Futile cycling of intermediates of fatty acid biosynthesis toward peroxisomal beta-oxidation in Saccharomyces cerevisiae.

The flux of fatty acids toward beta-oxidation was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate synthesis in the peroxisome from the polymerization, by a bacterial polyhydroxyalkanoate synthase, of the beta-oxidation intermediates 3-hydroxyacyl-CoAs. Synthesis of polyhydroxyalkanoate was dependent on the beta-oxidation enzymes acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional protein, which are involved in generating 3-hydroxyacyl-CoAs, and on the peroxin PEX5, which is involved in the import of proteins into the peroxisome. In wild type cells grown in media containing fatty acids, the polyhydroxyalkanoate monomer composition was largely influenced by the nature of the external fatty acid, such that even-chain monomers are generated from oleic acid and odd-chain monomers are generated from heptadecenoic acid. In contrast, polyhydroxyalkanoate containing predominantly 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate was synthesized in a mutant deficient in the peroxisomal 3-ketothiolase (fox3 Delta 0) growing either on oleic acid or heptadecenoic acid as well as in wild type and fox3 Delta 0 mutants grown on glucose or raffinose, indicating that 3-hydroxyacyl-CoAs used for polyhydroxyalkanoate synthesis were generated from the degradation of intracellular short- and medium-chain fatty acids by the beta-oxidation cycle. Inhibition of fatty acid biosynthesis with cerulenin blocked the synthesis of polyhydroxyalkanoate from intracellular fatty acids but still enabled the use of extracellular fatty acids for polymer production. Mutants affected in the synthesis of lipoic acid showed normal polyhydroxyalkanoate synthesis capacity. Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed toward the peroxisomal beta-oxidation pathway.

Use of recovered frying oils in chicken and rabbit feeds: effect on the fatty acid and tocol composition and on the oxidation levels of meat, liver and plasma

The addition of some fat co- and by-products to feeds is usual nowadays; however, the regulations of their use are not always clear and vary between countries. For instance, the use of recycled cooking oils is not allowed in the European Union, but they are used in other countries. However, oils recovered from industrial frying processes could show satisfactory quality for this purpose. Here we studied the effects of including oils recovered from the frying industry in rabbit and chicken feeds (at 30 and 60 g/kg, respectively) on the fatty acid (FA) and tocol (tocopherol + tocotrienol) compositon of meat, liver and plasma, and on their oxidative stability. Three dietary treatments (replicated eight times) were compared: fresh non-used oil (LOX); oil discarded from the frying industry, having a high content of secondary oxidation compounds (HOX); and an intermediate level (MOX) obtained by mixing 50 : 50 of LOX and HOX. The FA composition of oil diets and tissues was assessed by GC, their tocol content by HPLC, the thiobarbituric acid value was used to assess tissue oxidation status, and the ferrous oxidation-xylenol orange method was used to assess the susceptibility of tissues to oxidation. Our results indicate that FA composition of rabbit and chicken meat, liver and plasma was scarcely altered by the addition of recovered frying oils to feed. Differences were encountered in the FA composition between species, which might be attributed mainly to differences in the FA digestion, absorption and metabolism between species, and to some physiological dietary factors (i.e. coprophagy in rabbits that involves fermentation with FA structure modification). The α-tocopherol (αT) content of tissues was reduced in response to the lower αT content in the recovered frying oil. Differences in the content of other tocols were encountered between chickens and rabbits, which might be attributable to the different tocol composition of their feeds, as well as to species differences in the digestion and metabolism of tocols. Tissue oxidation and susceptibility to oxidation were in general low and were not greatly affected by the degree of oxidation of the oil added to the feeds. The relative content of polyunsaturated fatty acids/αT in these types of samples would explain the differences observed between species in the susceptibility of each tissue to oxidation. According to our results, oils recovered from the frying industry could be useful for feed uses.

Analysis of the contribution of the β-oxidation auxiliary enzymes in the degradation of the dietary conjugated linoleic acid 9-cis-11-trans-octadecadienoic acid in the peroxisomes of Saccharomyces cerevisiae

Beta-oxidation of the conjugated linoleic acid 9-cis,11-trans-octadecadienoic acid (rumenic acid) was analyzed in vivo in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanoate is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxyacyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The amount of polyhydroxyalkanaote synthesized from the degradation of rumenic acid was found to be similar to the amount synthesized from the degradation of 10-trans,12-cis-octadecadienoic acid, oleic acid or 10-cis-heptadecenoic acid. Furthermore, the degradation of 10-cis-heptadecenoic acid was found to be unaffected by the presence of rumenic acid in the media. Efficient degradation of rumenic acid was found to be independent of the Delta(3,5),Delta(2,4)-dienoyl-CoA isomerase but instead relied on the presence of Delta(3),Delta(2)-enoyl-CoA isomerase activity. The presence of the unsaturated monomer 3-hydroxydodecenoic acid in polyhydroxyalkanoate derived from rumenic acid degradation was found to be dependent on the presence of a Delta(3),Delta(2)-enoyl-CoA isomerase activity. Together, these data indicate that rumenic acid is mainly degraded in vivo in S. cerevisiae through a pathway requiring only the participation of the auxiliary enzymes Delta(3),Delta(2)-enoyl-CoA isomerase, along with the enzyme of the core beta-oxidation cycle.

Beta-oxidation in fatty acid degradation and beyond.

The degradation of fatty acids in plants occurs primarily in the peroxisomes through the beta-oxidation cycle. Enzymes that are involved in various aspects of beta-oxidation have been identified recently and shown to act biochemically on a diversity of fatty acids and derivatives. Analysis of several mutants has revealed essential roles for beta-oxidation in the breakdown of reserve triacylglycerols, seed development, seed germination and post-germinative growth before the establishment of photosynthesis. Beta-oxidation has also a considerable importance during the vegetative and reproductive growth phases, and plays a role in plant responses to stress, particularly in the synthesis of jasmonic acid.

Analysis of the contribution of the beta-oxidation auxiliary enzymes in the degradation of the dietary conjugated linoleic acid 9-cis-11-trans-octadecadienoic acid in the peroxisomes of Saccharomyces cerevisiae.

Beta-oxidation of the conjugated linoleic acid 9-cis,11-trans-octadecadienoic acid (rumenic acid) was analyzed in vivo in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanoate is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxyacyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The amount of polyhydroxyalkanaote synthesized from the degradation of rumenic acid was found to be similar to the amount synthesized from the degradation of 10-trans,12-cis-octadecadienoic acid, oleic acid or 10-cis-heptadecenoic acid. Furthermore, the degradation of 10-cis-heptadecenoic acid was found to be unaffected by the presence of rumenic acid in the media. Efficient degradation of rumenic acid was found to be independent of the Delta(3,5),Delta(2,4)-dienoyl-CoA isomerase but instead relied on the presence of Delta(3),Delta(2)-enoyl-CoA isomerase activity. The presence of the unsaturated monomer 3-hydroxydodecenoic acid in polyhydroxyalkanoate derived from rumenic acid degradation was found to be dependent on the presence of a Delta(3),Delta(2)-enoyl-CoA isomerase activity. Together, these data indicate that rumenic acid is mainly degraded in vivo in S. cerevisiae through a pathway requiring only the participation of the auxiliary enzymes Delta(3),Delta(2)-enoyl-CoA isomerase, along with the enzyme of the core beta-oxidation cycle.

Use of recovered frying oils in chicken and rabbit feeds: effect on the fatty acid and tocol composition and on the oxidation levels of meat, liver and plasma

The addition of some fat co- and by-products to feeds is usual nowadays; however, the regulations of their use are not always clear and vary between countries. For instance, the use of recycled cooking oils is not allowed in the European Union, but they are used in other countries. However, oils recovered from industrial frying processes could show satisfactory quality for this purpose. Here we studied the effects of including oils recovered from the frying industry in rabbit and chicken feeds (at 30 and 60 g/kg, respectively) on the fatty acid (FA) and tocol (tocopherol + tocotrienol) compositon of meat, liver and plasma, and on their oxidative stability. Three dietary treatments (replicated eight times) were compared: fresh non-used oil (LOX); oil discarded from the frying industry, having a high content of secondary oxidation compounds (HOX); and an intermediate level (MOX) obtained by mixing 50 : 50 of LOX and HOX. The FA composition of oil diets and tissues was assessed by GC, their tocol content by HPLC, the thiobarbituric acid value was used to assess tissue oxidation status, and the ferrous oxidation-xylenol orange method was used to assess the susceptibility of tissues to oxidation. Our results indicate that FA composition of rabbit and chicken meat, liver and plasma was scarcely altered by the addition of recovered frying oils to feed. Differences were encountered in the FA composition between species, which might be attributed mainly to differences in the FA digestion, absorption and metabolism between species, and to some physiological dietary factors (i.e. coprophagy in rabbits that involves fermentation with FA structure modification). The α-tocopherol (αT) content of tissues was reduced in response to the lower αT content in the recovered frying oil. Differences in the content of other tocols were encountered between chickens and rabbits, which might be attributable to the different tocol composition of their feeds, as well as to species differences in the digestion and metabolism of tocols. Tissue oxidation and susceptibility to oxidation were in general low and were not greatly affected by the degree of oxidation of the oil added to the feeds. The relative content of polyunsaturated fatty acids/αT in these types of samples would explain the differences observed between species in the susceptibility of each tissue to oxidation. According to our results, oils recovered from the frying industry could be useful for feed uses.

Use of recovered frying oils in chicken and rabbit feeds: effect on the fatty acid and tocol composition and on the oxidation levels of meat, liver and plasma

The addition of some fat co- and by-products to feeds is usual nowadays; however, the regulations of their use are not always clear and vary between countries. For instance, the use of recycled cooking oils is not allowed in the European Union, but they are used in other countries. However, oils recovered from industrial frying processes could show satisfactory quality for this purpose. Here we studied the effects of including oils recovered from the frying industry in rabbit and chicken feeds (at 30 and 60 g/kg, respectively) on the fatty acid (FA) and tocol (tocopherol1tocotrienol) compositon of meat, liver and plasma, and on their oxidative stability. Three dietary treatments (replicated eight times) were compared: fresh non-used oil (LOX); oil discarded from the frying industry, having a high content of secondary oxidation compounds (HOX); and an intermediate level (MOX) obtained by mixing 50 : 50 of LOX and HOX. The FA composition of oil diets and tissues was assessed by GC, their tocol content by HPLC, the thiobarbituric acid value was used to assess tissue oxidation status, and the ferrous oxidation-xylenol orange method was used to assess the susceptibility of tissues to oxidation. Our results indicate that FA composition of rabbit and chicken meat, liver and plasma was scarcely altered by the addition of recovered frying oils to feed. Differences were encountered in the FA composition between species, which might be attributed mainly to differences in the FA digestion, absorption and metabolism between species, and to some physiological dietary factors (i.e. coprophagy in rabbits that involves fermentation with FA structure modification). The a-tocopherol (aT) content of tissues was reduced in response to the lower aT content in the recovered frying oil. Differences in the content of other tocols were encountered between chickens and rabbits, which might be attributable to the different tocol composition of their feeds, as well as to species differences in the digestion and metabolism of tocols. Tissue oxidation and susceptibility to oxidation were in general low and were not greatly affected by the degree of oxidation of the oil added to the feeds. The relative content of polyunsaturated fatty acids/aT in these types of samples would explain the differences observed between species in the susceptibility of each tissue to oxidation. According to our results, oils recovered from the frying industry could be useful for feed uses.

Impact of unusual fatty acid synthesis on futile cycling through beta-oxidation and on gene expression in transgenic plants.

Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal beta-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward beta-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through beta-oxidation than the expression profile of genes involved in lipid metabolism.

Synthesis of polyhydroxyalkanoate in the peroxisome of Saccharomyces cerevisiae by using intermediates of fatty acid beta-oxidation.

Medium-chain-length polyhydroxyalkanoates (PHAs) are polyesters having properties of biodegradable thermoplastics and elastomers that are naturally produced by a variety of pseudomonads. Saccharomyces cerevisiae was transformed with the Pseudomonas aeruginosa PHAC1 synthase modified for peroxisome targeting by the addition of the carboxyl 34 amino acids from the Brassica napus isocitrate lyase. The PHAC1 gene was put under the control of the promoter of the catalase A gene. PHA synthase expression and PHA accumulation were found in recombinant S. cerevisiae growing in media containing fatty acids. PHA containing even-chain monomers from 6 to 14 carbons was found in recombinant yeast grown on oleic acid, while odd-chain monomers from 5 to 15 carbons were found in PHA from yeast grown on heptadecenoic acid. The maximum amount of PHA accumulated was 0.45% of the dry weight. Transmission electron microscopy of recombinant yeast grown on oleic acid revealed the presence of numerous PHA inclusions found within membrane-bound organelles. Together, these data show that S. cerevisiae expressing a peroxisomal PHA synthase produces PHA in the peroxisome using the 3-hydroxyacyl coenzyme A intermediates of the beta-oxidation of fatty acids present in the media. S. cerevisiae can thus be used as a powerful model system to learn how fatty acid metabolism can be modified in order to synthesize high amounts of PHA in eukaryotes, including plants.

Kinetics of selective oxidation of lactose to lactobionic acid over Pd-active carbon catalyst

Laktoosi eli maitosokeri on tärkein ainesosa useimpien nisäkkäiden tuottamassa maidossa. Sitä erotetaan herasta, juustosta ja maidosta. Laktoosia käytetään elintarvike- ja lääketeollisuuden raaka-aineena monissaeri tuotteissa. Lääketeollisuudessa laktoosia käytetään esimerkiksi tablettien täyteaineena. Hapettamalla laktoosia voidaan valmistaa laktobionihappoa, 2-keto-laktobionihappoa ja laktuloosia. Laktobionihappoa käytetään biohajoavien pintojen ja kosmetiikkatuotteiden valmistuksessa, sekä sisäelinten säilöntäliuoksissa, joissa laktobionihappo estää happiradikaalien aiheuttamien kudosvaurioiden syntymistä. Tässä työssä laktoosia hapetettiin laktobionihapoksi sekoittimella varustetussa laboratoriomittakaavaisessa panosreaktorissa käyttäenkatalyyttinä palladiumia aktiivihiilellä. Muutamissa kokeissa katalyytin promoottorina käytettiin vismuttia, joka hidastaa katalyytin deaktivoitumista. Työn tarkoituksena oli saada lisää tietoa laktoosin hapettamisen kinetiikasta. Laktoosin hapettumisessa laktobionihapoksi havaittiin selektiivisyyteen vaikuttavan muunmuassa reaktiolämpötila, paine, pH ja käytetyn katalyytin määrä. Katalyyttiä kierrättämällä eri kokeiden välillä saatiin paremmat konversiot, selektiivisyydet ja saannot. Parhaat koetulokset saatiin hapetettaessa synteettisellä ilmalla 60 oC lämpötilassa ja 1 bar paineessa. Tehdyissä kokeissa pH:n säätö tehtiin manuaalisesti, joten pH ei pysynyt koko ajan haluttuna. Laktoosin konversio oli parhaimmillaan 95 %. Laktobionihapon suhteellinen selektiivisyys oli 100% ja suhteellinen saanto 100 %. Kinetiikan matemaattinen mallinnus tehtiin Modest-ohjelmalla käyttäen kokeista saatuja mittaustuloksia.Ohjelman avulla estimoitiin parametreja ja saatiin matemaattinen malli reaktorille. Tässä työssä tehtiin kineettinen mallinnus myös ravistelureaktorissa tehdyille laktoosin hapetuskokeille, missä pH pysyi koko ajan haluttuna 'in-situ' titrauksen avulla. Työn yhteydessä selvitettiin myös mahdollisuutta käyttää monoliittikatalyyttejä laktoosin hapetusreaktiossa.

Indole ring oxidation by activated leukocytes prevents the production of hypochlorous acid

Hypochlorous acid (HOCl) released by activated leukocytes has been implicated in the tissue damage that characterizes chronic inflammatory diseases. In this investigation, 14 indole derivatives, including metabolites such as melatonin, tryptophan and indole-3-acetic acid, were screened for their ability to inhibit the generation of this endogenous oxidant by stimulated leukocytes. The release of HOCl was measured by the production of taurine-chloramine when the leukocytes (2 x 10(6) cells/mL) were incubated at 37ºC in 10 mM phosphate-buffered saline, pH 7.4, for 30 min with 5 mM taurine and stimulated with 100 nM phorbol-12-myristate acetate. Irrespective of the group substituted in the indole ring, all the compounds tested including indole, 2-methylindole, 3-methylindole, 2,3-dimethylindole, 2,5-dimethylindole, 2-phenylindole, 5-methoxyindole, 6-methoxyindole, 5-methoxy-2-methylindole, melatonin, tryptophan, indole-3-acetic acid, 5-methoxy-2-methyl-3-indole-acetic acid, and indomethacin (10 µM) inhibited the chlorinating activity of myeloperoxidase (MPO) in the 23-72% range. The compounds 3-methylindole and indole-3-acetic acid were chosen as representative of indole derivatives in a dose-response study using purified MPO. The IC50 obtained were 0.10 ± 0.03 and 5.0 ± 1.0 µM (N = 13), respectively. These compounds did not affect the peroxidation activity of MPO or the production of superoxide anion by stimulated leukocytes. By following the spectral change of MPO during the enzyme turnover, the inhibition of HOCl production can be explained on the basis of the accumulation of the redox form compound-II (MPO-II), which is an inactive chlorinating species. These results show that indole derivatives are effective and selective inhibitors of MPO-chlorinating activity.

Oxidation of the albumin thiol to sulfenic acid and its implications in the intravascular compartment

Human serum albumin (HSA) is the most abundant protein in the intravascular compartment. It possesses a single thiol, Cys34, which constitutes ~80% of the total thiols in plasma. This thiol is able to scavenge plasma oxidants. A central intermediate in this potential antioxidant activity of human serum albumin is sulfenic acid (HSA-SOH). Work from our laboratories has demonstrated the formation of a relatively stable sulfenic acid in albumin through complementary spectrophotometric and mass spectrometric approaches. Recently, we have been able to obtain quantitative data that allowed us to measure the rate constants of sulfenic acid reactions with molecules of analytical and biological interest. Kinetic considerations led us to conclude that the most likely fate for sulfenic acid formed in the plasma environment is the reaction with low molecular weight thiols to form mixed disulfides, a reversible modification that is actually observed in ~25% of circulating albumin. Another possible fate for sulfenic acid is further oxidation to sulfinic and sulfonic acids. These irreversible modifications are also detected in the circulation. Oxidized forms of albumin are increased in different pathophysiological conditions and sulfenic acid lies in a mechanistic junction, relating oxidizing species to final thiol oxidation products.

Oxidation of oleic acid at the air-water interface and its potential effects on cloud critical supersaturations

The oxidation of organic films on cloud condensation nuclei has the potential to affect climate and precipitation events. In this work we present a study of the oxidation of a monolayer of deuterated oleic acid (cis-9-octadecenoic acid) at the air-water interface by ozone to determine if oxidation removes the organic film or replaces it with a product film. A range of different aqueous sub-phases were studied. The surface excess of deuterated material was followed by neutron reflection whilst the surface pressure was followed using a Wilhelmy plate. The neutron reflection data reveal that approximately half the organic material remains at the air-water interface following the oxidation of oleic acid by ozone, thus cleavage of the double bond by ozone creates one surface active species and one species that partitions to the bulk (or gas) phase. The most probable products, produced with a yield of similar to(87 +/- 14)%, are nonanoic acid, which remains at the interface, and azelaic acid (nonanedioic acid), which dissolves into the bulk solution. We also report a surface bimolecular rate constant for the reaction between ozone and oleic acid of (7.3 +/- 0.9) x 10(-11) cm(2) molecule s(-1). The rate constant and product yield are not affected by the solution sub-phase. An uptake coefficient of ozone on the oleic acid monolayer of similar to 4 x 10(-6) is estimated from our results. A simple Kohler analysis demonstrates that the oxidation of oleic acid by ozone on an atmospheric aerosol will lower the critical supersaturation needed for cloud droplet formation. We calculate an atmospheric chemical lifetime of oleic acid of 1.3 hours, significantly longer than laboratory studies on pure oleic acid particles suggest, but more consistent with field studies reporting oleic acid present in aged atmospheric aerosol.

Synthesis of multicarboxylic acid appended imidazolium ionic liquids and their application in palladium-catalyzed selective oxidation of styrene

A series of multicarboxylic acid appended imidazolium ionic liquids ( McaILs) with chloride [ Cl](-) or bromide [ Br](-) as anions have been synthesized and characterized. Deprotonation of these ionic acids gives the corresponding zwitterions. Re-protonation of the zwitterions with strong Bronsted acids gives a series of new ionic acid-adducts, many of which remained as room-temperature ionic liquids. A new catalytic system, McaIL/PdCl2 for the selective catalytic oxidation of styrene to acetophenone with hydrogen peroxide as an oxidant has been attempted. In the presence of McaILs, it is found that the quantity of palladium chloride PdCl2 used can be greatly reduced while the activity ( TOF) and selectivity towards acetophenone are enhanced sharply. It is also shown that the catalytic properties of this system could be finely tuned through the molecular design of the McaILs. The best TOF value obtained so far is 146 h(-1) with 100% conversion of styrene at 93% selectivity to acetophenone. In addition, the catalytic activity has been maintained for at least ten catalytic cycles.

Oxovanadium(V) complexes of bis(phenolate) ligands with acetylacetone as co-ligand: synthesis, crystal structure, electrochemical and kinetics studies on the oxidation of ascorbic acid

Two vanadium(V) complexes, [VO(L-1)]acac)] (1) and [VO(L-2)(acac)] (2), where H2L1 = N,N-bis(2-hydroxy-3-5-di-tert-butyl-benzyl)propylamine and H2L2 = 2,2'-selenobis(4,6-di-tert-butylphenol), have been synthesized and characterized by elemental analyses, IR, V-51 NMR, both in the solid and in solution, and cyclic voltammetric studies. Single crystal X-ray studies reveal that in complex 1 the vanadium atom is octahedrally coordinated with an O5N donor environment, where the oxygen atom of the V-V=O moiety and the N atom of the ONO ligand occupy the axial sites while two oxygen atoms (O1 and O2) from the bisphenolate ligand and two oxygen atoms (O3 and O4) from the acac ligand occupy the equatorial plane. A similar bonding pattern has also been encountered for 2 with the exception that a Se atom instead of N is involved in weak bonding to the metal center. Both complexes showed reversible cyclic voltammeric responses and E-1/2 appears at -0.18 and 0.10 V versus NHE for complexes 1 and 2, respectively. The kinetics of oxidation of ascorbic acid by complex 1 were carried out in 50% MeCN-50% HO (v/v) at 25 degrees C. The high formation constant value, Q = 63 +/- 7 M-1, reveals that the reaction proceeds through the rapid formation of a H-bonded intermediate. The low k(2)Q(2)/k(1)Q(1) ratio (13.4) for 1 points out that there is extensive H-bonding between the oxygen atom of the V-V=O group and the OH group of ascorbic acid. (c) 2007 Published by Elsevier Ltd.