Oxidation of melatonin by oxoferryl hemoglobin: A mechanistic study

Reaction of melatonin with the hypervalent iron centre of oxoferryl hemoglobin, produced in aqueous solution from methemoglobin and H₂O₂, has been investigated at 37°C and pH 7.4, by absorption spectroscopy. The reaction results in reduction of the oxoferryl moiety with formation of a heme-ferric containing hemoprotein. Stopped-flow spectrophotometric measurements provide evidence that the reduction of oxoferryl-Hb by melatonin is first-order in oxoferryl-Hb and first-order in melatonin. The bimolecular reaction constant at pH 7.4 and 37°C is 112 ± 1.0 M^-1 s^-1.

Two major oxidation products from melatonin have been found by gas chromatography-mass spectroscopy: the cyclic compound 1,2,3,3a,8,8a-hexahydro-1-acetyl-5-methoxy-3a-hydroxypyrrolo[2,3-b]indole (cyclic 3-hydroxy-melatonin), and N-acetyl-N′-formyl 5-methoxykynuramine (AFMK). The percentage yield of the two major products appears dependent on the ratio [oxoferryl-Hb]: [melatonin]—the higher the ratio the higher the yield of AFMK. The observed stoichiometry oxoferryl-Hb_reduced:melatonin_consumed is 2, when the ratio [oxoferryl-Hb]:[melatonin] is 1:1, but appears >2 at higher molar ratios. The reduction of the hypervalent iron of the oxoferryl moiety may be consistent with an oxidation of melatonin by two one-electron steps.     

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.     

Oxidation of melatonin and tryptophan by an HRP cycle involving compound III

We recently described that horseradish peroxidase (HRP) and myeloperoxidase (MPO) catalyze the oxidation of melatonin, forming the respective indole ring-opening product N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) (Biochem. Biophys. Res. Commun. 279, 657-662, 2001). Although the classic peroxidatic enzyme cycle is expected to participate in the oxidation of melatonin, the requirement of a low HRP:H(2)O(2) ratio suggested that other enzyme paths might also be operative. Here we followed the formation of AFMK under two experimental conditions: predominance of HRP compounds I and II or presence of compound III. Although the consumption of substrate is comparable under both conditions, AFMK is formed in significant amounts only when compound III predominates during the reaction. Using tryptophan as substrate, N- formyl-kynurenine is formed in the presence of compound III. Both, melatonin and tryptophan efficiently prevents the formation of p-670, the inactive form of HRP. Since superoxide dismutase (SOD) inhibits the production of AFMK, we proposed that compound III acts as a source of O(-*)(2) or participates directly in the reaction, as in the case of enzyme indoleamine 2,3-dioxygenase.     

Radioimmunoassay of N-acetyl-N-formyl-5-methoxykynuramine (AFMK): a melatonin oxidative metabolite

N-acetyl-N-formyl-5-methoxykynuramine (AFMK) is a melatonin metabolite identified in rat brain by Hirata et al. (The Journal of Biological Chemistry 249 (1974) 1311). Since no assay has been described for its routine measurement, we have developed and validated such a radioimmunoassay. We synthesized AFMK and N-acetyl-5-methoxykynuramine (AMK), in order to produce anti-AFMK antibodies and to standardize the assay. The tracer [3H]-AFMK was obtained from [3H]-melatonin. The assay was preceded by a chromatographic step on Celite microcolumn in order to increase its specificity. The assay was suitable for the measurement of AFMK levels ranging from 59 to 1894 pmol/L. The detection limit of the assay was routinely set at 65 pmol/L. The intra- and inter-assay coefficients of variation were 3.5% and 11% respectively. Investigation of the 24 h plasma pattern in healthy volunteers did not reveal any AFMK levels in plasma samples. In rats, plasma AFMK showed a peak after melatonin injection, which confirmed the in vivo AFMK production as a melatonin metabolite. This AFMK assay is suitable for studies on melatonin metabolism.     

Structural characterization of the antigenic O-chain of the lipopolysaccharide of Escherichia coli serotype O65

The antigenic O-polysaccharide moiety of the lipopolysaccharide produced by Escherichia coli serotype O65 was investigated by composition, methylation, base hydrolysis, periodate oxidation, mass spectrometric methods, and by 1D and 2D NMR spectroscopy. The O-polysaccharide had [alpha]D + 108 degrees (water) and is a high-molecular-weight unbranched linear polymer of repeating pentasaccharide units composed of 1:1:1:1:1 D-galacturonic acid (D-GalA), D-galacturonamide (D-GalANH2), 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), 2-acetamido-2-deoxy-D-galactose (D-GalNAc), and 3-acetamido-3,6-dideoxy-D-glucose (D-Qui3NAc), and has the following structure: [formula: see text]     

Melatonin and its kynurenin-like oxidation products affect the microbicidal activity of neutrophils

Activated phagocytes oxidize the hormone melatonin to N1-acethyl-N2-formyl-5-methoxykynuramine (AFMK) in a superoxide anion- and myeloperoxidase-dependent reaction. We examined the effect of melatonin, AFMK and its deformylated-product N-acetyl-5-methoxykynuramine (AMK) on the phagocytosis, the microbicidal activity and the production of hypochlorous acid by neutrophils. Neither neutrophil and bacteria viability nor phagocytosis were affected by melatonin, AFMK or AMK. However these compounds affected the killing of Staphylococcus aureus. After 60 min of incubation, the percentage of viable bacteria inside the neutrophil increased to 76% in the presence of 1 mM of melatonin, 34% in the presence of AFMK and 73% in the presence of AMK. The sole inhibition of HOCl formation, expected in the presence of myeloperoxidase substrates, was not sufficient to explain the inhibition of the killing activity. Melatonin caused an almost complete inhibition of HOCl formation at concentrations of up to 0.05 mM. Although less effective, AMK also inhibited the formation of HOCl. However, AFMK had no effect on the production of HOCl. These findings corroborate the present view that the killing activity of neutrophils is a complex phenomenon, which involves more than just the production of reactive oxygen species. Furthermore, the action of melatonin and its oxidation products include additional activities beyond their antioxidant property. The impairment of the neutrophils' microbicidal activity caused by melatonin and its oxidation products may have important clinical implications, especially in those cases in which melatonin is pharmacologically administered in patients with infections.     

Copper-induced oxidation of epinephrine: protective effect of D-DAHK,a synthetic analogue of the high affinity copper binding site of human albumin

Epinephrine is known to be rapidly oxidized during sepsis. Ischemia and acidosis, which often accompany sepsis, are associated with the release of weakly bound cupric ions from plasma proteins. We investigated whether copper promotes oxidation of epinephrine at both physiological and acidic pH and whether D-Asp-D-Ala-D-His-D-Lys (D-DAHK), a human albumin (HSA) N-terminus synthetic peptide with a high affinity for cupric ions, attenuates this oxidation. Epinephrine alone [100 microM] or with CuCl(2) [10 microM], and with CuCl(2) [10 microM] and D-DAHK [20 microM] at pH 7.4, 7.0, 6.5, and 6.0 were incubated for 1h at 37 degrees C. Epinephrine oxidation was measured by the spectrophotometric quantification of its oxidation product, adrenochrome. We found that adrenochrome increased, suggesting copper-induced oxidation of epinephrine. At pH 7.4, 7.0, 6.5, and 6.0, adrenochrome increased by 47%, 53%, 24%, and 6% above baseline, respectively. D-DAHK attenuated the copper-induced oxidation of epinephrine to baseline levels. These in vitro results indicate that copper-induced epinephrine oxidation is greatest at the physiological pH 7.4 as well as in severe acidosis, pH 7.0, and that D-DAHK completely inhibits this oxidation.     

Kinetics of Reduction of Hypervalent Iron in Myoglobin by Crocin in Aqueous Solution

Crocin in aqueous solution is oxidized by ferrylmyoglobin, MbFe(IV)=O, in a second order reaction with k = 183 1 · mol^-1 · s^-1, AH^‡₂₉₈ = 55.0 kJ · mol^-1, and ΔLS^‡₂₉₈ = -17 J · mol^-1 K^-1 (pH = 6.8, ionic strength 0.16 (NaCl), 25°C), as studied by stopped-flow spectroscopy. The reaction has 1:1 stoichiometry to yield metmyoglobin, MbFe(III), and has AG^o = -11 kJ · mol^-1, as calculated from the literature value E⁰ = +0.85 V (pH = 7.4) vs. NHE for MbFe(IV)=O/MbFe(III) and from the half-peak potential +0.74 V (vs. NHE in aqueous 0.16 NaCl, pH = 7.4) determined by cyclic voltammetry for the one-electron oxidation product of crocin, for which a cation radical structure is proposed and which has a half-peak potential of +0.89 V for its formation from the two-electron oxidation product of crocin. The fer-rylmyoglobin protein-radical, MbFe(IV)=O, reacts with crocin with 2:l stoichiometq to yield MbFe(IV)= 0, as determined by ESR spectroscopy, in a reaction faster than the second order protein-radical generating reaction between H₂O₂ and MbFe(III), for which latter reaction k = 137 L · mol^-1 · s^-1, ΔH^‡₂₉₈ = 51.5 kJ · mol^-1, and ΔH^‡₂₉₈ = -31 J · mol^-1 · K^-1 (pH = 6.8, ionic strength = 0.16 (NaCI), 25°C) was determined. Based on the difference between the stoichiometry for the reaction between crocin and each of the two hypervalent forms of myoglobin, it is concluded in agreement with the determined half peak reduction potentials, that the crocin cation radical is less reducing compared to crocin, as the cation radical can reduce the protein radical but not the iron(IV) centre in hypervalent myoglobin.     

Oxidation of melatonin by AAPH-derived peroxyl radicals: Evidence of a pro-oxidant effect of melatonin

Background

Melatonin is well-established as a powerful reducing agent of oxidant generated in the cell medium. We aimed to investigate how readily melatonin is oxidized by peroxyl radicals ROO⋅ generated by the thermolysis of 2,2′-azobis(2-amidinopropane) hydrochloride (AAPH) and the role of glutathione (GSH) during the reaction course.

Methods

Chromatographic, mass spectroscopy, and UV–visible spectrometric techniques were used to study the oxidation of melatonin by ROO⋅ or horseradish peroxidase (HRP)/H₂O₂. Our focus was the characterization of products and the study of features of the reaction.

Results

We found that N¹-acetyl-N²-formyl-5-methoxykynuramine (AFMK) and a monohydroxylated derivative of melatonin were the main products of the reaction between melatonin and ROO⋅. Higher pH or saturation of the medium with molecular oxygen increased the yield of AFMK but did not affect the reaction rate. Melatonin increased the depletion of intracellular GSH mediated by AAPH. Using the HRP/H₂O₂ as the oxidant system, the addition of melatonin promoted the oxidation of GSH to GSSG.

Conclusions

These results show, for the first time, that melatonin radical is able to oxidize GSH.

General significance

We propose that this new property of melatonin could explain or be related to the recently reported pro-oxidant activities of melatonin.     

Rat liver mitochondrial malate dehydrogenase: purification, kinetic properties, and role in ethanol metabolism

Malate dehydrogenase was purified from the mitochondrial fraction of rat liver by ion-exchange chromatography with affinity elution. The kinetic parameters for the enzyme were determined at pH 7.4 and 37 degrees C, yielding the following values (microM): Ka, 72; Kia, 11; Kb, 110; Kp, 1600; Kip, 7100; Kq, 170; Kiq, 1100, where a = NADH, b = oxalacetate, p = malate, and q = NAD+. Kib was estimated to be about 100 microM. The maximum velocities for mitochondrial malate dehydrogenase in rat liver homogenates, at pH 7.4 and 37 degrees C, were 380 +/- 40 mumol/min per gram of liver, wet weight, for oxalacetate reduction and 39 +/- 3 mumol/min per gram of liver, wet weight, for malate oxidation. Rates of the reaction catalyzed by mitochondrial malate dehydrogenase under conditions similar to those in vivo were calculated using these kinetic parameters and were much lower than the maximum velocity of the enzyme. Since mitochondrial malate dehydrogenase is not saturated with malate at physiological concentrations, its kinetic parameters are probably important in the regulation of mitochondrial malate concentration during ethanol metabolism. For the mitochondrial enzyme to operate at a rate comparable to the flux through cytosolic malate dehydrogenase during ethanol metabolism (about 4 mumol min-1 per gram liver), the mitochondrial [malate] would need to be about 2 mM and the mitochondrial [oxalacetate] would need to be less than 1 microM.     

Identification of the factors affecting the rate of deactivation of hypochlorous acid by melatonin

It has been found that melatonin reacts rapidly with hypochlorous acid in phosphate-buffered, ethanol-water solutions to produce 2-hydroxymelatonin. The rate law, d[2 - HOMel]/dt - kHOCl[Mel][HOCl] - kOCl-[Mel][OCl-], was obtained. At 37 degrees C and at a water concentration of 23.5 M, kOCl- = 6.0 x 10(2) L. mol-1. s-1, and kHOCl was found to be a function of the water concentration, kHOCl = 11 +/- 3 L3. mol-3. s-1. [H2O]2, indicating that the availability of water at the site of the reaction plays a significant role. The part that the structural components of melatonin play in determining the reaction pathway was examined by comparing the rate of deactivation of HOCl by melatonin to that of the model compounds indole, 5-methoxyindole, and 3-methylindole. The relative reactivity is explained in terms of steric and electronic effects, and it was found that the presence of the substituent at the 3-position influences the nature of the oxidation product. Melatonin and 3-methylindole yielded hydroxylated products, whereas indole and 5-methoxyindole produce chlorinated products.     

Superoxide-dependent oxidation of melatonin by myeloperoxidase

Myeloperoxidase uses hydrogen peroxide to oxidize numerous substrates to hypohalous acids or reactive free radicals. Here we show that neutrophils oxidize melatonin to N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) in a reaction that is catalyzed by myeloperoxidase. Production of AFMK was highly dependent on superoxide but not hydrogen peroxide. It did not require hypochlorous acid, singlet oxygen, or hydroxyl radical. Purified myeloperoxidase and a superoxide-generating system oxidized melatonin to AFMK and a dimer. The dimer would result from coupling of melatonin radicals. Oxidation of melatonin was partially inhibited by catalase or superoxide dismutase. Formation of AFMK was almost completely eliminated by superoxide dismutase but weakly inhibited by catalase. In contrast, production of melatonin dimer was enhanced by superoxide dismutase and blocked by catalase. We propose that myeloperoxidase uses superoxide to oxidize melatonin by two distinct pathways. One pathway involves the classical peroxidation mechanism in which hydrogen peroxide is used to oxidize melatonin to radicals. Superoxide adds to these radicals to form an unstable peroxide that decays to AFMK. In the other pathway, myeloperoxidase uses superoxide to insert dioxygen into melatonin to form AFMK. This novel activity expands the types of oxidative reactions myeloperoxidase can catalyze. It should be relevant to the way neutrophils use superoxide to kill bacteria and how they metabolize xenobiotics.     

Inhibition of neuronal nitric oxide synthase activity by N1-acetyl-5-methoxykynuramine, a brain metabolite of melatonin

We assessed the effects of melatonin, N(1)-acetyl-N (2)-formyl-5-methoxykynuramine (AFMK) and N(1)-acetyl-5-methoxykynuramine (AMK) on neuronal nitric oxide synthase (nNOS) activity in vitro and in rat striatum in vivo. Melatonin and AMK (10(-11)-10(-3) m), but not AFMK, inhibited nNOS activity in vitro in a dose-response manner. The IC(50) value for AMK (70 microm) was significantly lower than for melatonin (>1 mm). A 20% nNOS inhibition was reached with either 10(-9) m melatonin or 10(-11) m AMK. AMK inhibits nNOS by a non-competitive mechanism through its binding to Ca(2+)-calmodulin (CaCaM). The inhibition of nNOS elicited by melatonin, but not by AMK, was blocked with 0.05 mm norharmane, an indoleamine-2,3-dioxygenase inhibitor. In vivo, the potency of AMK to inhibit nNOS activity was higher than that of melatonin, as a 25% reduction in rat striatal nNOS activity was found after the administration of either 10 mg/kg of AMK or 20 mg/kg of melatonin. Also, in vivo, the administration of norharmane blocked the inhibition of nNOS produced by melatonin administration, but not the inhibition produced by AMK. These data reveal that AMK rather than melatonin is the active metabolite against nNOS, which may be inhibited by physiological levels of AMK in the rat striatum.     

Stabilities of 7-alkylguanosines and 7-deoxyguanosines formed by phosphoramide mustard and nitrogen mustard

7-Alkylguanosine and 7-alkyldeoxyguanosine were prepared from phosphoramide mustard and nitrogen mustard in nonaqueous conditions. The guanosine products were N-(2-chloroethyl)-N-[2-(7-guanosinyl)ethyl] phosphorodiamidic acid, and N-(2-chloroethyl)-N-[2-(7-guanosinyl)ethyl]methylamine, respectively. These were also formed in aqueous reactions but they rapidly underwent secondary reactions. The half-life of the phosphoramide mustard-guanosine adduct was 3.1 h (37 degrees C, pH 7.4) and that of the nitrogen mustard adduct 1 h (25 degrees C, pH 7.4), as determined by HPLC. The half-lives of the respective adducts for imidazole ring-opening were 9.5 h and 0.78 h (37 degrees C, pH 7.4). The respective deoxyguanosine derivatives depurinated with half-lives of 8.5 h and 1.6 h (25 degrees C, pH 4.2). The mustard adducts are notably more labile than simple alkyl substituted guanosines and deoxyguanosines.     

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.     

Klebsiella pneumoniae nitrogenase. Inhibition of hydrogen evolution by ethylene and the reduction of ethylene to ethane.

Ethylene (C2H4) inhibited H2 evolution by the Mo-containing nitrogenase of Klebsiella pneumoniae. The extent of inhibition depended on the electron flux determined by the ratio of Fe protein (Kp2) to MoFe protein (Kp1) with KiC2H4 = 409 kPa ([Kp2]/[Kp1] = 22:1) and KC2H4i = 88 kPa ([Kp1]/[Kp2] = 21:1) at 23 degrees C at pH 7.4. At [Kp2]/[Kp1] = 1:1, inhibition was minimal with C2H4 (101 kPa). Extrapolation of data obtained when C2H4 was varied from 60 to 290 kPa indicates that at infinite pressure of C2H4 total inhibition of H2 evolution should occur. C2H4 inhibited concomitant S2O4(2-) oxidation to the same extent that it inhibited H2 evolution. Although other inhibitors of total electron flux such as CN- and CH3NC uncouple MgATP hydrolysis from electron transfer, C2H4 did not affect the ATP/2e ratio. Inhibition of H2 evolution by C2H4 was not relieved by CO. C2H4 was reduced to C2H6 at [Kp2]/[Kp1] ratios greater than or equal to 5:1 in a reaction that accounted for no more than 1% of the total electron flux. These data are discussed in terms of the chemistry of alkyne and alkene reduction on transition-metal centres.     

Insulin Binding to Human Astrocytoma Cells and Its Effect on Uridine Incorporation into Nucleic Acid

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.     

Electron transfer process in cytochrome bd-type ubiquinol oxidase from Escherichia coli revealed by pulse radiolysis

Cytochrome bd is a two-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and binds hemes b558, b595, and d as the redox metal centers. Taking advantage of spectroscopic properties of three hemes which exhibit distinct absorption peaks, we investigated electron transfer within the enzyme by the technique of pulse radiolysis. Reduction of the hemes in the air-oxidized, resting-state enzyme, where heme d exists in mainly an oxygenated form and partially an oxoferryl and a ferric low-spin forms, occurred in two phases. In the faster phase, radiolytically generated N-methylnicotinamide radicals simultaneously reduced the ferric hemes b558 and b595 with a second-order rate constant of 3 x 10(8) M-1 s-1, suggesting that a rapid equilibrium occurs for electron transfer between two b-type hemes long before 10 micros. In the slower phase, an intramolecular electron transfer from heme b to the oxoferryl and the ferric heme d occurred with the first-order rate constant of 4.2-5.6 x 10(2) s-1. In contrast, the oxygenated heme d did not exhibit significant spectral change. Reactions with the fully oxidized and hydrogen peroxide-treated forms demonstrated that the oxidation and/or ligation states of heme d do not affect the heme b reduction. The following intramolecular electron transfer transformed the ferric and oxoferryl forms of heme d to the ferrous and ferric forms, respectively, with the first-order rate constants of 3.4 x 10(3) and 5.9 x 10(2) s-1, respectively.     

A novel metabolic pathway of melatonin: oxidation by cytochrome C

The indoleamine melatonin is ubiquitously distributed, and because of its small size and amphiphilic nature, it is able to reach easily all cellular compartments. The highest intracellular melatonin concentrations are found in the mitochondria, suggestive of local metabolism and/or direct participation in organelle function. In mitochondria cytochrome c (cyt c) could represent a melatonin target since it has the capability to oxidize organic molecules in the presence of H2O2, and mitochondria are the main site of H2O2 production in nonphagocytic cells. Therefore, we investigated oxidation of melatonin by cyt c/H2O2 couple as a potential pathway for its metabolism in the mitochondria. We found melatonin conversion into N(1)-acetyl-N(2)-formyl-5-methoxykynuramine via sequential steps that generate the intermediates 2-hydroxymelatonin and 2,3-dihydroxymelatonin. We experimentally excluded mediation by a Fenton/Haber-Weiss-type reaction and documented the dependence on oxoferryl heme for melatonin oxidation. Given the high mitochondrial concentrations of both melatonin and cyt c as well as the continuous generation of H2O2 during respiration, it is entirely possible that mitochondrial cyt c-mediated oxidation of melatonin may be a plausible pathway of its biotransformation in vivo.     

Degradation of growth hormone releasing factor analogs in neutral aqueous solution is related to deamidation of asparagine residues. Replacement of asparagine residues by serine stabilizes

The incubation of a solution of the human growth hormone releasing factor analog, [Leu27] hGRF(1-32)NH2 at pH 7.4 and 37 degrees, resulted in extensive degradation of the sample. The major degradation products were identified as the peptides [beta-Asp8, Leu27] hGRF(1-32)NH2 and [alpha-Asp8, Leu27] hGRF(1-32)NH2, produced by deamidation of the Asn8 residue. When tested as growth hormone (GH) secretagogues in cultured bovine anterior pituitary cells, [beta-Asp8, Leu27] hGRF(1-32)NH2 was estimated to be 400-500 times less potent than the parent Asn8 peptide, while [alpha-Asp8, Leu27] hGRF(1-32)NH2 was calculated to be 25 times less potent than the parent Asn8 peptide. Three additional analogs of [Leu27] hGRF(1-32)NH2 containing either Ser or Asn at positions 8 and 28 were prepared and evaluated for their GH releasing activity and stability in aqueous phosphate buffer (pH 7.4, 37 degrees). Based on disappearance kinetics, [Leu27] hGRF(1-32)NH2 had a half-life of 202 h while the other analogs had the following half-lives: [Leu27, Asn28] hGRF(1-32)NH2 (150 h); [Ser8, Leu27, Asn28] hGRF(1-32)NH2 (746 h); and [Ser8, Leu27] hGRF(1-32)NH2 (1550 h). After 14 days, incubated samples of the Asn8 analogs lost GH releasing potency, while the Ser8 analogs retained full potency. The potential for loss of biological activity brought about by deamidation of other engineered peptides and proteins should be considered in their design.