links are shown by their fluorescence to be present in the oligomeric products. Addition of H(2)O(2) to myoglobin (Mb), followed by catalase to quench excess H(2)O(2) before the addition of LPO, still yields LPO cross-linked products. LPO oligomerization therefore requires radical transfer from Mb to LPO. In contrast to native LPO, recombinant LPO undergoes little self-dimerization in the absence of Mb but occurs normally in its presence. Simultaneous addition of 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS) and LPO to activated Mb produces a spin-trapped radical electron paramagnetic resonance signal located primarily on LPO, confirming the radical transfer. Mutation of Tyr-103 or Tyr-151 in SwMb decreased cross-linking with LPO, but mutation of Tyr-146, Trp-7, or Trp-14 did not. However, because DBNBS-trapped LPO radicals were observed with all the mutants, DBNBS traps LPO radicals other than those involved in protein oligomerization. The results clearly establish that radical transfer occurs from Mb to LPO and suggest that intermolecularly transferred radicals may reside on residues other than those that are generated by intramolecular reactions.     

The myoglobin protein radical. Coupling of Tyr-103 to Tyr-151 in the H2O2-mediated cross-linking of sperm whale myoglobin

Sperm whale metmyoglobin, which has tyrosine residues at positions 103, 146, and 151, dimerizes in the presence of H2O2. Equine metmyoglobin, which lacks Tyr-151, and red kangaroo metmyoglobin, which lacks Tyr-103 and Tyr-151, do not dimerize in the presence of H2O2. The dityrosine content of the sperm whale myoglobin dimer shows that it is primarily held together by dityrosine cross-links, although more tyrosine residues are lost than are accounted for by dityrosine formation. Digestion of the myoglobin dimer with chymotrypsin yields a peptide with the fluorescence spectrum of dityrosine. The amino acid composition, amino acid sequence, and mass spectrum of the peptide show that cross-linking involves covalent bond formation between Tyr-103 of one myoglobin chain and Tyr-151 of the other. Replacement of the prosthetic group of sperm whale myoglobin with zinc protoporphyrin IX prevents H2O2-induced dimerization even when intact horse metmyoglobin is present in the incubation. This suggests that the tyrosine radicals required for the dimerization reaction are generated by intra- rather than intermolecular electron transfer to the ferryl heme. Rapid electron transfer from Tyr-103 to the ferryl heme followed by slower electron transfer from Tyr-151 to Tyr-103 is most consistent with the present results.     

Spin trapping and protein cross-linking of the lactoperoxidase protein radical

Lactoperoxidase (LPO) reacts with H(2)O(2) to sequentially give two Compound I intermediates: the first with a ferryl (Fe(IV)=O) species and a porphyrin radical cation, and the second with the same ferryl species and a presumed protein radical. However, little actual evidence is available for the protein radical. We report here that LPO reacts with the spin trap 3,5-dibromo-4-nitroso-benzenesulfonic acid to give a 1:1 protein-bound radical adduct. Furthermore, LPO undergoes the H(2)O(2)-dependent formation of dimeric and trimeric products. Proteolytic digestion and mass spectrometric analysis indicates that the dimer is held together by a dityrosine link between Tyr-289 in each of two LPO molecules. The dimer retains full catalytic activity and reacts to the same extent with the spin trap, indicating that the spin trap reacts with a radical center other than Tyr-289. The monomeric protein recovered from incubations of LPO with H(2)O(2) is fully active but no longer forms dimers when incubated with H(2)O(2), clear evidence that it has also been structurally modified. Myeloperoxidase, a naturally dimeric protein, and eosinophil peroxidase do not undergo H(2)O(2)-dependent oligomerization. Analysis of the interface in the LPO dimers indicates that the same protein surface is involved in LPO dimerization as in the normal formation of myeloperoxidase dimers. Oligomerization of LPO alters its physical properties and may alter its ability to interact with macromolecular substrates.     

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.     

Formation of Isodityrosine by Peroxidase Isozymes

Fry, S. C. 1987. Formation of isodityrosine by peroxidase isozymes.—J.exp. Bot. 38: 853–862. Tyrosine residues of extensin are oxidatively coupled in vivoto form isodityrosine bridges, whereas treatment of purifiedextensin with H₂O₂+ peroxidase in vitro yields only dityrosine.Two explanations for the correct mode of coupling in vivo weretested. The first, that the pH of the cell wall is lower thanthat (pH 9-0) at which in vitro experiments have been conducted,provided part of the answer since treatment of L-tyrosine withH₂O₂+peroxidase in vitro at pH 37–5 yielded some isodityrosine.The second, that the wall contains other isozymes of peroxidasethan the basic isozyme usually studied in vitro, appeared unlikelybecause several sharply contrasting isozymes yielded similarisodityrosine: dityrosine ratios from L-tyrosine+ H₂O₂ at anygiven pH. The isozymes were also similar in their ability tooxidize tyrosine-dimers further to higher polymers. It is concludedthat the formation of isodityrosine in vivo is dictated by neighbouringwall molecules, possibly ionically-bound pectins, which modifythe local environment of the tyrosine residues of extensin. Key words: Isodityrosine, peroxidase isozymes, extensin     

Development of fluorescence and cross-links in eye lens crystallin by interaction with lipid peroxy radicals

Senile nuclear cataract formation is associated with the accumulation of fluorescent insoluble proteins. alpha-Crystallin from bovine eye lens was treated with the lipid peroxy radicals generated by interaction of linoleic acid 13-monohydroperoxide or phosphatidylcholine hydroperoxide with methemoglobin. A blue non-tryptophan fluorescence composed of at least two kinds of fluorophores, stable and unstable on borohydride treatment, was produced. One of the stable fluorophores was identified as dityrosine by comparison of its retention times in amino acid analysis and HPLC with those of authentic dityrosine. Dityrosine was formed by the radical reaction, and less than 0.3% of the total tyrosine residues of the protein were transformed into dityrosine. The unstable fluorophores may be produced on the amino groups of alpha-crystallin by the non-radical reaction of the decomposition products of the radicals including monofunctional aldehyde species. Extensive intermolecular cross-links could not be explained only by the dityrosine content, but by the other modifications. Formation of fluorescence and intermolecular cross-links in alpha-crystalline in reaction with the lipid peroxy radicals suggested the participation of lipid peroxidation in the cataractous process.     

Intramolecular translocation of the protein radical formed in the reaction of recombinant sperm whale myoglobin with H2O2.

A sperm whale myoglobin gene containing multiple unique restriction sites has been constructed in pUC 18 by sequential assembly of chemically synthesized oligonucleotide fragments. Expression of the gene in Escherichia coli DH5 alpha cells yields protein that is identical to native sperm whale myoglobin except that it retains the terminal methionine. Site-specific mutagenesis has been used to prepare all the possible tyrosine----phenylalanine mutants of the recombinant myoglobin, including the three single mutants at Tyr-103, -146, and -151, the three double mutants, and the triple mutant. All of the mutant proteins are stable except the Tyr-103 mutant. Introduction of a second mutation (Lys-102----Gln) stabilizes the Tyr-103 mutant. Absorption spectroscopy suggests that the active sites of the mutant proteins are intact. EPR and absorption spectroscopy show that all the proteins, including the triple mutant devoid of tyrosine residues, react with H2O2 to give a ferryl species and a protein radical. The presence of a protein radical in all the mutants suggests that the radical center is readily transferred from one amino acid to another. Cross-linking studies show, however, that protein dimers are only formed when Tyr-151 is present. Tyr-103, shown earlier to be the residue that primarily cross-links to Tyr-151 (Tew, D., and Ortiz de Montellano, P. R. (1988) J. Biol. Chem. 263, 17880-17886), is not essential for cross-linking. Electron transfer from Tyr-151 to the heme, which are 12 A apart, occurs in the absence of the intervening tyrosines at positions 103 and 146. The present studies show that the peroxide-generated myoglobin radical readily exchanges between remote loci, including non-tyrosine residues, but protein cross-linking only occurs when radical density is located on Tyr-151.     

Reaction between protein radicals and other biomolecules

The present study investigates the reactivity of bovine serum albumin (BSA) radicals towards different biomolecules (urate, linoleic acid, and a polypeptide, poly(Glu-Ala-Tyr)). The BSA radical was formed at room temperature through a direct protein-to-protein radical transfer from H(2)O(2)-activated immobilized horseradish peroxidase (im-HRP). Subsequently, each of the three different biomolecules was separately added to the BSA radicals, after removal of im-HRP by centrifugation. Electron spin resonance (ESR) spectroscopy showed that all three biomolecules quenched the BSA radicals. Subsequent analysis showed a decrease in the concentration of urate upon reaction with the BSA radical, while the BSA radical in the presence of poly(Glu-Ala-Tyr) resulted in increased formation of the characteristic protein oxidation product, dityrosine. Reaction between the BSA radical and a linoleic acid oil-in-water emulsion resulted in additional formation of lipid hydroperoxides and conjugated dienes. The results clearly show that protein radicals have to be considered as dynamic species during oxidative processes in biological systems and that protein radicals should not be considered as end-products, but rather as reactive intermediates during oxidative processes in biological systems hereby supporting recent data.     

Dimerisation of N-acetyl-L-tyrosine ethyl ester and Abeta peptides via formation of dityrosine

Alzheimer's disease (AD) is characterised by the formation of amyloid deposits composed primarily of the amyloid beta-peptide (Abeta). This peptide has been shown to bind redox active metals ions such as copper and iron, leading to the production of reactive oxygen species (ROS) and formation of hydrogen peroxide (H(2)O(2)). The generation of H(2)O(2) has been linked with Abeta neurotoxicity and neurodegeneration in AD. Because of the relative stability of a tyrosyl radical, the tyrosine residue (Tyr-10) is believed to be critical to the neurotoxicity of Abeta. This residue has also been shown to be important to Abeta aggregation and amyloid formation. It is possible that the formation of an Abeta tyrosyl radical leads to increased aggregation via the formation of dityrosine as an early aggregation step, which is supported by the identification of dityrosine in amyloid plaque. The role of dityrosine formation in Abeta aggregation and neurotoxicity is as yet undetermined, partly because there are no facile methods for the synthesis of Abeta dimers containing dityrosine. Here we report the use of horseradish peroxidase and H(2)O(2) to dimerise N-acetyl-L-tyrosine ethyl ester and apply the optimised conditions for dityrosine formation to fully unprotected Abeta peptides. We also report a simple fluorescent plate reader method for monitoring Abeta dimerisation via dityrosine formation.     

The hydrogen peroxide/copper ion system, but not other metal-catalyzed oxidation systems, produces protein-bound dityrosine

Dityrosine formation leads to the cross-linking of proteins intra- or intermolecularly. The formation of dityrosine in lens proteins oxidized by metal-catalyzed oxidation (MCO) systems was estimated by chemical and immunochemical methods. Among the four MCO systems examined (H(2)O(2)/Cu, H(2)O(2)/Fe-ethylenediaminetetraacetic acid (Fe-EDTA), ascorbate/Cu, ascorbate/Fe-EDTA), the treatment with H(2)O(2)/Cu preferentially caused dityrosine formation in the lens proteins. The success of oxidative protein modification with all the MCO systems was confirmed by carbonyl formation estimated using 2,4-dinitrophenylhydrazine. The loss of tyrosine by the MCO systems was partly due to the formation of protein-bound 3,4-dihydroxyphenylalanine. The formation of dityrosine specific to H(2)O(2)/Cu was confirmed by using poly-(Glu, Ala, Tyr) and N-acetyl-tyrosine as a substrate. The dissolved oxygen concentration in the H(2)O(2)/Cu system hardly affected the amount of dityrosine formation, suggesting that dityrosine generation by the H(2)O(2)/Cu system is independent of oxygen concentration. Moreover, the combination of copper ion with H(2)O(2) is the most effective system for dityrosine formation among various metal ions examined. The addition of reducing agents, glutathione or ascorbic acid, into the H(2)O(2)/Cu system suppressed the generation of the dityrosine moiety, suggesting effective quench of tyrosyl radicals by the reducing agents.     

Characterization of acatalasemic erythrocytes treated with low and high dose hydrogen peroxide. Hemolysis and aggregation

The effects of hydrogen peroxide on normal and acatalasemic erythrocytes were examined. Severe hemolysis of acatalasemic erythrocytes and a small tyrosine radical signal (g = 2.005) associated with the formation of ferryl hemoglobin were observed upon the addition of less than 0.25 mM hydrogen peroxide. However, when the concentration of hydrogen peroxide was increased to 0.5 mM, acatalasemic erythrocytes became insoluble in water and increased the tyrosine radical signal. Polymerization of hemoglobin and aggregation of the erythrocytes were observed. On the other hand, normal erythrocytes exhibited only mild hemolysis by the addition of hydrogen peroxide under similar conditions. From these results, the scavenging of hydrogen peroxide by hemoglobin generates the ferryl hemoglobin species (H-Hb-Fe(IV)=O) plus protein-based radicals (*Hb-Fe(IV)=O). These species induce hemolysis of erythrocytes, polymerization of hemoglobin, and aggregation of the acatalasemic erythrocytes. A mechanism for the onset of Takarara disease is proposed.     

Oxidation of 3-hydroxykynurenine catalyzed by methemoglobin with hydrogen peroxide.

Methemoglobin (metHb) with H2O2 catalyzed the oxidation of 3-hydroxykynurenine (3-HKY) in the reaction mixture of metHb, 3-HKY, and H2O2. The spectrophotometric experiments suggest the following mechanism for the 3-HKY oxidation by metHb with H2O2. MetHb first reacts with H2O2 to form the ferryl complex of Hb. This species then oxidizes 3-HKY, while it returns to metHb. 3-HKY was more reactive with the ferryl complex than glutathione but less reactive than ascorbic acid. Scavengers of the hydroxyl radical, dimethyl sulfoxide and ethanol, scarcely inhibited the 3-HKY oxidation by metHb with H2O2. Desferrioxamine, a metal chelator, hardly suppressed the 3-HKY oxidation. These results indicate that the hydroxyl radical is not involved in the 3-HKY oxidation by metHb with H2O2.     

Peroxyl adduct radicals formed in the iron/oxygen reconstitution reaction of mutant ribonucleotide reductase R2 proteins from Escherichia coli

Catalytically important free radicals in enzymes are generally formed at highly specific sites, but the specificity is often lost in point mutants where crucial residues have been changed. Among the transient free radicals earlier found in the Y122F mutant of protein R2 in Escherichia coli ribonucleotide reductase after reconstitution with Fe2+ and O2, two were identified as tryptophan radicals. A third radical has an axially symmetric EPR spectrum, and is shown here using 17O exchange and simulations of EPR spectra to be a peroxyl adduct radical. Reconstitution of other mutants of protein R2 (i.e. Y122F/W48Y and Y122F/W107Y) implicates W48 as the origin of the peroxyl adduct. The results indicate that peroxyl radicals form on primary transient radicals on surface residues such as W48, which is accessible to oxygen. However, the specificity of the reaction is not absolute since the single mutant W48Y also gives rise to a peroxyl adduct radical. We used density functional calculations to investigate residue-specific effects on hyperfine coupling constants using models of tryptophan, tyrosine, glycine and cysteine. The results indicate that any peroxyl adduct radical attached to the first three amino acid alpha-carbons gives similar 17O hyperfine coupling constants. Structural arguments and experimental results favor W48 as the major site of peroxyl adducts in the mutant Y122F. Available molecular oxygen can be considered as a spin trap for surface-located protein free radicals.     

Reaction of human myoglobin and H2O2. Electron transfer between tyrosine 103 phenoxyl radical and cysteine 110 yields a protein-thiyl radical

The sequence of human myoglobin (Mb) is similar to that of other species except for a unique cysteine at position 110 (Cys(110)). Adding hydrogen peroxide (H(2)O(2)) to human Mb affords Trp(14)-peroxyl, Tyr(103)-phenoxyl, and Cys(110)-thiyl radicals and coupling of Cys(110)-thiyl radicals yields a homodimer through intermolecular disulfide bond formation (Witting, P. K., Douglas, D. J., and Mauk, A. G. (2000) J. Biol. Chem. 275, 20391-20398). Treating a solution of wild type Mb and H(2)O(2) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) at DMPO:protein /= 100 mol/mol only DMPO-Tyr(103) radicals were present. The DMPO-dependent decrease in DMPO-Cys(110) was matched by a near 1:1 stoichiometric increase in DMPO-Tyr(103). In contrast, reaction of the Y103F human Mb with H(2)O(2) gave no DMPO-Cys(110) at DMPO:protein /= 100 mol/mol (i.e. conditions that consistently gave DMPO-Tyr(103) in the case of wild type Mb). No detectable homodimer was formed by incubation of the Y103F variant with H(2)O(2). However, the homodimer was detected in a mixture of both the Y103F and C110A variants of human Mb upon treatment with H(2)O(2) (C110A:Y103F:H(2)O(2) 2:1:5 mol/mol/mol); the yield of this homodimer increased with increasing ratios of C110A:Y103F. Together, these data suggest that addition of H(2)O(2) to human Mb can produce Cys(110)-thiyl radicals through an intermolecular electron transfer reaction from Cys(110) to a Tyr(103)-phenoxyl radical.     

Reactions of superoxide with the myoglobin tyrosyl radical

The contribution of superoxide-mediated injury to oxidative stress is not fully understood. A potential mechanism is the reaction of superoxide with tyrosyl radicals, which either results in repair of the tyrosine or formation of tyrosine hydroperoxide by addition. Whether these reactions occur with protein tyrosyl radicals is of interest because they could alter protein structure or modulate enzyme activity. Here, we have used a xanthine oxidase/acetaldehyde system to generate tyrosyl radicals on sperm whale myoglobin in the presence of superoxide. Using mass spectrometry we found that superoxide prevented myoglobin dimer formation by repairing the protein tyrosyl radical. An addition product of superoxide at Tyr151 was also identified, and exogenous lysine promoted the formation of this product. In our system, reaction of tyrosyl radicals with superoxide was favored over dimer formation with the ratio of repair to addition being approximately 10:1. Our results demonstrate that reaction of superoxide with protein tyrosyl radicals occurs and may play a role in free radical-mediated protein injury.     

Dityrosine formation outcompetes tyrosine nitration at low steady-state concentrations of peroxynitrite. Implications for tyrosine modification by nitric oxide/superoxide in vivo

Formation of peroxynitrite from NO and O-(*2) is considered an important trigger for cellular tyrosine nitration under pathophysiological conditions. However, this view has been questioned by a recent report indicating that NO and O-(*2) generated simultaneously from (Z)-1-(N-[3-aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino) diazen-1-ium-1,2-diolate] (SPER/NO) and hypoxanthine/xanthine oxidase, respectively, exhibit much lower nitrating efficiency than authentic peroxynitrite (Pfeiffer, S. and Mayer, B. (1998) J. Biol. Chem. 273, 27280-27285). The present study extends those earlier findings to several alternative NO/O-(*2)-generating systems and provides evidence that the apparent lack of tyrosine nitration by NO/O-(*2) is due to a pronounced decrease of nitration efficiency at low steady-state concentrations of authentic peroxynitrite. The decrease in the yields of 3-nitrotyrosine was accompanied by an increase in the recovery of dityrosine, showing that dimerization of tyrosine radicals outcompetes the nitration reaction at low peroxynitrite concentrations. The observed inverse dependence on peroxynitrite concentration of dityrosine formation and tyrosine nitration is predicted by a kinetic model assuming that radical formation by peroxynitrous acid homolysis results in the generation of tyrosyl radicals that either dimerize to yield dityrosine or combine with (*)NO(2) radical to form 3-nitrotyrosine. The present results demonstrate that very high fluxes (>2 microM/s) of NO/O-(*2) are required to render peroxynitrite an efficient trigger of tyrosine nitration and that dityrosine is a major product of tyrosine modification caused by low steady-state concentrations of peroxynitrite.     

Reaction of human myoglobin and H2O2. Involvement of a thiyl radical produced at cysteine 110

The human myoglobin (Mb) sequence is similar to other mammalian Mb sequences, except for a unique cysteine at position 110. Reaction of wild-type recombinant human Mb, the C110A variant of human Mb, or horse heart Mb with H(2)O(2) (protein/H(2)O(2) = 1:1.2 mol/mol) resulted in formation of tryptophan peroxyl (Trp-OO( small middle dot)) and tyrosine phenoxyl radicals as detected by EPR spectroscopy at 77 K. For wild-type human Mb, a second radical (g approximately 2. 036) was detected after decay of Trp-OO( small middle dot) that was not observed for the C110A variant or horse heart Mb. When the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was included in the reaction mixture at protein/DMPO ratios /=1:25 mol/mol, DMPO-tyrosyl radical adducts were detected. Mass spectrometry of wild-type human Mb following reaction with H(2)O(2) demonstrated the formation of a homodimer (mass of 34,107 +/- 5 atomic mass units) sensitive to reducing conditions. The human Mb C110A variant afforded no dimer under identical conditions. Together, these data indicate that reaction of wild-type human Mb and H(2)O(2) differs from the corresponding reaction of other myoglobin species by formation of thiyl radicals that lead to a homodimer through intermolecular disulfide bond formation.     

Isodityrosine, a new cross-linking amino acid from plant cell-wall glycoprotein.

1. Cell-wall hydrolysates from calli of all higher plants tested contained a new phenolic amino acid for which the trivial name isodityrosine is proposed. Isodityrosine was shown to be an oxidatively coupled dimer of tyrosine with the two tyrosine units linked by a diphenyl ether bridge. 2. The amount of isodityrosine in sodium dodecyl sulphate-insoluble cell-wall preparations was proportional to the amount of hydroxyproline. 3. Acidified chlorite split the diphenyl ether bridge of isodityrosine, and concomitantly solubilized the cell-wall glycoprotein. 4. Dithiothreitol inhibited isodityrosine synthesis in vivo, and suppressed in parallel the covalent binding of newly synthesized protein in the cell wall. 5. It is suggested that isodityrosine is an inter-polypeptide cross-link responsible for the insolubility of plant cell-wall glycoprotein.     

Protein radicals in the reaction between H2O2-activated metmyoglobin and bovine serum albumin

Hydrogen peroxide activation of MMb with and without the presence of BSA gave rise to rapid formation of hyper-valent myoglobin species, myoglobin ferryl radical (·MbFe(IV)=O) and/or ferrylmyoglobin (MbFe(IV)=O). Reduction of MbFe(IV)=O showed first-order kinetics for a 1-2 times stoichiometric excess of H₂O₂ to MMb while a 3-10 times stoichiometric excess of H₂O₂ resulted in a biphasic reaction pattern. Radical species formed in the reaction between MMb, H₂O₂ and BSA were influenced by [H₂O₂] as measured by electron spin resonance (ESR) spectroscopy and resulted in the formation of cross-linking between BSA and myoglobin which was confirmed by SDS-PAGE and subsequent amino acid sequencing. Moreover, dityrosine was formed in the initial phases of the reaction for all concentrations of H₂O₂. However, initially formed dityrosine was subsequently utilized in reactions employing stoichiometric excess of H₂O₂ to MMb. The observed breakdown of dityrosine was ascribed to additional radical species formed from the interaction between H₂O₂ and the hyper-valent iron-center of H₂O₂-activated MMb.