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.     

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.     

Electrochemical generation of ferrylmyoglobin during oxidation of styrene with films of DNA and a poly (ester sulfonic acid) ionomer.

The chemistry of electrochemically-driven myoglobin-catalyzed oxidation of styrene was investigated in films of DNA or Eastman AQ ionomer on optically transparent electrodes. Conversion of styrene to styrene oxide proceeded via a ferrylmyoglobin radical intermediate. Ferrylmyoglobins were clearly detected by spectroelectrochemistry in films of 1-4 mm thick. The ferrylmyoglobin radical is produced by reaction of metmyoglobin (Mb) in the films with hydrogen peroxide formed by electrochemical catalytic reduction of oxygen catalyzed by Mb. Thus, electrochemically-driven styrene oxidation with these films proceeds by a 'doubly catalytic' electrode-driven reduction-oxidation pathway. Ferrylmyoglobin formation during electrolysis of Mb-DNA films in aerobic solutions was much faster, and styrene oxidation occurred with less Mb decomposition compared to the Mb-AQ films. The better performance of Mb-DNA films is correlated with a larger fraction of electroactive Mb and better stability than for the Mb-AQ films.     

Free fatty acids enhance the oxidation of oxymyoglobin and inhibit the peroxidase activity of metmyoglobin

The effect of low concentrations of sodium oleate on the oxidation of oxymyoglobin to metmyoglobin has been examined. This long chain fatty acid results in a tripling of the initial rate (1.5-4.3 h-1) at which oxymyoglobin is converted to metmyoglobin and more than doubling of the rate of the long-term reaction (0.12-0.33 h-1). Examination of rate constant enhancement over a range of oleate concentrations (0-0.215 mM) has allowed an estimate of association constants for both phases of the reaction system. The peroxidase activity expressed by metmyoglobin towards hydrogen peroxide is inhibited by the presence of sodium oleate by a fivefold increase in the apparent Km value (0.33-1.77 mM). The observed changes in oxymyoglobin concentration over time are discussed in terms of competition between metmyoglobin, which acts as a peroxidase decreasing in situ concentrations of H2O2, and oxymyoglobin, which also is oxidized by the peroxide. It is shown that oleate can bind to metmyoglobin and azidometmyoglobin, but not oxymyoglobin. Catalase reduces the oxidation rates of oxymyoglobin in the presence or in the absence of oleate, substantiating the involvement of H2O2. The results are discussed in relation to the potential increase in tissue peroxidations in the presence of ischaemically elevated fatty acid concentrations.     

Nitric oxide as an antioxidant.

Benzoate monohydroxy compounds, and in particular salicylate, were produced during interaction of ferrous complexes with hydrogen peroxide (Fenton reaction) in a N2 environment. These reactions were inhibited when Fe complexes were flushed, prior to the addition in the model system, by nitric oxide. Methionine oxidation to ethylene by Fenton reagents was also inhibited by nitric oxide. Myoglobin in several forms such as metmyoglobin, oxymyoglobin, and nitric oxide-myoglobin were interacted with an equimolar concentration of hydrogen peroxide. Spectra changes in the visible region and the changes in membrane (microsomes) lipid peroxidation by the accumulation of thiobarbituric acid-reactive substances (TBA-RS) were determined. The results showed that metmyoglobin and oxymyoglobin were activated by H2O2 to ferryl myoglobin, which initiates membrane lipid peroxidation; but not nitric oxide-myoglobin, which, during interaction with H2O2, did not form ferryl but metmyoglobin which only poorly affected lipid peroxidation. It is assumed that nitric oxide, liganded to ferrous complexes, acts to prevent the prooxidative reaction of these complexes with H2O2.     

The Crystal-Solution Problem of Sperm Whale Myoglobin

The central question to be discussed in this paper is whether the structure established for sperm whale myoglobin in the crystalline state is the same as that of the protein in solution. As judged by its ultraviolet optical rotatory dispersion, the helical content of metmyoglobin in solution does not differ from that in the crystal, 77 per cent. Although an uncertainty of about ±5 per cent must attach to this result, it excludes many alternative arrangements of the polypeptide chain. The folding of the chain may be further restricted to the basic form seen in the crystal if the dimensions of the molecule in solution and the interactions of specific chemical groups are taken into account. Since the rotatory dispersion of metmyoglobin is constant with respect to ionic strength, and since the dispersions of reduced and oxymyoglobin reveal no change in helical content upon their formation from metmyoglobin, one may infer that the structure of the protein is largely maintained both as it dissolves and during its reversible combination with oxygen. The crystallographic model of myoglobin thus offers a structural basis for attempting to explain its physiological function in solution. The relevance of this conclusion to the crystal-solution problems presented by other species of protein is then best seen in the light of common factors that govern the equilibrium of all proteins between crystal and solution.     

The Respiratory Chain of Plant Mitochondria: XIV. Ordering of Ubiquinone, Flavoproteins, and Cytochromes in the Respiratory Chain

The effect of initial oxygen concentration on the rate and extent of oxidation of the respiratory chain carriers of anaerobic mitochondria from mung bean (Phaseolus aureus) seedlings was examined. The substrate was succinate, with malonate added to give malonate to succinate ratios of 6 to 12, thereby minimizing the flow of reducing equivalents from substrate and insuring maximal extent of oxidation of the carriers. The ratio of oxidizing equivalents available from oxygen to reducing equivalents available from reduced ubiquinone, designated the equivalents ratio, varied from 30 to 1. Cytochromes aa₃ and c₅₄₇ have unaltered oxidation half-times, designated t_{½ on}, as the equivalents ratio is reduced from 30 to 3, and the extent of oxidation is decreased by about 25%. The time of the oxidation-reduction cycle induced by the oxygen pulse, calculated from the point of half oxidation to that of half reduction and designated t_{½ off}, decreases 200 fold with this reduction in equivalents ratio. The oxidation half-time, t_{½ on}, for ubiquinone is unaltered by decreasing the equivalents ratio from 6 to 1; the value of t_{½ off} decreases only 30% while the extent of oxidation decreases 50%. The values of t_{½ on} and t_{½ off} and the extent of oxidation of cytochrome b₅₅₃ and flavoprotein Fp_ha were all much reduced at low equivalents ratios. The results, plus results from previous studies, indicate that there is the following linear sequence of components in the plant respiratory chain:     

Reduction of ferrylmyoglobin by the spin trap N-tert-butyl-alpha-phenylnitrone (PBN) in aqueous solution and during freezing

The hypervalent muscle pigment ferrylmyoglobin, formed by activation of metmyoglobin by hydrogen peroxide, was found to be reduced in a second-order reaction by N-tert-butyl-alpha-phenylnitrone (PBN, often used as a spin trap). In acidic aqueous solution at ambient temperature, the reduction is relatively slow (deltaH++ = 65+/-2kJ x mol(-1) and deltaS++ = -54+/-7 J x mol(-1). K(-1) for pH = 5.6), but phase transitions during freezing of the buffered solutions accelerates the reaction between ferrylmyoglobin and PBN. In these heterogenous systems at low temperature (but not when ice-formation was inhibited by glycerol), a PBN-derived radical intermediate was detected by ESR-spectroscopy, identified as a nitroxyl radical by a parallel nitrogen hyperfine coupling constant of 31.8 G, and from microwave power saturation behavior concluded not to be located in the heme-cleft of the protein. The acceleration of the reaction is most likely caused by a lowering of the pH during the freezing of the buffered solutions whereby ferrylmyoglobin becomes more oxidizing.     

The reactivity of thiols and disulfides with different redox states of myoglobin. Redox and addition reactions and formation of thiyl radical intermediates.

The reactivity of several thiols, including glutathione, dihydrolipoic acid, cysteine, N-acetyl cysteine, and ergothioneine, as well as several disulfides, toward different redox states of myoglobin, mainly met-myoglobin (HX-FeIII) and ferrylmyoglobin (HX-FeIV=O), was evaluated by optical spectral analysis, product formation, and thiyl free radical generation. Only dihydrolipoic acid reduced met-myoglobin to oxy-myoglobin, whereas all the other thiols tested did not interact with met-myoglobin. Although the redox transitions involved in the former reduction were expected to yield the dihydrolipoate thiyl radical, the reaction was EPR silent. Conversely, all thiols interacted to different extent with the high oxidation state of myoglobin, i.e. ferrylmyoglobin, via two processes. First, direct electron transfer to heme iron in ferrylmyoglobin (HX-FeIV=O) with formation of met-myoglobin (HX-FeIII) or oxymyoglobin (HX-FeIIO2); the former transition was effected by all thiols except dihydrolipoate, which facilitated the latter, i.e. the formation of the two-electron reduction product of ferrylmyoglobin. Second, nucleophilic addition onto a pyrrole in ferrylmyoglobin with subsequent formation of sulfmyoglobin. The contribution of either direct electron transfer to the heme iron or nucleophilic addition depended on the physicochemical properties of the thiol involved and on the availability of H2O2 to reoxidize met-myoglobin to ferrylmyoglobin. The thiyl radicals of glutathione, cysteine, and N-acetylcysteine were formed during the interaction of the corresponding thiols with ferrylmyoglobin and detected by EPR in conjunction with the spin trap 5,5'-dimethyl-1-pyroline-N-oxide. The intensity of the EPR signal was insensitive to superoxide dismutase and it was decreased, but not suppressed, by catalase. The disulfides of glutathione and cysteine did not react with ferrylmyoglobin, but the disulfide bridge in lipoic acid interacted efficiently with the ferryl species by either reducing directly the heme iron to form met-myoglobin or adding onto a pyrrole ring to form sulfmyoglobin; either process depended on the presence or absence of catalase (to eliminate the excess of H2O2) in the reaction mixture, respectively. The biological significance of the above results is discussed in terms of the occurrence and distribution of high oxidation states of myoglobin, its specific participation in cellular injury, and its potential interaction with biologically important thiols leading to either recovery of myoglobin or generation of nonfunctional forms of the hemoprotein as sulfmyoglobin.     

Participation of tyrosine residues of myoglobin in the disproportionateness reaction of heme iron (II) and (IV)]

By means of the comparison of the constant oxidation reactions of both the myoglobin modified by N-acetylimidazole and the intact myoglobin in the presence of H2O2 or ferrylmyoglobin we characterized the role of the tyrosine residues (Tyr) of myoglobin in the synproportionation reaction between heme iron (II) of one molecule and heme iron (IV)--of another. It was demonstrated that Tyr derivatization resulted in the decrease of the velocity of redox interaction between deoxymyoglobin (II) and ferrylmyoglobin (IV) and led to the decrease of the efficiency of oxymyoglobin deoxygenation. The effects were shown to be independent on Tyr quantity in myoglobin molecule and to have the same character for both the sperm-whale myoglobin and the horse myoglobin.     

Reduction of ferryl- and metmyoglobin to ferrous myoglobin by menadione-glutathione conjugate. Spectrophotometric studies under aerobic and anaerobic conditions

Both metmyoglobin (MbIII) and ferrylmyoglobin (MbIV) are reduced by the menadiol-glutathione conjugate (GS-Q2-) to oxymyoglobin (MbIIO2) or deoxymyoglobin (MbII), depending whether the assay is carried out under aerobic or anaerobic conditions, respectively. Under aerobic conditions, the reduction of MbIII to MbIIO2 by GS-Q2- is associated with O2 consumption. The latter process is accounted for by (a) the autoxidation of the conjugate yielding H2O2 and (b) the rapid binding of O2 to MbII to yield MbIIO2. The ratio [O2]consumed/[MbIIO2]formed is approximately 1.5 at the time when MbIIO2 formation is maximal (at about 0.8 min). This ratio, higher than the unit, indicates that there is more than one O2-consuming reaction in this experimental model. The ratio of initial rates of O2 consumption and MbIIO2 formation is close to the unit [(-dO2/dt)/(+ dMbIIO2/dt) = 1.1]. The formation of H2O2 originating during the autoxidation of the GS-Q2- is substantially lower in the presence of MbIII, probably due to the heterolytic cleavage of the O--O bond of the peroxide by the hemoprotein. Although the latter reaction should yield MbIV, this species is not observed in the absorption spectrum, probably due to its rapid reduction by GS-Q2-. MbIV is reduced to MbIIO2 by the GS-Q2-. Whether this reaction takes place in one-electron transfer steps, that is, the sequence: MbIV----MbIII----MbIIO2 is difficult to evaluate by absorption spectral analysis, due to the rapid rate of the [MbIV----MbIIO2] transition. Under anaerobic conditions, the reduction of either MbIII or MbIV by GS-Q2- yields MbII as a stable molecular product. Anaerobic conditions prevent any further interaction of MbII with intermediates of O2 reduction derived from GS-Q2- autoxidation.     

The purification and some properties of the polyphenol oxidase from tea (Camellia sinensis L.)

1. Polyphenol oxidase (EC 1. 10. 3.–) from the shoots of the tea plant was purified about 5000-fold on a dry-weight basis. 2. At an intermediate stage of purification four soluble yellow fractions were obtained. They are believed to represent complexes of a basic enzyme protein with acidic phenolic oxidation products and nucleic acids. After removal of the complex-forming materials the fractions were blue and similar to each other. About 40% of the activity could not be extracted from the acetone-dried powder. 3. Each of the four blue fractions was resolved further into two species, A and B. The following results refer to species A. 4. The enzyme showed absorption maxima at 279mμ (E^1%_1cm., 13·5) and 611mμ (E^1%_1cm., 0·84) with a shoulder at 330mμ. The enzyme was bleached by substrate under anaerobic conditions and the colour was restored by oxygen. 5. The molecular weight measured by sedimentation and diffusion was 144000±16000. The copper content was 0·32% (w/w). 6. Kinetic constants are given for a number of substrates and inhibitors, including the natural substrates of the tea leaf. The specific activity towards pyrogallol was 373 units/mg. at 30°. 7. The best substrates were o-dihydric phenols. Quinol and p-phenylenediamine were slowly oxidized. Monohydric phenols and ascorbic acid were not oxidized. 8. The kinetics of oxidation of most substrates are consistent with a mechanism in which oxidized and reduced forms of the enzyme form binary complexes with phenol and oxygen respectively. A modified mechanism is postulated for the oxidation of chlorogenic acid. 9. The relation of the results to the mechanism of tea fermentation is discussed.     

Absorption and Screening in Phycomyces

In vivo absorption measurements were made through the photosensitive zones of Phycomyces sporangiophores and absorption spectra are presented for various growth media and for wavelengths between 400 and 580 mµ. As in mycelia, β-carotene was the major pigment ordinarily found. The addition of diphenylamine to the growth media caused a decrease in β-carotene and an increase in certain other carotenoids. Growth in the dark substantially reduced the amount of β-carotene in the photosensitive zone; however, growth on a lactate medium failed to suppress β-carotene in the growing zone although the mycelia appeared almost colorless. Also when diphenylamine was added to the medium the absorption in the growing zone at 460 mµ was not diminished although the colored carotenoids in the bulk of the sporangiophore were drastically reduced. Absorption which is characteristic of the action spectra was not found. Sporangiophores immersed in fluids with a critical refractive index show neither positive nor negative tropism. Measurements were made of the critical refractive indices for light at 495 and 510 mµ. The critical indices differed only slightly. Assuming primary photoreceptors at the cell wall, the change in screening due to absorption appears too large to be counterbalanced solely by a simple effect of the focusing change. The possibility is therefore advanced that the receptors are internal to most of the cytoplasm; i.e., near the vacuole.     

The reversible reduction of horse metmyoglobin by the iron(II) complex of trans-1,2-diaminocyclohexane-N,N,N,n-tetraacetate.

The reduction of metmyoglobin by the iron(II) complex of trans-1,2-diaminocyclohexane-N,N,N'N'-tetraacetate (FeCDTA2-) has been investigated. The equilibrium constant, measured spectrophotometrically, is 0.21 with a resulting reduction potential of 0.050 V for Mb0. The rate constant for the reduction is 28 M-1 sec-1 with a deltaH ++ of 13 kcal M-1 and deltaS ++ of -11 eu. Both CN- and OH- inhibit the reduction because of the relatively low reactivity of cyanometmyoglobin (Mb+CN-) and ionized metmyglobin (Mb+OH-). The rate constant for the reduction of Mb+CN- by FeCDTA2- is 4.0 X 10(-2) M-1 sec-1 and that for reduction of Mb+OH- is 4.8 M-1 sec-1. The nitric oxide complex of metmyoglobin is reduced with a rate constant of 10 M-1 sec-1. The kinetics of oxidation of oxymyoglobin by FeCDTA- were studied. The data are consistent with a mechanism where oxidation takes place entirely through the deoxy form. A rate constant of 1.45 X 10(2) M-1 sec-1 was calculated for the oxidation of deoxymyoglobin by FeCDTA-, in equilibrium constant and rate constant for reduction. The above data are discussed in terms of a simple outer-sphere reduction reaction.     

Rapid Myoglobin Aggregation through Glucosamine-Induced α-Dicarbonyl Formation

The extent of glycation and conformational changes of horse myoglobin (Mb) upon glycation with N-acetyl-glucosamine (GlcNAc), glucose (Glc) and glucosamine (GlcN) were investigated. Among tested sugars, the rate of glycation with GlcN was the most rapid as shown by MALDI and ESI mass spectrometries. Protein oxidation, as evaluated by the amount of carbonyl groups present on Mb, was found to increase exponentially in Mb-Glc conjugates over time, whereas in Mb-GlcN mixtures the carbonyl groups decreased significantly after maximum at 3 days of the reaction. The reaction between GlcN and Mb resulted in a significantly higher amount of α-dicarbonyl compounds, mostly glucosone and 3-deoxyglucosone, ranging from and 27 to 332 mg/L and from 14 to 304 mg/L, respectively. Already at 0.5 days, tertiary structural changes of Mb-GlcN conjugate were observed by altered tryptophan fluorescence. A reduction of metmyoglobin to deoxy-and oxymyoglobin forms was observed on the first day of reaction, coinciding with the greatest amount of glucosone produced. In contrast to native α-helical myoglobin, 41% of the glycated protein sequence was transformed into a β-sheet conformation, as determined by circular dichroism spectropolarimetry. Transmission electron microscopy demonstrated that Mb glycation with GlcN causes the formation of amorphous or fibrous aggregates, started already at 3 reaction days. These aggregates bind to an amyloid-specific dye thioflavin T. With the aid of α-dicarbonyl compounds and advanced products of reaction, this study suggests that the Mb glycation with GlcN induces the unfolding of an initially globular protein structure into amyloid fibrils comprised of a β-sheet structure.     

Evidences from Action Spectra for a Specific Participation of Chlorophyll b in Photosynthesis

Rate of oxygen evolution in photosynthesis was measured as the current from a polarized platinum electrode covered by a thin layer of Chlorella. The arrangement gave a reproducibly measurable rate of photosynthesis proportional to light intensity at the low levels used and gave rapid response to changes in illumination. Two phenomena have been explored. The Emerson effect was observed as an enhancement of photosynthesis in long wavelength red light (700 mµ) when shorter wavelengths were added. Two light beams of wavelengths 653 and 700 mµ when presented together gave a photosynthetic rate about 25 per cent higher than the sum of the rates obtained separately. Large and reproducible transients in rate of oxygen evolution were observed accompanying change in illumination between two wavelengths adjusted in intensity to support equal steady rates of photosynthesis. The transients were found not to be specifically related to long wavelength red light. Both enhancement and the transients have identical action spectra which are interpreted as demonstrating a specific photochemical participation of chlorophyll b.     

A Fast-Start Pacing Strategy Speeds Pulmonary Oxygen Uptake Kinetics and Improves Supramaximal Running Performance

The focus of the present study was to investigate the effects of a fast-start pacing strategy on running performance and pulmonary oxygen uptake () kinetics at the upper boundary of the severe-intensity domain. Eleven active male participants (28±10 years, 70±5 kg, 176±6 cm, 57±4 mL/kg/min) visited the laboratory for a series of tests that were performed until exhaustion: 1) an incremental test; 2) three laboratory test sessions performed at 95, 100 and 110% of the maximal aerobic speed; 3) two to four constant speed tests for the determination of the highest constant speed (HS) that still allowed achieving maximal oxygen uptake; and 4) an exercise based on the HS using a higher initial speed followed by a subsequent decrease. To predict equalized performance values for the constant pace, the relationship between time and distance/speed through log-log modelling was used. When a fast-start was utilized, subjects were able to cover a greater distance in a performance of similar duration in comparison with a constant-pace performance (constant pace: 670 m±22%; fast-start: 683 m±22%; P = 0.029); subjects also demonstrated a higher exercise tolerance at a similar average speed when compared with constant-pace performance (constant pace: 114 s±30%; fast-start: 125 s±26%; P = 0.037). Moreover, the mean response time was reduced after a fast start (constant pace: 22.2 s±28%; fast-start: 19.3 s±29%; P = 0.025). In conclusion, middle-distance running performances with a duration of 2–3 min are improved and response time is faster when a fast-start is adopted.     

Reduction of ferrylmyoglobin by the spin trap N-tert-butyl-α-phenylnitrone (PBN) in aqueous solution and during freezing

The hypervalent muscle pigment ferrylmyoglobin, formed by activation of metmyoglobin by hydrogen peroxide, was found to be reduced in a second-order reaction by N-tert-butyl-α-phenylnitrone (PBN, often used as a spin trap). In acidic aqueous solution at ambient temperature, the reduction is relatively slow (δH^? = 65 ± 2 kJ · mol^-1 and δS^? = -54 ± 7 J · mol^-1. K^-1 for pH = 5.6), but phase transitions during freezing of the buffered solutions accelerates the reaction between ferrylmyoglobin and PBN. In these heterogenous systems at low temperature (but not when ice-formation was inhibited by glycerol), a PBN-derived radical intermediate was detected by ESR-spectroscopy, identified as a nitroxyl radical by a parallel nitrogen hyperfine coupling constant of 31.8 G, and from microwave power saturation behavior concluded not to be located in the heme-cleft of the protein. The acceleration of the reaction is most likely caused by a lowering of the pH during the freezing of the buffered solutions whereby ferrylmyoglobin becomes more oxidizing.     

Pro-oxidant and antioxidant potential of catecholestrogens against ferrylmyoglobin-induced oxidative stress

Ferryl heme proteins may play a major role in vivo under certain pathological conditions. Catecholestrogens, the estradiol-derived metabolites, can act either as antioxidants or pro-oxidants in iron-dependent systems. The aim of the present work was (1) to determine the effects of ferrylmyoglobin on hepatocyte cytotoxicity, and (2) to assess the pro/antioxidant potential of a series of estrogens (phenolic, catecholic and stilbene-derived) against ferrylmyoglobin induced lipid peroxidation in rat hepatocytes. Cells were exposed to metmyoglobin plus hydrogen peroxide to form ferrylmyoglobin in the presence of the transition metal chelator diethylentriaminepentaacetic acid. Results showed that ferrylmyoglobin induced an initial oxidative stress, mainly reflected in an early lipid peroxidation and further decrease in GSH and ATP. However, cells gradually adapted to this situation, by recovering the endogenous ATP and GSH levels at longer incubation times. Phenolic and stilbene-derived estrogens inhibited ferrylmyoglobin-induced lipid peroxidation to different degrees: diethylstilbestrol>estradiol>resveratrol. Catecholestrogens at concentrations higher than 1 microM also inhibited lipid peroxidation with similar efficacy. The ability of estrogens to reduce ferrylmyoglobin to metmyoglobin may account for their antioxidant activity. In contrast, physiological concentrations (100 pM-100 nM) of the catecholestrogens exerted pro-oxidant activities, 4-hydroxyestradiol being more potent than 2-hydroxyestradiol. The implications of these interactions should be considered in situations where local myoglobin or hemoglobin microbleeding takes place.     

The Reaction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Provide an Understanding of the para-Hydroxylation Enzyme Catalytic Cycle

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that catalyzes the para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on results from stopped-flow spectrophotometry, the reduced enzyme-3HB complex reacts with oxygen to form a C4a-peroxy flavin with a rate constant of 1.13 ± 0.01 × 10⁶ m⁻¹ s⁻¹ (pH 8.0, 4 °C). This intermediate is subsequently protonated to form a C4a-hydroperoxyflavin with a rate constant of 96 ± 3 s⁻¹. This step shows a solvent kinetic isotope effect of 1.7. Based on rapid-quench measurements, the hydroxylation occurs with a rate constant of 36 ± 2 s⁻¹. 3HB6H does not exhibit substrate inhibition on the flavin oxidation step, a common characteristic found in most ortho-hydroxylation enzymes. The apparent k_cat at saturating concentrations of 3HB, NADH, and oxygen is 6.49 ± 0.02 s⁻¹. Pre-steady state and steady-state kinetic data were used to construct the catalytic cycle of the reaction. The data indicate that the steps of product release (11.7 s⁻¹) and hydroxylation (36 ± 2 s⁻¹) partially control the overall turnover.