Erv2p: characterization of the redox behavior of a yeast sulfhydryl oxidase

The FAD prosthetic group of the ERV/ALR family of sulfhydryl oxidases is housed at the mouth of a 4-helix bundle and communicates with a pair of juxtaposed cysteine residues that form the proximal redox active disulfide. Most of these enzymes have one or more additional distal disulfide redox centers that facilitate the transfer of reducing equivalents from the dithiol substrates of these oxidases to the isoalloxazine ring where the reaction with molecular oxygen occurs. The present study examines yeast Erv2p and compares the redox behavior of this ER luminal protein with the augmenter of liver regeneration, a sulfhydryl oxidase of the mitochondrial intermembrane space, and a larger protein containing the ERV/ALR domain, quiescin-sulfhydryl oxidase (QSOX). Dithionite and photochemical reductions of Erv2p show full reduction of the flavin cofactor after the addition of 4 electrons with a midpoint potential of -200 mV at pH 7.5. A charge-transfer complex between a proximal thiolate and the oxidized flavin is not observed in Erv2p consistent with a distribution of reducing equivalents over the flavin and distal disulfide redox centers. Upon coordination with Zn2+, full reduction of Erv2p requires 6 electrons. Zn2+ also strongly inhibits Erv2p when assayed using tris(2-carboxyethyl)phosphine (TCEP) as the reducing substrate of the oxidase. In contrast to QSOX, Erv2p shows a comparatively low turnover with a range of small thiol substrates, with reduced Escherichia coli thioredoxin and with unfolded proteins. Rapid reaction studies confirm that reduction of the flavin center of Erv2p is rate-limiting during turnover with molecular oxygen. This comparison of the redox properties between members of the ERV/ALR family of sulfhydryl oxidases provides insights into their likely roles in oxidative protein folding.     

Decrease in heart mitochondrial creatine kinase activity due to oxygen free radicals.

This study was undertaken to examine the effects of oxygen free radicals on mitochondrial creatine kinase activity in rat heart. Xanthine plus xanthine oxidase (superoxide anion radical generating system) reduced mitochondrial creatine kinase activity both in a dose- and a time-dependent manner. Superoxide dismutase showed a protective effect on depression in creatine kinase activity due to xanthine plus xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a dose-dependent manner, this inhibition was protected by the addition of catalase. In order to understand the detailed mechanisms by which oxygen free radicals inhibit mitochondrial creatine kinase activity, the effects of oxygen free radicals on mitochondrial sulfhydryl groups were examined. Mitochondrial sulfhydryl groups contents were decreased by xanthine plus xanthine oxidase or hydrogen peroxide; this depression in sulfhydryl groups contents was prevented by the addition of superoxide dismutase or catalase. N-Ethylmaleimide (sulfhydryl group reagent) expressed inhibitory effects on the creatine kinase activity both in a dose- and a time-dependent manner; dithiothreitol or cysteine (sulfhydryl group reductant) showed protective effects on the creatine kinase activity depression induced by N-ethylmaleimide. Dithiothreitol or cysteine also blocked the depression of mitochondrial creatine kinase activity caused by xanthine plus xanthine oxidase or hydrogen peroxide. These results lead us to conclude that oxygen free radicals may inhibit mitochondrial creatine kinase activity by modifying sulfhydryl groups in the enzyme protein.     

Yeast ERV2p is the first microsomal FAD-linked sulfhydryl oxidase of the Erv1p/Alrp protein family

Saccharomyces cerevisiae Erv2p was identified previously as a distant homologue of Erv1p, an essential mitochondrial protein exhibiting sulfhydryl oxidase activity. Expression of the ERV2 (essential for respiration and vegetative growth 2) gene from a high-copy plasmid cannot substitute for the lack of ERV1, suggesting that the two proteins perform nonredundant functions. Here, we show that the deletion of the ERV2 gene or the depletion of Erv2p by regulated gene expression is not associated with any detectable growth defects. Erv2p is located in the microsomal fraction, distinguishing it from the mitochondrial Erv1p. Despite their distinct subcellular localization, the two proteins exhibit functional similarities. Both form dimers in vivo and in vitro, contain a conserved YPCXXC motif in their carboxyl-terminal part, bind flavin adenine dinucleotide (FAD) as a cofactor, and catalyze the formation of disulfide bonds in protein substrates. The catalytic activity, the ability to form dimers, and the binding of FAD are associated with the carboxyl-terminal domain of the protein. Our findings identify Erv2p as the first microsomal member of the Erv1p/Alrp protein family of FAD-linked sulfhydryl oxidases. We propose that Erv2p functions in the generation of microsomal disulfide bonds acting in parallel with Ero1p, the essential, FAD-dependent oxidase of protein disulfide isomerase.     

New water-soluble phosphines as reductants of peptide and protein disulfide bonds: reactivity and membrane permeability

Tris(2-carboxyethyl)phosphine (TCEP) is a widely used substitute for dithiothreitol (DTT) in the reduction of disulfide bonds in biochemical systems. Although TCEP has been recently shown to be a substrate of the flavin-dependent sulfhydryl oxidases, there is little quantitative information concerning the rate by which TCEP reduces other peptidic disulfide bonds. In this study, mono-, di-, and trimethyl ester analogues of TCEP were synthesized to evaluate the role of carboxylate anions in the reduction mechanism, and to expand the range of phosphine reductants. The effectiveness of all four phosphines relative to DTT has been determined using model disulfides, including a fluorescent disulfide-containing peptide (H(3)N(+)-VTWCGACKM-NH(2)), and with protein disulfide bonds in thioredoxin and sulfhydryl oxidase. Mono-, di-, and trimethyl esters exhibit phosphorus pK values of 6.8, 5.8, and 4.7, respectively, extending their reactivity with the model peptide to correspondingly lower pH values relative to that of TCEP (pK = 7.6). At pH 5.0, the order of reactivity is as follows: trimethyl- > dimethyl- > monomethyl- > TCEP > DTT; tmTCEP is 35-fold more reactive than TCEP, and DTT is essentially unreactive. Esterification also increases lipophilicity, allowing tmTCEP to penetrate phospholipid bilayers rapidly (>30-fold faster than DTT), whereas the parent TCEP is impermeant. Although more reactive than DTT toward small-molecule disulfides at pH 7.5, all phosphines are markedly less reactive toward protein disulfides at this pH. Molecular modeling suggests that the nucleophilic phosphorus of TCEP is more sterically crowded than the thiolate of DTT, contributing to the lower reactivity of the phosphine with protein disulfides. In sum, these data suggest that there is considerable scope for the synthesis of phosphine analogues tailored for specific applications in biological systems.     

Augmenter of liver regeneration: a flavin-dependent sulfhydryl oxidase with cytochrome c reductase activity

Augmenter of liver regeneration (ALR; hepatopoietin) is a recently discovered enigmatic flavin-linked sulfhydryl oxidase. An N-terminal His-tagged construct of the short form of the human protein has been overexpressed in Escherichia coli. Several lines of evidence suggest that, contrary to a recent report, human ALR is a disulfide-bridged dimer (linked via C15-C124) with two free cysteine residues (C74 and 85) per monomer. The C15-124 disulfides are not critical for dimer formation and have insignificant impact on the dithiothreitol (DTT) oxidase activity of ALR. Although the crystal structure of rat ALR shows a proximal disulfide (C62-C65) poised to interact with the FAD prosthetic group [Wu, C. K., Dailey, T. A., Dailey, H. A., Wang, B. C., and Rose, J. P. (2003) Protein Sci. 12, 1109-1118], only flavin reduction is evident during redox titrations of the enzyme. ALR forms large amounts of neutral semiquinone during aerobic turnover with DTT. This semiquinone arises, in part, by comproportionation between flavin centers within the dimer. Surprisingly, cytochrome c is about a 100-fold better electron acceptor for ALR than oxygen when DTT is the reducing substrate. These data suggest that this poorly understood flavoenzyme may not function as a sulfhydryl oxidase within the mitochondrial intermembrane space but may communicate with the respiratory chain via the mediation of cytochrome c.     

Modification of contractile proteins by oxygen free radicals in rat heart

This study was undertaken to investigate the effects of oxygen free radicals on myofibrillar creatine kinase activity. Isolated rat heart myofibrils were incubated with xanthine+xanthine oxidase (a superoxide anion radical-generating system) or hydrogen peroxide and assayed for creatine kinase activity. To clarify the involvement of changes in sulfhydryl groups in causing alterations in myofibrillar creatine kinase activity, 1) effects of N-ethylmaleimide (sulfhydryl groups reagent) on myofibrillar creatine kinase activity, 2) effect of oxygen free radicals on myofibrillar sulfhydryl groups content, and 3) protective effects of dithiothreitol (sulfhydryl groups-reducing agent) on the changes in myofibrillar creatine kinase activity due to oxygen free radicals were also studied. Xanthine+xanthine oxidase inhibited creatine kinase activity both in a time-and a concentration-dependent manner. Superoxide dismutase (SOD) showed a protective effect on the depression in creatine kinase activity caused by xanthine+xanthine oxidase. Hydrogen peroxide inhibited creatine kinase activity in a concentration-dependent manner; this inhibition was prevented by the addition of catalase. N-ethylmaleimide reduced creatine kinase activity in a dose-dependent manner. The content of myofibrillar sulfhydryl groups was decreased by xanthine+xanthine oxidase; this reduction was protected by SOD. Furthermore, the depression in myofibrillar creatine kinase activity by xanthine+xanthine oxidase was protected by the addition of dithiothreitol. Oxygen free radicals may inhibit myofibrillar creatine kinase activity by modifying sulfhydryl groups in the enzyme protein. The reduction of myofibrillar creatine kinase activity may lead to a disturbance of energy utilization in the heart and may contribute to cardiac dysfunction due to oxygen free radicals.     

Disulfide Bond Formation: Sulfhydryl Oxidase ALR Controls Mitochondrial Biogenesis of Human MIA40

The conserved MIA pathway is responsible for the import and oxidative folding of proteins destined for the intermembrane space of mitochondria. In contrast to a wealth of information obtained from studies with yeast, the function of the MIA pathway in higher eukaryotes has remained enigmatic. Here, we took advantage of the molecular understanding of the MIA pathway in yeast and designed a model of the human MIA pathway. The yeast model for MIA consists of two critical components, the disulfide bond carrier Mia40 and sulfhydryl oxidase Erv1/ALR. Human MIA40 and ALR substituted for their yeast counterparts in the essential function for the oxidative biogenesis of mitochondrial intermembrane space proteins. In addition, the sulfhydryl oxidases ALR/Erv1 were found to be involved in the mitochondrial localization of human MIA40. Furthermore, the defective accumulation of human MIA40 in mitochondria underlies a recently identified disease that is caused by amino acid exchange in ALR. Thus, human ALR is an important factor that controls not only the ability of MIA40 to bind and oxidize protein clients but also the localization of human MIA40 in mitochondria.     

Disulfide relays between and within proteins: the Ero1p structure

The essential flavoenzyme Ero1p both creates de novo disulfide bonds and transfers these disulfides to the folding catalyst protein disulfide isomerase (PDI). The recently solved crystal structure of Ero1p, in combination with previous biochemical, genetic and structural data, provides insight into the mechanism by which Ero1p accomplishes these tasks. A comparison of Ero1p with the smaller flavoenzyme Erv2p highlights important structural elements that are shared by these flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases and suggests some general themes that might be common to proteins that generate disulfide bonds.     

QSOX contains a pseudo-dimer of functional and degenerate sulfhydryl oxidase domains

Quiescin sulfhydryl oxidase (QSOX) catalyzes formation of disulfide bonds between cysteine residues in substrate proteins. Human QSOX1 is a multi-domain, monomeric enzyme containing a module related to the single-domain sulfhydryl oxidases of the Erv family. A partial QSOX1 crystal structure reveals a single-chain pseudo-dimer mimicking the quaternary structure of Erv enzymes. However, one pseudo-dimer “subunit” has lost its cofactor and catalytic activity. In QSOX evolution, a further concatenation to a member of the protein disulfide isomerase family resulted in an enzyme capable of both disulfide formation and efficient transfer to substrate proteins.     

Structure of a baculovirus sulfhydryl oxidase, a highly divergent member of the erv flavoenzyme family

Genomes of nucleocytoplasmic large DNA viruses (NCLDVs) encode enzymes that catalyze the formation of disulfide bonds between cysteine amino acid residues in proteins, a function essential for the proper assembly and propagation of NCLDV virions. Recently, a catalyst of disulfide formation was identified in baculoviruses, a group of large double-stranded DNA viruses considered phylogenetically distinct from NCLDVs. The NCLDV and baculovirus disulfide catalysts are flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases related to the cellular Erv enzyme family, but the baculovirus enzyme, the product of the Ac92 gene in Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is highly divergent at the amino acid sequence level. The crystal structure of the Ac92 protein presented here shows a configuration of the active-site cysteine residues and bound cofactor similar to that observed in other Erv sulfhydryl oxidases. However, Ac92 has a complex quaternary structural arrangement not previously seen in cellular or viral enzymes of this family. This novel assembly comprises a dimer of pseudodimers with a striking 40-degree kink in the interface helix between subunits. The diversification of the Erv sulfhydryl oxidase enzymes in large double-stranded DNA viruses exemplifies the extreme degree to which these viruses can push the boundaries of protein family folds.     

Avian sulfhydryl oxidase is not a metalloenzyme: adventitious binding of divalent metal ions to the enzyme

Metal- and flavin-dependent sulfhydryl oxidases catalyze the generation of disulfide bonds with reduction of oxygen to hydrogen peroxide. The mammalian skin enzyme has been reported to be copper-dependent, but a recent protein sequence shows it belongs to the Quiescin/sulfhydryl oxidase (QSOX) flavoprotein family. This work demonstrates that avian QSOX is not a metalloenzyme, and that copper and zinc ions inhibit the oxidation of reduced pancreatic ribonuclease by the enzyme. Studies with Zn(2+), as a redox inactive surrogate for copper, show that one Zn(2+) binds to four-electron-reduced QSOX by diverting electrons away from the flavin and into two of the three redox active disulfide bridges in the enzyme. The resulting zinc complex is modestly air-stable, reverting to a spectrum of the native protein with a t(1/2) of 40 min, whereas the four-electron-reduced native QSOX is reoxidized in less than a second under comparable conditions. Using tris(2-carboxyethyl)phosphine hydrochloride (TCEP), an alternate substrate of QSOX that binds Zn(2+) relatively weakly (unlike dithiothreitol), allows rapid inhibition of oxidase activity to be demonstrated at low micromolar metal levels. Zinc binding was followed by rapid-scanning spectrophotometry. Copper also binds the four-electron-reduced form of QSOX with a visible spectrum suggestive of active site occupancy. In addition to interactions with the reduced enzyme, dialysis experiments show that multiple copper and zinc ions can bind to the oxidized enzyme without the perturbation of the flavin spectrum seen earlier. These data suggest that a reinvestigation of the metal content of skin sulfhydryl oxidases is warranted. The redox-modulated binding of zinc to QSOX is considered in light of evidence for a role of zinc-thiolate interactions in redox signaling and zinc mobilization.     

An essential function of the mitochondrial sulfhydryl oxidase Erv1p/ALR in the maturation of cytosolic Fe/S proteins

Biogenesis of Fe/S clusters involves a number of essential mitochondrial proteins. Here, we identify the essential Erv1p of Saccharomyces cerevisia mitochondria as a novel component that is specifically required for the maturation of Fe/S proteins in the cytosol, but not in mitochondria. Furthermore, Erv1p was found to be important for cellular iron homeostasis. The homologous mammalian protein ALR (‘augmenter of liver regeneration’), also termed hepatopoietin, can functionally replace defects in Erv1p and thus represents the mammalian orthologue of yeast Erv1p. Previously, a fragment of ALR was reported to exhibit an activity as an extracellular hepatotrophic growth factor. Both Erv1p and full-length ALR are located in the mitochondrial intermembrane space and represent the first components of this compartment with a role in the biogenesis of cytosolic Fe/S proteins. It is likely that Erv1p/ALR operates downstream of the mitochondrial ABC transporter Atm1p/ABC7/Sta1, which also executes a specific task in this essential biochemical process.     

Egg white sulfhydryl oxidase: kinetic mechanism of the catalysis of disulfide bond formation

The flavin-dependent sulfhydryl oxidase from chicken egg white catalyzes the oxidation of sulfhydryl groups to disulfides with reduction of oxygen to hydrogen peroxide. The oxidase contains FAD and a redox-active cystine bridge and accepts a total of 4 electrons per active site. Dithiothreitol (DTT; the best low molecular weight substrate known) reduces the enzyme disulfide bridge with a limiting rate of 502/s at 4 degrees C, pH 7.5, yielding a thiolate-to-flavin charge-transfer complex. Further reduction to EH4 is limited by the slow internal transfer of reducing equivalents from enzyme dithiol to oxidized flavin (3.3/s). In the oxidative half of catalysis, oxygen rapidly converts EH4 to EH2, but Eox appearance is limited by the slow internal redox equilibration. During overall turnover with DTT, the thiolate-to-flavin charge-transfer complex accumulates with an apparent extinction coefficient of 4.9 mM-1 cm-1 at 560 nm. In contrast, glutathione (GSH) is a much slower reductant of the oxidase to the EH2 level and shows a kcat/Km 100-fold smaller than DTT. Full reduction of EH2 by GSH shows a limiting rate of 3.6/s at 4 degrees C comparable to that seen with DTT. Reduced RNase is an excellent substrate of the enzyme, with kcat/Km per thiol some 1000- and 10-fold better than GSH and DTT, respectively. Enzyme-monitored steady-state turnover shows that RNase is a facile reductant of the oxidase to the EH2 state. This work demonstrates the basic similarity in the mechanism of turnover between all of these three substrates. A physiological role for sulfhydryl oxidase in the formation of disulfide bonds in secreted proteins is discussed.     

Effect of a prolonged superoxide flux on transferrin and ferritin

The involvement of "free" iron in damage caused by oxidative stress is well recognized. Superoxide generated in a short burst and at a relatively high flux by the xanthine/xanthine oxidase couple is known to release iron from ferritin in the presence of phenanthroline derivatives as iron chelators. However, superoxide generation via xanthine oxidase is accompanied by the simultaneous direct generation of hydrogen peroxide and, in the presence of ferritin, there is also a superoxide-independent release of iron. In this study it was found that the iron chelator employed attenuates superoxide formation from the xanthine/xanthine oxidase couple. The reaction of ferritin and transferrin with a clean chemical source of superoxide, di(4-carboxybenzyl)hyponitrite (SOTS-1) was therefore investigated. The efficiency of superoxide-induced iron release from ferritin increases dramatically as the superoxide flux is decreased, reaching as high as 0.5 Fe per O2*-. Treatment of ferritin for 16 h with SOTS-1 yielded as many as 130 Fe atoms/ferritin molecule, which greatly exceeds the amount of possible "contaminating" iron absorbed on the protein shell.     

Sarco/endoplasmic reticulum Ca2+-pump isoform SERCA3a is more resistant to superoxide damage than SERCA2b

Endo/sarcoplasmic reticulum (ER) Ca²⁺-pumps are important for cell survival and communication but they are inactivatedby reactive oxygen species (ROS).We have previously reported that the Ca²⁺-pump isoform SERCA3a is more resistant than SERCA2b to damage by peroxide. Since peroxide and superoxide differ in their redox potentials, we now report the effects of superoxide on the two Ca²⁺-pump isoforms. We isolated microsomes from HEK293 cells transiently transfected with SERCA2b or SERCA3a cDNA. We exposed these microsomes to superoxide which was generated using xanthine plus xanthine oxidase and catalase to prevent accumulation of peroxide due to superoxide dismutation. Superoxide damaged the Ca²⁺- transport activity of both isoforms but SERCA3a was damaged at higher concentrations of superoxide and upon longer periods of exposures than was SERCA2b. Thus the SERCA3a isoform is more resistant than SERCA2b to inactivation by both superoxide and peroxide. (Mol Cell Biochem 000: 000-000, 1999)     

Thioureas react with superoxide radicals to yield a sulfhydryl compound. Explanation for protective effect against paraquat

Thiourea and superoxide dismutase were effective antidotes to paraquat toxicity in an HL60 cell culture system, whereas other hydroxyl scavengers were ineffective. The efficacy of thioureas was not due to blockage of intracellular paraquat uptake, inhibition of NADPH-P-450 reductase, or reaction with the paraquat radical. Thiourea also competitively inhibited the reduction of cytochrome c by the xanthine/xanthine oxidase superoxide-generating system, and the release of iron from ferritin by superoxide radicals. The reaction of superoxide with thiourea produced a sulfhydryl compound distinct from products formed by hydrogen peroxide or hydroxyl radicals. Spectrophotometric and chromatographic studies indicated the carbon-sulfide double bond was converted to a sulfhydryl group which reacted with Ellman's reagent. Additional confirmatory evidence for the sulfhydryl compound was obtained with carbon-13 NMR and mass spectroscopies. Thus, thioureas are direct scavengers of superoxide radicals as well as hydroxyl radicals and hydrogen peroxide. The rate constant for the reduction of thiourea by superoxide was estimated at 1.1 x 10(3) M-1 s-1. The implication of this finding on free radical studies, the mechanism of paraquat toxicity, and the metabolism of thioureas is discussed.     

Up-regulation of rat adipose tissue adiponectin gene expression by long-term but not by short-term food restriction

3H-1,2-Dithiole-3-thione (D3T), a potent member of dithiolethiones, induces phase 2 enzymes by activating an Nrf2/Keap1-dependent signaling pathway. It was proposed that interaction between D3T and two adjacent sulfhydryl groups of Keap1 might cause dissociation of Keap1 from Nrf2, leading to Nrf2 activation. This study was undertaken to investigate the reactions between D3T and thiols, including the dithiol compound, dithiothreitol (DTT), and the monothiol, glutathione (GSH). We reported here that under physiologically relevant conditions incubation of D3T with DTT caused remarkable oxygen consumption, indicating a redox reaction between D3T and the dithiol molecule. Incubation of D3T with GSH also led to oxygen consumption, but to a less extent. Electron paramagnetic resonance (EPR) studies showed that the redox reaction between D3T and DTT generated superoxide. Superoxide was also formed from the redox reaction of D3T with GSH. These findings demonstrate that D3T reacts with thiols, particularly a dithiol, generating superoxide, which may provide a mechanistic explanation for induction of Nrf2-dependent phase 2 enzymes by D3T.     

Nuclear and mitochondrial interaction involving mt-Nd2 leads to increased mitochondrial reactive oxygen species production

NADH dehydrogenase subunit 2, encoded by the mtDNA, has been associated with resistance to autoimmune type I diabetes (T1D) in a case control study. Recently, we confirmed a role for the mouse ortholog of the protective allele (mt-Nd2(a)) in resistance to T1D using genetic analysis of outcrosses between T1D-resistant ALR and T1D-susceptible NOD mice. We sought to determine the mechanism of disease protection by elucidating whether mt-Nd2(a) affects basal mitochondrial function or mitochondrial function in the presence of oxidative stress. Two lines of reciprocal conplastic mouse strains were generated: one with ALR nuclear DNA and NOD mtDNA (ALR.mt(NOD)) and the reciprocal with NOD nuclear DNA and ALR mtDNA (NOD.mt(ALR)). Basal mitochondrial respiration, transmembrane potential, and electron transport system enzymatic activities showed no difference among the strains. However, ALR.mt(NOD) mitochondria supported by either complex I or complex II substrates produced significantly more reactive oxygen species when compared with both parental strains, NOD.mt(ALR) or C57BL/6 controls. Nitric oxide inhibited respiration to a similar extent for mitochondria from the five strains due to competitive antagonism with molecular oxygen at complex IV. Superoxide and hydrogen peroxide generated by xanthine oxidase did not significantly decrease complex I function. The protein nitrating agents peroxynitrite or nitrogen dioxide radicals significantly decreased complex I function but with no significant difference among the five strains. In summary, mt-Nd2(a) does not confer elevated resistance to oxidative stress; however, it plays a critical role in the control of the mitochondrial reactive oxygen species production.     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O2.- and H2O2 during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-beta-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O2 consumption, only slightly but substantially inhibited in a competitive manner the O2.- -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two mutations convert mammalian xanthine oxidoreductase to highly superoxide-productive xanthine oxidase

Reactive oxygen species are generated by various systems, including NADPH oxidases, xanthine oxidoreductase (XOR) and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide in a molar ratio of about 1:3, depending upon the conditions. Here, we present a mutant of rat XOR that displays mainly XO activity with a superoxide:hydrogen peroxide production ratio of about 6:1. In the mutant, tryptophan 335, which is a component of the amino acid cluster crucial for switching from the XDH to the XO conformation, was replaced with alanine, and phenylalanine 336, which modulates FAD's redox potential through stacking interactions with the flavin cofactor, was changed to leucine. When the mutant was expressed in Sf9 cells, it was obtained in the XO form, and dithiothreitol treatment only partially restored the pyridine nucleotide-binding capacity. The crystal structure of the dithiothreitol-treated mutant at 2.3 Angstroms resolution showed the enzyme's two subunits to be quite similar, but not identical: the cluster involved in conformation-switching was completely disrupted in one subunit, but remained partly associated in the other one. The chain trace of the active site loop in this mutant is very similar to that of the bovine XO form. These results are consistent with the idea that the XDH and XO forms of the mutant are in an equilibrium that greatly favours the XO form, but the equilibrium is partly shifted towards the XDH form upon incubation with dithiothreitol.