The NADPH oxidase of professional phagocytes--prototype of the NOX electron transport chain systems

The NADPH oxidase is an electron transport chain in "professional" phagocytic cells that transfers electrons from NADPH in the cytoplasm, across the wall of the phagocytic vacuole, to form superoxide. The electron transporting flavocytochrome b is activated by the integrated function of four cytoplasmic proteins. The antimicrobial function of this system involves pumping K+ into the vacuole through BKCa channels, the effect of which is to elevate the vacuolar pH and activate neutral proteases. A number of homologous systems have been discovered in plants and lower animals as well as in man. Their function remains to be established.     

Alkalinity of Neutrophil Phagocytic Vacuoles Is Modulated by HVCN1 and Has Consequences for Myeloperoxidase Activity

The NADPH oxidase of neutrophils, essential for innate immunity, passes electrons across the phagocytic membrane to form superoxide in the phagocytic vacuole. Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced. Using the pH indicator SNARF, we measured changes in pH in the phagocytic vacuole and cytosol of neutrophils. In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly. By contrast, in Hvcn1 knock out mouse neutrophils, the vacuolar pH rose above 11, vacuoles swelled, and the cytosol acidified excessively, demonstrating that ordinarily this channel plays an important role in charge compensation. Proton extrusion was not diminished in Hvcn1^-/- mouse neutrophils arguing against its role in maintaining pH homeostasis across the plasma membrane. Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH.     

The Ca2+-Regulation of the Mitochondrial External NADPH Dehydrogenase in Plants Is Controlled by Cytosolic pH

NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca²⁺ and pH in vitro, but concentrations of Ca²⁺ needed are not known. We have determined the K_0.5(Ca²⁺) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O₂ and decylubiquinone as electron acceptors. The K_0.5(Ca²⁺) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K_0.5(Ca²⁺) for NADPH oxidation was high (≈100 μM), yet 20-fold lower K_0.5(Ca²⁺) values were determined at pH 6.8. Lower K_0.5(Ca²⁺) values were observed with decylubiquinone than with O₂ as terminal electron acceptor. NADPH oxidation responded to changes in Ca²⁺ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca²⁺-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene.     

The cytosolic subunit p67 of the NADPH-oxidase complex does not bind NADPH

The NADPH-oxidase of phagocytic cells is a multicomponent enzyme that generates superoxide. It comprises a membrane flavocytochrome b₅₅₈ and four cytosolic proteins; p67^phox, p47^phox, p40^phox and Rac. The NADPH-binding site of this complex was shown to be located on the flavocytochrome b₅₅₈. However, a number of studies have suggested the presence of another site on the p67^phox subunit which is the key activating component. Using several approaches like tryptophan quenching fluorescence measurement, inhibition by 2′,3′-dialdehyde NADPH, and free/bound NADPH concentration measurements, we demonstrate that no NADPH binds on p67^phox, thus definitively solving the controversy on the number and location of the NADPH-binding sites on this complex.     

The NADPH oxidase of phagocytic cells is an electron pump that alkalinises the phagocytic vacuole

Summary Phagocytic cells of the immune system contain an oxidase that is important for the killing and digestion of engulfed microbes. This is an electron transport chain that transfers electrons from NADPH in the cytosol to oxygen to form superoxide and hydrogen peroxide in the phagocytic vacuole. Absence or abnormality of this oxidase results in the syndrome of CGD, characterised by a profound predisposition to infection. The electron transport chain consists of a flavocytochrome b located in the plasma membrane and membrane of the specific granules. It is composed of a and b-subunits, with apparent molecular masses of 23 kDa and 76–92 kDa, respectively. The b-subunit is a member of the FNR family of reductases with FAD and NADPH binding sites. Based upon the crystal structure of FNR we have constructed a model of the more hydrophilic C terminal half of this b-subunit, which acts as a guide to the organisation of the molecule, and provides a template on which to map mutations in CGD. The location of the heme is uncertain. Electron transport is dependent upon an activation complex of cytosolic proteins including p40 ^phox , p47 ^phox , and p67 ^phox , and the small GTP binding protein, p21 ^rac . This oxidase system is important for the killing and digestion of bacteria and fungi. This might be accomplished in a number of ways. The oxidase produces superoxide and hydrogen which might be toxic themselves. The hydrogen peroxide can act as substrate for myeloperoxidase which can oxidise chloride and iodide to chlorine and iodine and their hypohalous acids. The proteins contained within the cytoplasmic granules are also very important in the killing process. These are neutral proteinases that require a neutral or slightly alkaline pH for optimal activity. The oxidase transports electrons, unaccompanied by protons, across the wall of the phagocytic vacuole, resulting in an elevation of the vacuolar pH, thereby optimising conditions for killing and digestion of engulfed organisms by these neutral proteinases.     

Swell activated chloride channel function in human neutrophils

Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I_e) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I_e has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I_e must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.     

Purification and Characterization of a Ferredoxin-NADP Oxidoreductase-Like Enzyme from Radish Root Tissues

An enzyme able to reduce cytochrome c via ferredoxin in the presence of NADPH, was isolated, purified from radish (Raphanus sativus var acanthiformis cultivar miyashige) roots and characterized. The enzyme was purified by DEAE-cellulose, Blue-Cellulofine, Ferredoxin-Sepharose 4B, and Sephadex G-100 column chromatography. Molecular mass of the enzyme was estimated to be 33,000 and 35,000 daltons by Sephadex G-100 gel filtration and SDS-PAGE, respectively. Its absorption spectrum suggested that the enzyme contains flavin as a prosthetic group. The K_m values for NADPH and ferredoxin were calculated to be 9.2 and 1.2 micromolar, respectively. The enzyme required NADPH and did not use NADH as an electron donor. The optimal pH was 8.4. The enzyme also catalyzed the photoreduction of NADP⁺ in the spinach leaf thylakoid membranes depleted of ferredoxin and ferredoxin-NADP⁺ oxidoreductase. The effect of NaCl and MgCl₂ concentration on the activity and amino acid composition of the enzyme were demonstrated. The results suggest that the enzyme is similar to ferredoxin-NADP⁺ oxidoreductase from chloroplasts and cyanobacteria and is the key enzyme catalyzing the electron transport between NADPH, generated by the pentose phosphate pathway, and ferredoxin in plastids of plant heterotrophic tissues.     

Nitrofuran enhancement of microsomal electron transport, superoxide anion production and lipid peroxidation

Addition of nifurtimox (a nitrofuran derivative used for the treatment of Chagas' disease) to rat liver microsomes produced an increase of (a) electron flow from NADPH to molecular oxygen, (b) generation of both superoxide anion radical (O₂^?) and hydrogen peroxide, and (c) lipid peroxidation. The nifurtimox-stimulated NADPH oxidation was greatly inhibited by NADP⁺ and p-chloromercuribenzoate, and to a lesser extent by SKF-525-A and metyrapone. These inhibitions reveal the function of both the NADPH-cytochrome P-450 (c) reductase and cytochrome P-450 in nifurtimox reduction. Superoxide dismutase, catalase (in the presence of superoxide dismutase), and hydroxyl radical scavengers (mannitol, 5,5-dimethyl-1-pyrroline-1-oxide) inhibited the nifurtimox-stimulated NADPH oxidation, in accordance with the additional operation of a reaction chain including the hydroxyl radical. Further evidence supporting the role of superoxide anion and hydroxyl radicals in the nifurtimox-induced NADPH oxidation resulted from the effect of specific inhibitors on NADPH oxidation by O₂^? (generated by the xanthine oxidase reaction) and by OH. (generated by an iron chelate or the Fenton reaction). Production of O₂^? by rat kidney, testes and brain microsomes was significantly stimulated by nifurtimox in the presence of NADPH. It is postulated that enhanced formation of free radicals is the basis for nifurtimox toxicity in mammals, in good agreement with the postulated mechanism of the trypanocide effect of nifurtimox on Trypanosoma cruzi.     

Oxidant Production by Human B Lymphocytes: Detection of Activity and Identification of Components Involved

The O⁻₂-generating NADPH oxidase, originally thought to be expressed only in phagocytic cells of the immune system, can also be expressed in B lymphocytes. Epstein–Barr virus-transformed B cell lines (EBV-BL) can generate O⁻₂at rates corresponding to between 1 and 5% of the rates obtained by activated neutrophils. The composition of the NADPH oxidase of EBV-BL appears to be identical to that of phagocytic cells. In this report, methods are described for the establishment of EBV-BL, together with assays for the measurement of reactive oxidant production and detection of the constituent components of the NADPH oxidase in these cells.     

Calcium alginate-Immobilized hepatic microsomes: Effect of NADPH cofactor on oxidation rates

An important function of the liver is detoxification of drugs, toxins and foreign compounds. Within the liver cell, the endoplasmic reticulum, isolated as the microsomal fraction, is especially active. Microsomal oxidation is the major oxidation pathway for many compounds, and the requirement for NADPH, an expensive cofactor, is an important consideration in bioreactor design. This paper presents design information for NADPH- and substrate-dependent oxidation rates for free and immobilized microsomes. The primary goal of this paper is determining NADPH requirements for oxidation. The effect of various initial levels of nicotinamide adenine dinucleotide phosphate (NADPH) on chlorpromazine oxidation rate has been studied for a crude hepatic microsomal fraction immobilized in calcium alginate gel. At an initial NADPH concentration of 600 nmoles/ml, immobilized microsomes accelerate to a maximal velocity of ≈ 240 nmoles min⁻¹ ml⁻¹ of oxygen consumption. In comparison, free microsomes reach a maximal velocity of approximately 150 nmoles min⁻¹ ml⁻¹ at an initial NADPH concentration of 220 nmoles/ml. By fitting the “initial” rate as a function of NADPH concentration to Michaelis-Menten kinetics, the apparent half-saturation coefficients (K_m)app are 3.5 nmole/ml for free microsomes and 134.4 nmole/ml for immobilized microsomes, however the maximum reaction velocity, V_max, for immobilized microsomes is calculated to be 322 nmoles min⁻¹ ml⁻¹ compared with 145 nmols min⁻¹ ml⁻¹ for free microsomes. Preliminary studies indicate that is is possible to obtain significant reaction rates using calcium alginate immobilized microsomes and that this system may offer advantages due to its simplicity and lower cost.     

Characterisation of electron currents generated by the human neutrophil NADPH oxidase

Electron transport by the human neutrophil NADPH oxidase is an important microbicidal weapon for phagocytes. The electron current (I_e) generated by the neutrophil NADPH oxidase is poorly characterised due to the lack of appropriate electrophysiological data. In this study, I fully characterise the neutrophil generated I_e when the NADPH oxidase is activated by NADPH and GTPγS. The neutrophil I_e was markedly voltage-dependent in the entire voltage range in comparison to those electron currents measured after chloride was removed from the external bath solution. The difference in I_e measured in chloride free conditions was not due to a change in the activation kinetics of voltage-gated proton channels. The I_e depolarises the neutrophil plasma membrane at a rate of 2.3 V s⁻¹ and this depolarisation was opposed when voltage-gated proton channels are activated. 3 mM ZnCl₂ depolarised the membrane potential to +97.8 ± 2.5 mV (n = 4), and this depolarisation was abolished after NADPH oxidase inhibition.     

Cyanide-resistant respiration in yeast. II. Characterization of a cyanide-insensitive NAD(P)H oxidoreductase

A single protein species isolated from yeast (preceding paper) which catalyzes the cyanide-resistant reduction of molecular oxygen by reduced pyridine nucleotides has been characterized using spectral and chemical criteria. This NAD(P)H:O₂ oxidoreductase is a metalloflavoprotein containing FMN as a prosthetic group and Cu²⁺ as the metal ion. The enzyme which is devoid of nonheme iron centers is inhibited by the chelators m-chlorohydroxamic acid; salicylhydroxamic acid, and bathophenathroline sulfonate. Of the many substrates tested only NADH and NADPH were found to be active with the latter having a higher apparent K_m (2.1 of 10^?5 vs 1.4 × 10^?6m). Stopped-flow kinetics established that the $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for these substrates was 0.41 and 0.32, respectively. It is suggested that the function of this enzyme is associated with the nonmitochondrial P-450 system. A tentative pathway of electron flow has been proposed to NADPH → FMN → Cu²⁺ → H₂O₂.     

Ferredoxin:NADP+ Oxidoreductase Association with Phycocyanin Modulates Its Properties

In photosynthetic organisms, ferredoxin:NADP⁺ oxidoreductase (FNR) is known to provide NADPH for CO₂ assimilation, but it also utilizes NADPH to provide reduced ferredoxin. The cyanobacterium Synechocystis sp. strain PCC6803 produces two FNR isoforms, a small one (FNR_S) similar to the one found in plant plastids and a large one (FNR_L) that is associated with the phycobilisome, a light-harvesting complex. Here we show that a mutant lacking FNR_L exhibits a higher NADP⁺/NADPH ratio. We also purified to homogeneity a phycobilisome subcomplex comprising FNR_L, named FNR_L-PC. The enzymatic activities of FNR_L-PC were compared with those of FNR_S. During NADPH oxidation, FNR_L-PC exhibits a 30% decrease in the Michaelis constant K_m(NADPH), and a 70% increase in K_{m(ferredoxin)}, which is in agreement with its predicted lower activity of ferredoxin reduction. During NADP⁺ reduction, the FNR_L-PC shows a 29/43% decrease in the rate of single electron transfer from reduced ferredoxin in the presence/absence of NADP⁺. The increase in K_{m(ferredoxin)} and the rate decrease of single reduction are attributed to steric hindrance by the phycocyanin moiety of FNR_L-PC. Both isoforms are capable of catalyzing the NADP⁺ reduction under multiple turnover conditions. Furthermore, we obtained evidence that, under high ionic strength conditions, electron transfer from reduced ferredoxin is rate limiting during this process. The differences that we observe might not fully explain the in vivo properties of the Synechocystis mutants expressing only one of the isoforms. Therefore, we advocate that FNR localization and/or substrates availability are essential in vivo.     

Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic Clostridia

The physiological function of the clostridial NADH- and NADPH-ferredoxin oxidoreductases was investigated with Clostridium pasteurianum and Clostridium butyricum.The NADH-ferredoxin oxidoreductases are concluded to be catabolic enzymes required for the reduction of ferredoxin by NADH. The conclusion is based on the finding that during the entire growth phase the fermentation of glucose can be formally represented by the weighted sum of Eqns 1 and 2, Glucose + 2 H₂O → 1 butyrate^? + 2 HCO₃^? + 3 H⁺ + 2 H₂ (1) Glucose + 4 H₂O → 2 acetate^? + 2 HCO₃^? + 4 H⁺ + 4 H₂ (2) and that in these redox processes NADH rather than NADPH is specifically formed during glyceraldehyde phosphate dehydrogenation. This NADH can be consumed by substrate reduction in Process 1 only, while it must be reoxidized in Process 2 by the ferredoxin-dependent proton reduction to hydrogen which involves the NADH-ferredoxin oxidoreductases.The kinetic and regulatory properties of these enzymes are in line with their catabolic role: they are found with high specific activities typical for other catabolic enzymes; essentially they catalyze electron flow from NADH to ferredoxin only because the back reaction is very effectively inhibited by low concentrations of NADH. These enzymes have a key role in the coupling of the two partial processes and in regulating the overall thermodynamic efficiency of the fermentations.The NADPH-ferredoxin oxidoreductases are concluded to participate in anabolism; they are required for the regeneration of NADPH. The conclusion is based on the finding that in the two clostridia all catabolic oxidations-reductions are specific for NAD(H) and that the usual NADPH-producing processes such as the glucose 6-phosphate dehydrogenase or malate enzyme reactions are absent. The kinetic properties of the enzymes are in agreement with their anabolic function: the NADPH-ferredoxin oxidoreductases are found with sufficient specific activities; they preferentially catalyze electron transfer from reduced ferredoxin to NADP⁺.     

Distribution and some properties of NADPH and NADH oxidase in parenchymal and nonparenchymal liver cells

The activities of NADPH and NADH oxidase were determined in homogenates of isolated pure parenchymal and nonparenchymal rat liver cells at neutral (7.4) and acid (5.5) pH. The NADPH oxidase at pH 7.4 is about equally active in parenchymal and nonparenchymal cells and in both cell types is rather insensitive to KCN (1 mm) inhibition. By lowering the pH to 5.5, the NADPH oxidase of the nonparenchymal cells is stimulated (twofold) while the activity in parenchymal cells is decreased. The NADH consumption at neutral pH in parenchymal cells is 75% inhibited by KCN, while this activity in nonparenchymal cells is relatively insensitive to KCN. The NADH oxidase in both parenchymal and nonparenchymal liver cells is less active when the pH is lowered from 7.4 to 5.5. The distribution of NAD(P)H oxidases between parenchymal and nonparenchymal liver cells and the effect of pH on their activities suggest that in the nonparenchymal cells, the NADPH oxidase might play a role in the synthesis of H₂O₂ within the phagocytic vacuole. A scheme is proposed which describes the metabolic events involved in H₂O₂ formation and catabolism of endo(phago)cytosed particles in nonparenchymal liver cells.     

Yeast glutathione reductase. Steady-state kinetic studies of its transhydrogenase activity.

Yeast glutathione reductase catalyzes a pyridine nucleotide transhydrogenase reaction using either NADPH or NADH as the electron donor and thionicotinamideadenine dinucleotide phosphate as the electron acceptor. Competitive substrate inhibition of the transhydrogenase reaction by NADPH (K_i = 11 μM) is observed when NADPH is the electron donor. Competitive substrate inhibition by thionicotinamide-adenine dinucleotide phosphate (K_i = 58 μM) is observed with NADH as the electron donor. The turnover numbers of the two transhydrogenase reactions are similar and are equal to about 1% of the turnover number for the NADPH-dependent reduction of oxidized glutathione catalyzed by the enzyme. The transhydrogenase kinetics are analyzed in terms of a pingpong mechanism. It is concluded that the substrate inhibition results from formation of abortive complexes of NADPH with the reduced form of the enzyme and of thionicotinamide-adenine dinucleotide phosphate with the oxidized form of the enzyme. With NADPH as the electron donor, the apparent Michaelis constant for thionicotinamide-adenine dinucleotide phosphate is sensitive to the ionic composition of the assay medium. The data are interpreted to support the existence of a general pyridine nucleotide-binding site at the active site of the enzyme and separate from the binding site for oxidized glutathione.     

Targeted Delivery of Amoxicillin to C. trachomatis by the Transferrin Iron Acquisition Pathway

Weak intracellular penetration of antibiotics makes some infections difficult to treat. The Trojan horse strategy for targeted drug delivery is among the interesting routes being explored to overcome this therapeutic difficulty. Chlamydia trachomatis, as an obligate intracellular human pathogen, is responsible for both trachoma and sexually transmitted diseases. Chlamydia develops in a vacuole and is therefore protected by four membranes (plasma membrane, bacterial inclusion membrane, and bacterial membranes). In this work, the iron-transport protein, human serum-transferrin, was used as a Trojan horse for antibiotic delivery into the bacterial vacuole. Amoxicillin was grafted onto transferrin. The transferrin-amoxicillin construct was characterized by mass spectrometry and absorption spectroscopy. Its affinity for transferrin receptor 1, determined by fluorescence emission titration [K_affTf-amox = (1.3 ± 1.0) x 10⁸], is very close to that of transferrin [4.3 x 10⁸]. Transmission electron and confocal microscopies showed a co-localization of transferrin with the bacteria in the vacuole and were also used to evaluate the antibiotic capability of the construct. It is significantly more effective than amoxicillin alone. These promising results demonstrate targeted delivery of amoxicillin to suppress Chlamydia and are of interest for Chlamydiaceae and maybe other intracellular bacteria therapies.     

Kinetic and structural insight into a role of the re-face Tyr328 residue of the homodimer type ferredoxin-NADP+ oxidoreductase from Rhodopseudomonas palustris in the reaction with NADP+/NADPH

Among the thioredoxin reductase-type ferredoxin-NAD(P)⁺ oxidoreductase (FNR) family, FNR from photosynthetic purple non‑sulfur bacterium Rhodopseudomonas palustris (RpFNR) is distinctive because the predicted residue on the re-face of the isoalloxazine ring portion of the FAD prosthetic group is a tyrosine. Here, we report the crystal structure of wild type RpFNR and kinetic analyses of the reaction of wild type, and Y328F, Y328H and Y328S mutants with NADP⁺/NADPH using steady state and pre-steady state kinetic approaches.The obtained crystal structure of wild type RpFNR confirmed the presence of Tyr328 on the re-face of the isoalloxazine ring of the FAD prosthetic group through the unique hydrogen bonding of its hydroxyl group. In the steady state assays, the substitution results in the decrease of K_d for NADP⁺ and K_M for NADPH in the diaphorase assay; however, the k_cat values also decreased significantly. In the stopped-flow spectrophotometry, mixing oxidized RpFNRs with NADPH and reduced RpFNRs with NADP⁺ resulted in rapid charge transfer complex formation followed by hydride transfer. The observed rate constants for the hydride transfer in both directions were comparable (>400 s⁻¹). The substitution did not drastically affect the rate of hydride transfer, but substantially slowed down the subsequent release and re-association of NADP⁺/NADPH in both directions. The obtained results suggest that Tyr328 stabilizes the stacking of C-terminal residues on the isoalloxazine ring portion of the FAD prosthetic group, which impedes the access of NADP⁺/NADPH on the isoalloxazine ring portions, in turn, enhancing the release of the NADP⁺/NADPH and/or reaction with electron transfer proteins.     

Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae

Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP⁺ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/NADP⁺ ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H₂O₂ exposure, implying that Yhm2p has an antioxidant function.