13C-NMR spectroscopy of acetyltyrosyl-guanidinated horse heart cytochrome c

The tyrosine residues of guanidinated horse heart cytochrome c have been specifically acetylated by reaction with N-[1-13C]acetylimidazole (90 atom%). Acetylation was monitored by 13C-NMR spectroscopy. The tyrosine residues were found to show widely varying reactivities ranging from one that is completely and exclusively acetylated at low reagent concentration (residue 67) to one that is acetylated only when the protein is unfolded (residue 97). Homogeneous derivatives were prepared containing one (either residue 67 or 97), three 48, 67 and 74), or four (residues 48, 67, 74 and 97) O-[1-13C]acetyl groups. 13C-NMR spectra of selected derivatives were obtained at pH 5.8, in the presence of cyanide ion, in the ferrous and ferric oxidation states, and after denaturation with 6M guanidine hydrochloride. The O-[1-13C]acetyltyrosyl resonances gave chemical shift values ranging from 171.8 to 176.0 ppm. These resonances were assigned to specific groups based on the known order of reactivity of the tyrosyl side chains toward N-acetylimidazole. The chemical shift of O-[1-13C]acetyltyrosyl 67 was found to be particularly sensitive to changes in protein structure. The proximity of this group to the heme makes it subject to distance-dependent paramagnetic and ring current effects. Acetylation of tyrosyl 74 gives rise to a pH-dependent equilibrium between conformers in the ferric state and a conformation change in the ferrous state. Acetylation of this residue also leads to an absorbance decrease at 695 nm that can be related to the 13C-NMR-detected conformational equilibrium. Addition of cyanide ion abolished this equilibrium.     

Fluorescence and the structure of proteins. XXI. Fluorescence of aminotyrosyl residues in peptides and helical proteins.

1. Five peptides containing tyrosine were converted to the 3-aminotyrosyl peptides by nitration with tetranitromethane and subseuqent reduction of the nitro groups to amino groups. The fluorescence of these aminotyrosyl residues was found to be quite similar to that of 3-aminotyrosine and it is concluded that the fluorescence is not sensitive to incorporation of the amino acid into the peptide chain. 2. Fluorescence of 3-aminotyrosine derivatives was sensitive, however, to the nature of the solvent; as the dielectric constant decreased, fluorescence was enhanced ten fold and the emission maximum shifted from the 350-370 nm value in aqueous solution to 320 nm. It is predicted that similar differences might be expected for exposed and buried aminotyrosyl residues in a protein. 3. Exposed tyrosyl residues on the helical protein tropomyosin and a helical segment of paramyosin were aminated in part (39% and 34% of the total tyrosyl residues, respectively). The fluorescence of the aminated tyrosyl residues on these proteins was similar to that of the aminotyrosyl peptides in an aqueous medium. Although the fluorescence efficiency of an aminotyrosyl residue was much lower than that of a tyrosyl residue, it was easy to distinguish the fluorescence of the aminotyrosyl residues (350-355 nm) on the protein from that arising from unmodified tyrosyl residues (305 nm).     

Behaviour of tyrosyl residues of calf-thymus histone F3. Difference-spectroscopy studies.

We have studied the behaviour of microenvironments containing tyrosine of calf thymus histone F3 (or histone H3) by using the difference spectroscopy techniques of thermal and solvent perturbation. By comparison of the parameters found for the models L-tyrosine methyl ester and N-acetyl-L-tyrosine ethyl ester with those for the protein at various conditions, several aspects of the tertiary structure of histone F3 become apparent. The raising of ionic strength produces a general burial of tyrosyl residues of the histone, whereas low pH or urea treatment causes a complete exposure of tyrosyl groups with respect to the solvent. Anomalously high values can also be observed of accessibility of the perturbants sucrose and ethylene glycol at low concentrations of phosphate buffer. The relevance of these findings towards a better understanding of the tertiary structure of histone F3 and of its interactions with DNA is discussed.     

Structural studies of human chorionic gonadotropin and its subunits using tyrosine fluorescence.

The fluorescence properties of the tyrosyl residues of human chorionic gonadotropin (hCG) and its α and β subunits have been examined. The effects of pH, guanidine, and disulfide cleavage on the intensity and polarization of the fluorescence suggest that the isolated subunits possess little, if any, tertiary structure beyond that which is stabilized by the disulfide bonds. Essentially all of the fluorescence of hCG and its subunits was accessible to quenching by iodide ions. Similar results were observed for several other proteins whose fluorescence originates from tyrosyl residues. Thus, we have confirmed and extended the conclusion of R. W. Cowgill ((1966) Biochim. Biophys. Acta120, 196) that the buried tyrosyl residues in ribonuclease fluoresce with a much lower quantum yield than those which are exposed. The dissociation of hCG into its subunits was accompanied by an increase in fluorescence, suggesting the exposure of tyrosyl residues. This was confirmed by difference absorption measurements which indicate a net exposure of two to three tyrosyl residues upon dissociation of the subunits. An additional 0.6 tyrosine was exposed when the disulfide bonds of the β-subunit were cleaved. The polarization of the fluorescence of hCG-β was high (P = 0.19) and, unlike several other proteins with high polarization, could not be lowered by denaturing conditions. Only by cleavage of the disulfide bonds could the fluorescence polarization of either subunit be lowered to a value (P = 0.08) characteristic of a random polypeptide. It appears that the disulfide bonds play an important role in maintaining the rigidity of the fluorescent tyrosyl residues, located at or near the surface of the protein.     

Conformational changes in pantetheine hydrolase as a function of guanidinium chloride concentration

The denaturation of pantetheinase (pantetheine hydrolase, EC 3.5.1.-) was followed in guanidinium chloride using tyrosyl and tryptophanyl residues as probes in connection with change in enzymatic activity. Movements of tryptophanyl and tyrosyl residues during denaturation were studied by second-derivative and fluorescence spectroscopy and the number of these amino acids present in the protein was calculated from spectroscopic data. Pantetheinase shows a very high resistance to denaturation, being completely unfolded at guanidinium chloride concentration higher than 6.5 M. Monitoring enzymatic activity shows that inactivation of the enzyme occurred before noticeable conformational changes were detected and it is suggested that the conformation of the active site is flexible and easily perturbable compared to the protein as a whole. This inactivation is reversible, as shown by renaturation experiments. Second-derivative and fluorescence spectra showed also that tyrosyl and tryptophanyl residues are largely exposed in the native protein, confirming its hydrophobic behavior.     

Specific perturbation by Ca2+ of tyrosyl residue 138 of calmodulin.

Calmodulin that was selectively nitrated at tyrosyl residue 99 or 138 was used as a regional spectral probe of the structural changes associated with Ca2+ or Mg2+ binding to the protein. Perturbations of the spectral properties of the nitrotyrosyl residues correlated well with those of the tyrosyl residues in unmodified calmodulin. The spectral properties of tyrosyl residue 99 were sensitive to ionic strength, but not to Ca2+ binding to the protein. The spectrum of tyrosyl residue 138, in contrast, was markedly affected both by ionic strength and Ca2+ binding. Although Mg2+ caused spectral changes, they were clearly different from those produced by Ca2+ and resembled instead the less specific changes elicited by Na+. Conformational changes associated with Ca2+ but not Mg2+ binding to calmodulin seem to affect selectively the microenvironment around tyrosyl residue 138.     

Ultraviolet resonance Raman and absorption difference spectroscopy of myoglobins: titration behavior of individual tyrosine residues

The UV resonance Raman spectra of horse and sperm whale myoglobin excited at 240 nm show bands between 600 and 1700 cm-1 which derive from tyrosyl and tryptophyl residues. No significant contribution from phenylalanine and peptide backbone vibrations occurs at this excitation wavelength. We examine the pH dependence of the UV resonance Raman and UV absorption difference spectra of these myoglobins to correlate the local protein environment of the tyrosyl residues as given by the protein crystal structure to their pKa values, molar absorptivities, and Raman cross sections. Some of our pKa values for the tyrosinate residues of horse Mb differ from those of previous studies. We show that the lambda max values, the molar absorptivities, and the Raman cross sections are sensitive to the local environment of the tyrosinate residues in the protein. We relate differences in the tyrosyl absorption spectra to differences in Raman cross sections. In addition, we discuss the importance to the Raman cross sections of the local electromagnetic field enhancement due to the dielectric environment of the tyrosinate residues in the protein. This local field should scale the Raman cross sections in a way useful as a probe of the average aromatic amino acid residue environment.     

Estimation of tryptophyl and tyrosyl exposure in tryptophan-rich proteins by ultraviolet difference spectrophotometry. Lysozyme and Chymotrypsinogen.

Ultraviolet difference absorption spectra produced by ethylene glycol were measured for hen lysozyme [EC 3.2.1.17] and bovine chymotrypsinogen. N-Acetyl-L-tryptophanamide and N-acetyl-L-tyrosinamide were employed as model compounds for tryptophyl and tyrosyl residues, respectively, and their ultraviolet difference spectra were also measured as a function of ethylene glycol concentration. By comparison of the slopes of plots of molar difference extinction coefficients (delta epsilon) versus ethylene glycol concentration for the proteins with those of the model compounds at peak positions (291-293 and 284-287 nm) in the difference spectra, the average number of tyrosyl as well as tryptophyl residues in exposed states could be estimated. The results gave 2.7 tryptophyl and 1.9 tyrosyl residues exposed for lysozyme at pH 2.1 and 2.6 tryptophyl and 3.4 tyrosyl residues exposed for chymotrypsinogen at pH 5.4. The somewhat higher tyrosyl exposure of chymotrypsinogen, compared with the findings from spectrophotometric titration and chemical modification, was not unexpected, because delta epsilon285 was larger than delta epsilon292, and the situation is discussed with reference to preferential interaction of ethylene glycol with the tyrosyl residues and/or side chains in the vicinity of the chromophore in the protein. The procedure employed in the present work seems to be suitable for estimation of the average number of exposed tryptophyl and tyrosyl residues in tryptophan-rich proteins. The effects of ethylene glycol on the circular dichroism spectra of lysozyme at pH 2.1 and chymotrypsinogen at pH 5.4 were also investigated. At high ethylene glycol concentrations, both proteins were found to undergo conformational changes in the direction of more ordered structures, presumably more helical for lysozyme and more beta-structured for chymotrypsinogen.     

Spectrophotometric titration of tyrosyl residues in alkaline mesentericopeptidase.

At pH 7.0 the alkaline mesentericopeptidase has ultraviolet absorption spectrum with a minimum at 251 nm and a maximum at 280 nm and no visible absorption. From the tyrosine to tryptophan ratio a value of 3 tryptophyl residues per mole of protein is obtained. The molar extinction coefficient at 280 nm is 3.55 X 10(4)M-1cm-1. Spectrophotometric titration studies show that the molecule of mesentericopeptidase contains seven phenolic groups with a pKapp - 9.92 and four to five groups with a pKapp = 11.96. Denaturing agents, such as 5 M guanidine hydrochloride or alkali, normalize the ionization of the tyrosyl residues. There is a good correlation between the spectrophotometric titration data and the results for the reactivities of the tyrosines in mesentericopeptidase towards tetranitromethane. The correlation is explained by the mechanism of nitration. Conclusions about the state of the tyrosyl residues and the three-dimensional structure of mesentericopeptidase are made.     

Spectrophotometric titration of phenolic groups of pepsin.

The ionization of tyrosine residues in diazotized pepsin under various solvent conditions was studied. All tyrosyl residues of the protein titrated normally with a pK of 10.02 in 6 M guanidine hydrochloride solution. On the other hand, two stages in the phenolic group titration curve were observed for the inactivated protein in the absence of guanidine hydrochloride; only about 10 tyrosine residues ionized reversibly up to pH 11, above which titration was irreversible. The irreversible titration zone corresponds to the pH range 11--13 in which unfolding, leading to the random coil state, was shown to occur by circular dichroism and viscosity measurements. The number of tyrosine residues exposed in the native and alkali-denatured (pH 7.5) states of diazotized protein were also studied by solvent perturbation techniques; 10 and 12 groups are exposed in the native and denatured states, respectively.     

Fluorimetric and spectrophotometric studies of DPN-linked isocitrate dehydrogenase from bovine heart. Properties of tyrosyl and tryptophyl residues.

The emission maximum of DPN-linked isocitrate dehydrogenase in pH 7.07 buffer is shifted from 317 to 324 nm and fluorescence intensity is decreased when the excitation wave-length is varied from 270 to 290 nm; in 0.2 M KOH, where the fluorescence of tyrosyl residues is almost completely quenched, a further substantial decline in quantum yield of protein fluorescence and a red shift of the emission peak to 339 nm occur. The latter should be due mainly to tryptophyl residues. The enzyme contains 9.4 tyrosyl residues per subunit of molecular weight 42,000 determined spectrophotometrically (295 nm) at pH 13, in good agreement with a tyrosine content of 9.7 by amino acid analysis. No more than 1.1 tyrosyl residues per subunit can be detected up to pH 10.6 at 7 degrees upon prolonged incubation. The increase in absorption at 295 nm with increasing pH is related to loss of enzyme activity and results in a red shift of the emission maximum, and decreased fluorescence intensity. Treatment of the enzyme in a Li+-containing buffer at pH 7.5 with an excess of N-acetylimidazole results in (a) modification of 1.1 tyrosyl residues per subunit, (b) a 30% decrease in enzyme activity, (c) a 6-nm red shift in emission maximum, and (d) a decrease in fluorescence intensity. Manganous DL-isocitrate (1.06 mM) prevents the acetylation of the enzyme. Deacetylation of the O-acetylated enzyme by hydroxylamine completely restores the enzyme activity and reverses the spectral changes. The acetylation studies indicate that the reactive tyrosyl residue does not participate directly in catalysis but may be involved in maintaining the proper conformation of the active enzyme center. A net of 1 of the 2 tryptophyl residues per subunit is perturbed immediately by a number of solvents. This perturbation is not affected by manganous isocitrate, whereas exposure of tyrosyl residues occurs only with time and is prevented by the substrate. The perturbation of the tryptophyl residue is accompanied by a red shift of the fluorescence emission maximum. The more exposed tryptophyl residue may contribute to the energy transfer from protein to nucleotides since the quenching of protein fluorescence upon binding of DPN+, DPNH, or ADP by enzyme results in a blue shift of the emission maximum. Manganous DL-isocitrate (1.06 mM) quenches protein fluorescence by 16% without a shift in emission peak and does not affect the relative extent of fluorescence quenching induced by the nucleotides.     

Double-headed protease inhibitors from black-eyed peas. II. Structural studies by optical absorption and circular dichroism.

Two new double-headed protease inhibitors from black-eyed peas have amino acid compositions typical of the low molecular weight protease inhibitors from legume seeds. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) contains no tryptophan, 1 tyrosine, and 14 half-cystines out of 83 amino acid residues per monomer. Black-eyed pea trypsin inhibitor (BEPTI) contains no tryptophan, 1 tyrosine, and 14 half-cystines out of 75 residues per monomer. The molar extinctions at 280 nm are 2770 for BEPCI and 3440 for BEPTI. The single tyrosyl residue is very inaccessible to solvent in native BEPCI and BEPTI at neutral pH and titrates anomalously with an apparent pK = 12. Ionization of tyrosine is complete in 13 hours above pH 12. No heterogeneity of the local environment of the tyrosyl residues in different subunits can be detected spectrophotometrically. The large number of cystine residues leads to an intense and complex near-ultraviolet CD spectrum with cystine contributions in the regions of 248 and 280 nm and tyrosine contributions at 233 and 280 nm. An intact disulfide structure is required for appearance of the tyrosyl CD bands. The inhibitors are unusually resistant to denaturation when compared with similar low molecular weight proteins of high disulfide content. All observations are consistent with a far more rigid structure for BEPCI and BEPTI than for a typical protein.     

Ligand binding and enzymic catalysis coupled through subunits in tyrosyl-tRNA synthetase.

The interaction of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus with its substrates in the aminoacyl adenylation reaction has been studied by stopped-flow fluorescence. The observed changes have been assigned to their chemical and physical processes by comparison with equilibrium dialysis, pyrophosphate exchange kinetics and rapid quenching and sampling techniques to give the rate constants for ligand binding, the formation of tyrosyl adenylate, and the reverse reaction. The stoichiometry of tyrosine and ATP binding in the catalytic process has been determined directly by equilibrium dialysis and equilibrium gel filtration under pyrophosphate exchange conditions, i.e., where a steady state has been set up in which the equilibrium position favors starting materials. It is shown that the rate-determining step in the formation of tyrosyl adenylate involves 1 mole each of tyrosine and ATP. A second mole of tyrosine and ATP bind to the aminoacyl adenylate complex stabilizing the high-energy intermediate. The enzyme tyrosyl adenylate complex that is isolated by gel filtration is in a different conformational state from that in the presence of tyrosine and ATP.     

Phosphorylation and activation of p40 tyrosine kinase by casein kinase-1

Because examination of regulatory trans-phosphorylations can help elucidate the cellular functions of tyrosyl protein kinases, we have investigated the effects of phosphorylation by casein kinase-1 on the activity of the p40 tyrosyl protein kinase. We find that casein kinase-1 can phosphorylate the p40 tyrosyl kinase on serine and threonine residues, in part on a unique tryptic peptide. The phosphorylation induces a substantial increase in the tyrosyl protein kinase activity of p40, in contrast to most instances in which serine/threonine phosphorylation inhibits activity of tyrosyl protein kinases. These findings raise the possibility that p40 might be part of a protein phosphorylation network in which casein kinase-1 participates.     

The states of tyrosyl and tryptophyl residues in a protein proteinase inhibitor (Streptomyces subtilisin inhibitor

The states of tyrosyl and tryptophyl residues of a dimeric protein proteinase inhibitor, Streptomyces subtilisin inhibitor (Sato, S & Murao, S. (1973), Agric. Biol. Chem. 37, 1067) were studies by solvent perturbation difference spectroscopy with methanol, ethylene glycol, polyethylene glycol, and deuterium oxide as perturbants, and by spectrophotometric titration at alkaline pH. It appeared that all three tyrosyl residues per monomer of the inhibitor were exposed on the surface of the molecule, and their apparent pK values were estimated separately to be 9.58, 11.10, and 12.42. The single tryptophyl residue per monomer of the inhibitor appeared to be partially buried in the protein molecule.     

The phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus aureus. Complete tyrosine assignments in the 1H nuclear-magnetic-resonance spectrum of the phosphocarrier protein HPr.

Upon nitration of the phosphocarrier protein HPr three nitrated derivatives of the protein were isolated: mononitrated HPr, dinitrated HPr and trinitrated HPr. Tryptic digestion of the derivatives leads to nitrotyrosine-containing peptides which were isolated and characterized by amino acid analysis. This resulted in the determination of the positions of the nitrated tyrosyl residues in the amino acid sequence. In mononitrated HPr only Tyr-56 was modified, in dinitrated HPr both Tyr-56 and Tyr-37 had reacted with the nitrating agent; modification of all three tyrosyl residues in trinitrated HPr required more drastic reaction conditions. The nuclear magnetic resonance spectra of the three derivatives allowed the assignments of the tyrosine resonances as follows: Tyr-A and Tyr-B with pK values of 10.5 and 11.5 were designated Tyr-56 and Tyr-37 whereas Tyr-C, whose protons are not titratable before denaturation of the protein, was assigned to Tyr-6 in the amino acid sequence. The nitration studies, together with the titration behaviour of the three tyrosines, indicate the topology of the tyrosyl residues to be as follows: Tyr-56 is located at the surface, Tyr-37 is slightly buried, Tyr-6 is deeply buried. The nitrotyrosyl derivatives retain their biological activity.     

The stereospecificity of protein kinases

To test whether cellular protein kinases exist that phosphorylate D-amino acid residues, a method was developed for separating O-phospho-D-serine from O-phospho-L-serine and O-phospho-L-tyrosine from O-phospho-D-tyrosine. This was accomplished by converting these amino acids to the L-leucyl dipeptide derivatives followed by separation of the diastereomers by anion-exchange high-performance liquid chromatography. The enantiomeric content of these D- and L-residues were measured in hydrolysates of 32P-labeled proteins produced by the protein kinases of human erythrocytes and the tyrosyl protein kinase of the Abelson leukemia virus. We found no measurable D-phosphoserine in erythrocyte membrane proteins under conditions where a 1% content of this residue relative to L-phosphoserine would have been detected. These values can be used to place an upper hypothetical limit on the fraction of erythrocyte protein kinase activity that is specific for serine residues in the D-configuration. In separate experiments, we examined the specificity of the tyrosyl protein kinases. We found that all of the phosphotyrosine that we isolated from the erythrocyte band 3 NH2-terminal fragment and from the autophosphorylation of the Abelson virus tyrosyl kinase was in the L-configuration.     

Chemical accessibility of tyrosyl and lysyl residues in turnip yellow mosaic virus capsids.

The chemical accessibility of tyrosyl residues in TYMV capsids was studied by spectrophotometric titration and with the nitrating agent tetranitromethane. That of the lysyl residues was probed with trinitrobenzenesulfonate. Attempts to test their accessibility in virions were also made. Since some of these reactions were accompanied by structural changes, degradation of the particles were monitored with ultracentrifugation and light-scattering measurements. Alkaline titration of TYMV capsids induced ionization of two of the three tyrosyl residues per subunit at pH 11.3, but the third tyrosyl ionized with an apparent pK of 12.65, concomitantly with the degradation of the capsids. Reaction with tetranitromethane suggested that one tyrosyl residue per subunit can easily be nitrated and initiates degradation, after which the remaining residues also react. In intact capsids, five out of seven lysyl residues per subunit reacted readily with trinitrobenzenesulfonate. The other two lysyl residues were trinitrophenylated only after degradation of the capsids. On the other hand, all seven lysyl residues per subunit were easily trinitrophenylated in virions, during which reaction the virions disintegrated. The demonstrated chemical inaccessibility of specific numbers of tyrosyl and lysyl residues in TYMV capsids and the observed structural consequences to the capsids when the residues were made to react are consistent with previously published properties of the cysteinyl and tryptophanyl residues. The findings suggest that in the capsid the central region of the TYMV polypeptide chain is buried and might represent a site of contact between neighboring subunits.     

Chemical Nature and Mode of Stabilization of Egg-shell in Helicometra pulchella (Rudolphi, 1819) (Digenea: Opecoelidae)

The chemical nature and mode of stabilization of egg-shell protein in digenetic trematode Helicometra pulchella (Rudolphi, 1819) have been investigated using histochemical techniques. It was found that the egg-shell is stabilized by quinone-tanning together with dityrosine. Other structural proteins (elastin, collagen and keratin-like proteins), glycogen and acid mucopolysaccharides were absent in egg-shell. Tyrosine was present in vitelline cells and immature egg-shell indicating that the proteins involved in quinone-tanning were tyrosine rich and tyrosyl residues are modified to form dityrosine in mature egg-shell.     

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.