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.     

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.     

Intra- and intermolecular transfers of protein radicals in the reactions of sperm whale myoglobin with hydrogen peroxide

Reaction of sperm whale metmyoglobin (SwMb) with H2O2 produces a ferryl (MbFeIV=O) species and a protein radical and leads to the formation of oligomeric products. The ferryl species is maximally formed with one equivalent of H2O2, and the maximum yields of the dimer (28%) and trimer (17%) with 1 or 2 eq. Co-incubation of the SwMb Y151F mutant with native apoSwMb and H2O2 produced dimeric products, which requires radical transfer from the nondimerizing Y151F mutant to apoSwMb. Autoreduction of ferryl SwMb to the ferric state is biphasic with t = 3.4 and 25.9 min. An intramolecular autoreduction process is implicated at low protein concentrations, but oligomerization decreases the lifetime of the ferryl species at high protein concentrations. A fraction of the protein remained monomeric. This dimerization-resistant protein was in the ferryl state, but after autoreduction it underwent normal dimerization with H2O2. Proteolytic digestion established the presence of both dityrosine and isodityrosine cross-links in the oligomeric proteins, with the isodityrosine links primarily forged by Tyr151-Tyr151 coupling. The tyrosine content decreased by 47% in the dimer and 14% in the recovered monomer, but the yields of isodityrosine and dityrosine in the dimer were only 15.2 and 6.8% of the original tyrosine content. Approximately 23% of the lost tyrosines therefore have an alternative but unknown fate. The results clearly demonstrate the concurrence of intra- and intermolecular electron transfer processes involving Mb protein radicals. Intermolecular electron transfers that generate protein radicals on bystander proteins are likely to propagate the cellular damage initiated by the reaction of metalloproteins with H2O2.     

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 interaction of Trolox C, a water-soluble vitamin E analog, with ferrylmyoglobin: reduction of the oxoferryl moiety.

The oxidation of the heme iron of metmyoglobin by H2O2 yields an oxo ferryl complex (FeIV = O), similar to Compound II of peroxidases, as well as a protein radical; this high oxidation state of myoglobin is known as ferrylmyoglobin. The interaction of Trolox, a water-soluble vitamin E analog, with ferrylmyoglobin entailed two sequential one-electron oxidations of the phenolic antioxidant with intermediate formation of a phenoxyl radical and accumulation of a quinone end product. These oxidation reactions were linked to individual reductions of ferrylmyoglobin to metmyoglobin, as indicated by the value of the relationship [metmyoglobin]formed/[Trolox]consumed: 1.92 +/- 0.28. The Trolox-mediated reduction of ferrylmyoglobin to metmyoglobin could proceed directly, i.e., electron transfer from the phenolic-OH group in Trolox to the oxoferryl moiety, or indirectly, i.e., sequential electron transfer from Trolox to a protein radical to the oxoferryl moiety. The former mechanism is supported by the finding that the high oxidation heme iron is reduced under conditions where the tyrosyl residues are blocked by o-acetylation and when hemin is substituted for myoglobin. The latter mechanism is consistent with the following observations: (a) the EPR signal ascribed to the protein radical is suppressed by Trolox, with the concomitant appearance of the EPR spectrum of the Trolox phenoxyl radical and (b) the rate of ferrylmyoglobin reduction by Trolox is decreased with increasing number of tyrosyl residues in the proteins of horse myoglobin (titrated by o-acetylation) and sperm whale myoglobin. The apparent discrepancy between these observations can be reconciled by considering that both electrophilic centers in ferrylmyoglobin--the oxoferryl heme moiety and the protein radical--function independently of each other and that recovery of ferrylmyoglobin by Trolox could be effected through the tyrosyl residues, albeit at slower rates. The mechanistic aspects of these results are discussed in terms of the two main redox transitions in the myoglobin molecule encompassing valence changes of the heme iron and electron transfer of the tyrosyl residue in the protein and linked to the two sequential one-electron oxidations of Trolox.     

Reactions of the protein radical in peroxide-treated myoglobin. Formation of a heme-protein cross-link

Reaction of horse myoglobin with H2O2 oxidizes the iron to the ferryl (Fe(IV) = O) state and produces a protein radical that is rapidly dissipated by poorly understood mechanisms. As reported here, the reaction with H2O2 results in covalent binding of up to 18% of the prosthetic heme group to the protein. The chromophore of the protein-bound prosthetic group is very similar to that of heme itself. High performance liquid chromatography of tryptic digests indicates that the formation of heme-bound peptides is associated with disappearance of the peptide with the sequence YLE-FISDAIIHVLHSK corresponding to residues 103-118 of horse myoglobin. Amino acid analysis, terminal amino acid sequencing, and liquid secondary ion mass spectrometry establish that the heme is primarily attached to this peptide. The heme appears to be bound to the tyrosine residue because the tyrosine is the only amino acid that disappears from the amino acid analysis. The mass spectrometric data indicates that the heme-peptide is formed without addition or loss of an oxygen or other major structural fragment. The site of attachment to the heme group has not been unambiguously determined, but the heme vinyl groups are not essential for the reaction because equal cross-linking is observed in H2O2-treated mesoheme-reconstituted myoglobin. The results are most consistent with binding of tyrosine 103 to a meso-carbon of the prosthetic heme group.     

Titration behavior of individual tyrosine residues of myoglobins from sperm whale, horse, and red kangaroo.

The titration behavior of individual tyrosine residues of myoglobins has been studied by observing the pH dependence of the chemical shifts of Czeta and Cgamma of these residues in natural abundance of 13C Fourier transform NMR spectra (at 15.18 MHz, in 20-mm sample tubes, at 37 degrees) of cyanoferrimyoglobins from sperm whale, horse, and red kangaroo. A comparison of the pH dependence of the spectra of the three proteins yielded specific assignments for the resonance of Tyr-151 (sperm whale) and Tyr-103 (sperm whale and horse). Selective proton decoupling yielded specific assignments for Czeta of Tyr-146 of the cyanoferrimyoglobins from horse and kangaroo, but not the corresponding assignment for sperm whale. The pH dependence of the chemical shifts indicated that only Tyr-151 and Tyr-103 are titratable tyrosine residues. Even at pH 12, Tyr-146 did not begin to titrate. The titration behavior of C zeta and Cgamma of Tyr-151 of sperm whale cyanoferrimyoglobin yielded a single pK value of 10.6. The pH dependence of the chemical shift of each of the resonances of Tyr-103 of the cyanoferrimyoglobins from horse and sperm whale could not be fitted with the use of a single pK value, but was consistent with two pK values (about 9.8 and 11.6). Furthermore, the resonances of Czeta and Cgamma of Tyr-103 broadened at high pH. The titration behavior of the tyrosines of sperm whale carbon monoxide myoglobin and horse ferrimyoglobin was also examined. A comparison of all the experimental results indicated that Tyr-151 is exposed to solvent, Tyr-146 is not exposed, and Tyr-103 exhibits intermediate behavior. These results for myoglobins in solution are consistent with expectations based on the crystal structure.     

Tyrosine residues as redox cofactors in human hemoglobin: implications for engineering nontoxic blood substitutes

Respiratory proteins such as myoglobin and hemoglobin can, under oxidative conditions, form ferryl heme iron and protein-based free radicals. Ferryl myoglobin can safely be returned to the ferric oxidation state by electron donation from exogenous reductants via a mechanism that involves two distinct pathways. In addition to direct transfer between the electron donor and ferryl heme edge, there is a second pathway that involves "through-protein" electron transfer via a tyrosine residue (tyrosine 103, sperm whale myoglobin). Here we show that the heterogeneous subunits of human hemoglobin, the alpha and beta chains, display significantly different kinetics for ferryl reduction by exogenous reductants. By using selected hemoglobin mutants, we show that the alpha chain possesses two electron transfer pathways, similar to myoglobin. Furthermore, tyrosine 42 is shown to be a critical component of the high affinity, through-protein electron transfer pathway. We also show that the beta chain of hemoglobin, lacking the homologous tyrosine, does not possess this through-protein electron transfer pathway. However, such a pathway can be engineered into the protein by mutation of a specific phenylalanine residue to a tyrosine. High affinity through-protein electron transfer pathways, whether native or engineered, enhance the kinetics of ferryl removal by reductants, particularly at low reductant concentrations. Ferryl iron has been suggested to be a major cause of the oxidative toxicity of hemoglobin-based blood substitutes. Engineering hemoglobin with enhanced rates of ferryl removal, as we show here, is therefore likely to result in molecules better suited for in vivo oxygen delivery.     

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.     

Assignment of 1H NMR resonances of histidine and other aromatic residues in met-, cyano-, oxy-, and (carbon monoxy)myoglobins

The resolved 1H NMR resonances of the aromatic region in the 270-MHz NMR spectrum of sperm whale, horse, and pig metmyoglobin (metMb) have been assigned, including the observable H-2 and H-4 histidine resonances, the tryptophan H-2 resonances, and upfield-shifted resonances from one tyrosine residue. The use of different Mb species, carboxymethylation, and matching of pK values allows the assignment of the H-4 resonances, which agree in only three cases out of seven with scalar-correlated two-dimensional NMR spectroscopy assignments by others. The conversion to hydroxymyoglobin at high pH involves rearrangements throughout the molecule and is observed by many assigned residues. In sperm whale ferric cyanomyoglobin, nine H-2 and eight H-4 histidine resonances have been assigned, including the His-97 H-2 resonance and tyrosine resonances from residues 103 and 146. The hyperfine-shifted resonances from heme and near-heme protons observe a shift with a pK = 5.3 +/- 0.3 (probably due to deprotonation of His-97, pK = 5.6) and another shift at pK = 10.8 +/- 0.3. The spectrum of high-spin ferrous sperm whale deoxymyoglobin is very similar to that of metMb, which allows the assignment of seven surface histidine H-2 and H-4 resonances and also resonances from the two tryptophan residues and one tyrosine. In diamagnetic sperm whale (carbon monoxy)myoglobin (COMb), 10 His H-2 and 11 His H-4 resonances are observed, and 8 H-2 and 9 H-4 resonances are assigned, including His-64 H-4, the distal histidine. This important resonance is not observed in sperm whale oxymyoglobin, which in general shows very similar titration curves to COMb. Histidine-36 shows unusual titration behavior in the paramagnetic derivatives but normal behavior in the diamagnetic derivatives, which is discussed in the accompanying paper [Bradbury, J. H., & Carver, J. A. (1984) Biochemistry (following paper in this issue)].     

Identification of the myoglobin tyrosyl radical by immuno-spin trapping and its dimerization

5,5-Dimethyl-1-pyrroline N-oxide (DMPO) spin trapping in conjunction with antibodies specific for the DMPO nitrone epitope was used on hydrogen peroxide-treated sperm whale and horse heart myoglobins to determine the site of protein nitrone adduct formation. The present study demonstrates that the sperm whale myoglobin tyrosyl radical, formed by hydrogen peroxide-dependent self-peroxidation, can either react with another tyrosyl radical, resulting in a dityrosine cross-linkage, or react with the spin trap DMPO to form a diamagnetic nitrone adduct. The reaction of sperm whale myoglobin with equimolar hydrogen peroxide resulted in the formation of a myoglobin dimer detectable by electrophoresis/protein staining. Addition of DMPO resulted in the trapping of the globin radical, which was detected by Western blot. The location of this adduct was demonstrated to be at tyrosine-103 by MS/MS and site-specific mutagenicity. Interestingly, formation of the myoglobin dimer, which is known to be formed primarily by cross-linkage of tyrosine-151, was inhibited by the addition of DMPO.     

Autoxidation of myoglobin from bigeye tuna fish (Thunnus obesus)

Native oxymyoglobin (MbO2) was isolated directly from the skeletal muscle of bigeye tuna (Thunnus obesus) with complete separation from metmyoglobin (metMb) on a CM-cellulose column. It was examined for its stability properties over a wide range of pH values (pH 5-12) in 0.1 M buffer at 25 degrees C. When compared with sperm whale MbO2 as a reference, the tuna MbO2 was found to be much more susceptible to autoxidation. Kinetic analysis has revealed that the rate constant for a nucleophilic displacement of O2- from MbO2 by an entering water molecule is 10-times higher than the corresponding value for sperm whale MbO2. The magnitude of the circular dichroism of bigeye tuna myoglobin at 222 nm was comparable to that of sperm whale myoglobin, but its hydropathy profile revealed the region corresponding to the distal side of the heme iron to be apparently less hydrophobic. The kinetic simulation also demonstrated that accessibility of the solvent water molecule to the heme pocket is clearly a key factor in the stability properties of the bound dioxygen.     

Reduction of sperm whale ferrylmyoglobin by endogenous reducing agents: potential reducible loci of ferrylmyoglobin.

The reactivity of the endogenous antioxidants ascorbate, ergothioneine, and urate toward the high oxidation state of sperm whale myoglobin, ferrylmyoglobin-formed upon oxidation of metmyoglobin by H2O2--was evaluated by optical spectroscopy and SDS-PAGE analysis. Depending on whether these antioxidants were present in the reaction mixture before or after the addition of H2O2 to a metmyoglobin suspension, two different effects were observed: (a) In the former instances, ascorbate, ergothioneine, and urate reduced efficiently the oxoferryl moiety in ferrylmyoglobin to metmyoglobin and prevented dimer formation, a process which requires intermolecular cross-link involving specific tyrosyl residues. In addition, all the reducing compounds inhibited--albeit with different efficiencies--dityorosine-dependent fluorescence build up produced via dimerization of photogenerated tyrosyl radicals. (b) In the latter instances, the antioxidants reduced the preformed sperm whale ferrylmyoglobin to a modified metmyoglobin, the spectral profile of which was characterized by a blue shift of the typical 633 nm absorbance of native metmyoglobin. In addition, under these experimental conditions, the antioxidants did not affect dimer formation, thus indicating the irreversible character of the process. The dimeric form of sperm whale myoglobin--separated from the monomeric form by gel electrophoresis of a solution in which ergothioneine was added to preformed ferrylmyoglobin--revealed optical spectral properties in the visible region identical to that of the modified myoglobin. This suggests that the dimeric form of the hemoprotein is redox active, inasmuch as the oxoferryl complex can be reduced to its ferric form. These results are discussed in terms of the potential reactivity of these endogenous antioxidants toward the reducible loci of ferrylmyoglobin, the oxoferryl moiety, and the apoprotein radical.     

New transient species of sperm whale myoglobin in photodissociation of dioxygen from oxymyoglobin

We carried out the flash photolysis of oxy complexes of sperm whale myoglobin, cobalt-substituted sperm whale myoglobin, and Aplysia myoglobin. When the optical absorption spectral changes associated with the O2 rebinding were monitored on the nanosecond to millisecond time scale, we found that the transient spectra of the O2 photoproduct of sperm whale myoglobin were significantly different from the static spectra of deoxy form. This was sharply contrasted with the observations that the spectra of the CO photoproduct of sperm whale myoglobin and of the O2 photoproducts of cobalt-substituted sperm whale myoglobin and Aplysia myoglobin are identical to the corresponding spectra of their deoxy forms. These results led us to suggest the presence of a fairly stable transient species in the O2 photodissociation from the oxy complex of sperm whale myoglobin, which has a protein structure different from the deoxy form. We denoted the O2 photo-product to be Mb*. In the time-resolved resonance Raman measurements, the nu Fe-His mode of Mb* gave the same value as that of the deoxy form, indicating that the difference in the optical absorption spectra is possibly due to the structural difference at the heme distal side rather than those of the proximal side. The structure of Mb* is discussed in relation to the dynamic motion of myoglobin in the O2 entry to or exit from the heme pocket. Comparing the structural characteristics of several myoglobins employed, we suggested that the formation of Mb* relates to the following two factors: a hydrogen bonding of O2 with the distal histidine, and the movement of iron upon the ligation of O2.     

A comparison of the intrinsic fluorescence of red kangaroo, horse and sperm whale metmyoglobins

Several metmyoglobins (red kangaroo, horse and sperm whale), containing different numbers of tyrosines, but with invariant tryptophan residues (Trp-7, Trp-14), exhibit intrinsic fluorescence when studied by steady-state front-face fluorometry. The increasing tyrosine content of these myoglobins correlates with a shift in emission maximum to shorter wavelengths with excitation at 280 nm: red kangaroo (Tyr-146) emission maximum 335 nm; horse (Tyr-103, -146) emission maximum 333 nm; sperm whale (Tyr-103, -146, -151) emission maximum 331 nm. Since 280 nm excites both tyrosine and tryptophan, this strongly suggests that tyrosine emission is not completely quenched but also contributes to this fluorescence emission. Upon titration to pH 12.5, there is a reversible shift of the emission maximum to longer wavelengths with an increase greater than 2-fold in fluorescence intensity. With excitation at 305 nm, a tyrosinate-like emission is detected at a pH greater than 12. These studies show that: (1) metmyoglobins, Class B proteins containing both tyrosine and tryptophan residues, exhibit intrinsic fluorescence; (2) tyrosine residues also contribute to the observed steady-state fluorescence emission when excited by light at 280 nm; (3) the ionization of Tyr-146 is likely coupled to protein unfolding.     

The Role of H2O2-Generated Myoglobin Radical in Crosslinking of Myosin

The mechanism of myoglobin/H₂O₂ derived peroxidation of myosin was studied by comparing the catalytic activity of myoglobin and horseradish peroxidase using O-dianisidine, N-acetyl tyrosine and myosin as substrates. It was found that both hemoproteins induced myosin crosslinking and concomitant tyrosines oxidation to bityrosines, suggesting inter-molecular coupling of tyrosines in the crosslinking. The enzymatic activity of both hemoproteins on myosin was weak compared to small substrates. While horseradish peroxidase was much more active than myoglobin on small substrates, the reverse was true for myosin peroxidation. Since the suicidal interaction of myoglobin with H₂O₂ forms unstable tyrosine radicals, we suggest that the increased activity of myoglobin on myosin results from an efficient electron transfer between surface tyrosines of myosin and myoglobin but not horseradish peroxidase. These conclusions were supported by evidence that sperm whale myoglobin, which contains two active tyrosines -the heme-adjacent (tyrosine-103) and the surface (tyrosine-151), is more active as a mediator of myosin peroxidation than horse heart myoglobin which is devoid of the surface tyrosine.     

A 1H NMR comparison of the met-cyano complexes of elephant and sperm whale myoglobin. Assignment of labile proton resonances in the heme cavity and determination of the distal glutamine orientation from relaxation data

The met-cyano complex of elephant myoglobin has been investigated by high field 1H NMR spectroscopy, with special emphasis on the use of exchangeable proton resonances in the heme cavity to obtain structural information on the distal glutamine. Analysis of the distance dependence of relaxation rates and the exchange behavior of the four hyperfine shifted labile proton resonances has led to the assignment of the proximal His-F8 ring and peptide NHs and the His-FG3 ring NH and the distal Gln-E7 amide NH. The similar hyperfine shift patterns for both the apparent heme resonances as well as the labile proton peaks of conserved resonances in elephant and sperm whale met-cyano myoglobins support very similar electronic/molecular structures for their heme cavities. The essentially identical dipolar shifts and dipolar relaxation times for the distal Gln-E7 side chain NH and the distal His-E7 ring NH in sperm whale myoglobin indicate that those labile protons occupy the same geometrical position relative to the iron and heme plane. This geometry is consistent with the distal residue hydrogen bonding to the coordinated ligand. The similar rates and identical mechanisms of exchange with bulk water of the labile protons for the three conserved residues in the elephant and sperm whale heme cavity indicate that the dynamic stability of the proximal side of the heme pocket is unaltered upon the substitution (His----Gln). The much slower exchange rate (by greater than 10(4] of the distal NH in elephant relative to sperm whale myoglobin supports the assignment of the resonance to the intrinsically less labile amide side chain.     

The complete amino acid sequence of the major component myoglobin of Amazon river dolphin (Inia geoffrensis).

The complete amino acid sequence of the major component myoglobin from Amazon River dolphin, Inia geoffrensis, was determined by specific cleavage of the protein to obtain large peptides which are readily degraded by the automatic sequencer. Three easily separable peptides were obtained by cleaving the protein with cyanogen bromide at the methionine residues and four peptides were obtained by cleaving the methyl-acetimidated protein with trypsin at the arginine residues. From these peptides over 85% of the sequence was completed. The remainder of the sequence was obtained by fragmentation of the large cyanogen bromide peptide with trypsin. This protein differs from that of the common porpoise, Phocoena phocoena, at seven positions, from that of the common dolphin, Delphinus delphis, at 11 positions, and from that of the sperm whale, Physeter catodon, at 15 positions. By comparison of this sequence with the three-dimensional structure of sperm whale myoglobin it appears that those residues close to the heme group are most conserved followed by those in nonhelical regions and lastly by those in the helical segments. All of the substitutions observed in this sequence fit easily into the three-dimensional structure of the sperm whale myoglobin.     

Redox cycling of myoglobin and ascorbate: a potential protective mechanism against oxidative reperfusion injury in muscle

Metmyoglobin catalyzes the decomposition of H2O2 as well as other hydroperoxides by using ascorbic acid as a substrate. The ratio of H2O2 reduced to ascorbate oxidized is close to one, whereas the rate of oxidation is directly proportional to both H2O2 and metmyoglobin concentrations. Ascorbate also prevents the protein modifications and the O2 evolution that accompany the reaction of metmyoglobin with hydroperoxides. In the absence of ascorbate, myoglobin and H2O2 promote the peroxidation of unsaturated fatty acids and, thus, may cause damage to cellular constituents. However, lipid peroxidation is inhibited in the presence of ascorbate and, for this reason, it is suggested that this heme protein functions in the opposite manner. The redox cycling of myoglobin by ascorbate may act as an important electron "sink" and defense mechanism against peroxides during oxidative challenge to muscle.