Reconstitution and characterization of the human neutrophil respiratory burst oxidase using recombinant p47-phox, p67-phox and plasma membrane.

Human neutrophil respiratory burst oxidase (NADPH-oxidase) activity can be reconstituted in a cell-free system consisting of plasma membrane, cytosol and an anionic amphiphile [e.g., sodium dodecyl sulfate (SDS) or arachidonate]. Herein, we report reconstitution of oxidase activity using isolated neutrophil plasma membrane together with purified recombinant p47-phox and p67-phox which had been produced using a baculovirus expression system. Activity required an anionic amphiphile (SDS or arachidonate) and was potentiated by diacylglycerol and GTP gamma S. Serial washes of the plasma membrane failed to affect its ability to reconstitute activity, indicating that a dissociable membrane component was not present. The Km for NADPH, 43 microM, was the same as that determined using cytosol in place of recombinant factors. The EC50 values for p47-phox and p67-phox under optimal activation conditions were 220 nM and 80 nM, respectively, indicating a relatively high affinity of these components in an activation complex. Since neither cytosolic component contains a nucleotide binding consensus sequence, these data indicate that the NADPH binding component of the oxidase resides in the plasma membrane.     

Two cytosolic components of the neutrophil NADPH oxidase, P47-phox and P67-phox, are not flavoproteins

Two cytosolic proteins, p47-phox and p67-phox, have been shown to be essential components of the NADPH-dependent oxidase of human neutrophils, although the specific role of each of these proteins in the multicomponent electron transport complex is undetermined. The superoxide-generating activity of this oxidase can be reproduced in a cell-free system, combining cytosol and membranes from unstimulated neutrophils in the presence of fatty acid and NADPH. In the present studies, cytosol was treated with myristic acid, arachidonic acid, or sodium dodecyl sulfate in the absence of membranes and the resultant precipitate collected by centrifugation and analyzed. Both p47-phox and p67-phox precipitated in the presence of fatty acid. However, neither FAD nor FMN was localized in the precipitates, even though substantial amounts of p47-phox and p67-phox precipitated. These results suggest that neither p47-phox nor p67-phox is a flavoprotein and that neither, therefore, is the oxidase component which accepts electrons from NADPH.     

Complementation of NADPH oxidase in p67-phox-deficient CGD patients p67-phox/p40-phox interaction.

Chronic granulomatous disease (CGD) is due to a functional defect of the O2- generating NADPH oxidase of phagocytes. Epstein-Barr-virus-immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein-Barr-virus-immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67-phox-deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell-free assay consisting of membrane-suspended or solubilized and purified cytochrome b558 that was associated with cytosol or with the isolated cytosolic-activating factors (p67-phox, p47-phox, p40-phox) from healthy or CGD patients. In p67-phox-deficient CGD patients, two cytosolic factors are deficient or missing: p67-phox and p40-phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67-phox-deficient patients with recombinant p67-phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell-free assay after isolation in combination with cytochrome b558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67-phox and recombinant p40-phox reversed the previous complementation in a p40-phox dose-dependent process. These results suggest that in the reconstitution of oxidase activity, p67-phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40-phox from p67-phox for efficient oxidase activity. In the process, p40-phox could function as a negative regulatory factor and stabilize the resting state.     

NADPH oxidase is involved in angiotensin II-induced apoptosis in H9C2 cardiac muscle cells: effects of apocynin

Angiotensin II stimulates NADPH oxidase activity in vascular cells. However, it is not fully understood whether angiotensin II, which plays an important role in heart failure, stimulates NADPH oxidase activation and expression in cardiac myocytes. Previous studies have shown that angiotensin II induces myocyte apoptosis, but whether the change is mediated via NADPH oxidase remains to be elucidated. In this study we proposed to determine whether angiotensin II stimulated NADPH oxidase activation and NADPH oxidase subunit p47-phox expression in H9C2 cardiac muscle cells. If so, we would determine whether the NADPH oxidase inhibitor apocynin prevented angiotensin II-induced apoptosis. The results showed that angiotensin II increased NADPH oxidase activity, p47-phox protein and mRNA expression, intracellular reactive oxygen species, and apoptosis in H9C2 cells. Angiotensin II elevated p38 mitogen-activated protein kinase (MAPK) activity, decreased Bcl-2 protein, and increased Bax protein and caspase-3 activity. Apocynin treatment inhibited angiotensin II-induced NADPH oxidase activation and increases in p47-phox expression, intracellular reactive oxygen species, and apoptosis. The effect of apocynin on apoptosis was associated with reduced p38 MAPK activity, increased Bcl-2 protein, and decreased Bax protein and caspase-3 activity. These results suggest that angiotensin II-induced apoptosis is mediated via NADPH oxidase activation probably through p38 MAPK activation, a decrease in Bcl-2 protein, and caspase activation.     

New insights into the membrane topology of the phagocyte NADPH oxidase: characterization of an anti-gp91-phox conformational monoclonal antibody

Cytochrome b(558) is the catalytic core of the phagocyte NADPH oxidase that mediates the production of bactericidal reactive oxygen species. Cytochrome b(558) is formed by two subunits gp91-phox and p22-phox (1/1), non-covalently associated. Its activation depends on the interaction with cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac) leading to an electron transfer from NADPH to molecular oxygen and to the release of superoxide anions. Several studies have suggested that the activation process was linked to a change in cytochrome b(558) conformation. Recently, we confirmed this hypothesis by isolating cytochrome b(558) in a constitutively active form. To characterize active and inactive cytochrome b(558) conformations, we produced four novel monoclonal antibodies (7A2, 13B6, 15B12 and 8G11) raised against a mixture of cytochrome b(558) purified from both resting and stimulated neutrophils. The four antibodies labeled gp91-phox and bound to both native and denatured cytochrome b(558). Interestingly, they were specific of extracellular domains of the protein. Phage display mapping combined to the study of recombinant gp91-phox truncated forms allowed the identification of epitope regions. These antibodies were then employed to investigate the NADPH oxidase activation process. In particular, they were shown to inhibit almost completely the NADPH oxidase activity reconstituted in vitro with membrane and cytosol. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils pointed out the capacity of the monoclonal antibody 13B6 to bind preferentially to the active form of cytochrome b(558). All these data suggested that the four novel antibodies are potentially powerful tools to detect the expression of cytochrome b(558) in intact cells and to analyze its membrane topology. Moreover, the antibody 13B6 may be conformationally sensitive and used as a probe for identifying the active NADPH oxidase complex in vivo.     

Characterization of neutrophil NADPH oxidase factors p47-phox and p67-phox from recombinant baculoviruses

Superoxide production by phagocytic blood cells involves assembly of an active NADPH oxidase complex from components found both in membrane and cytosolic locations in resting cells. We recently cloned cDNAs encoding two cytosolic components (p47-phox and p67-phox) of the oxidase that are deficient in distinct forms of autosomal recessive chronic granulomatous disease. The precise roles of p47-phox and p67-phox were explored further using purified factors produced in large quantities using recombinant baculoviruses to infect cultured Sf9 insect cells. Neither p47-phox nor p67-phox are thought to represent the flavoprotein components of the oxidase, since neither of the purified recombinant factors contained or bound FAD. Recombinant p47-phox and p67-phox are capable of restoring the deficient cytosol from chronic granulomatous disease patient neutrophils to nearly normal levels in a cell-free reconstitution system. Both p47-phox and p67-phox, used together in the absence of neutrophil cytosol, are incapable of supporting cell free production of superoxide, confirming the involvement of other soluble factor(s) in the assembly of an active oxidase in vitro.     

Differential effects of NADPH oxidase and xanthine oxidase inhibition on sympathetic reinnervation in postinfarct rat hearts

Superoxide has been shown to play a major role in ventricular remodeling and arrhythmias after myocardial infarction. However, the source of increased myocardial superoxide production and the role of superoxide in sympathetic innervation remain to be further characterized. Male Wistar rats, after coronary artery ligation, were randomized to vehicle, allopurinol, or apocynin for 4weeks. To determine the role of peroxynitrite in sympathetic reinnervation, we also used 3-morpholinosydnonimine (a peroxynitrite generator). The postinfarction period was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine, xanthine oxidase activity, NADPH oxidase activity, and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham. Sympathetic hyperinnervation was blunted after administration of allopurinol. Arrhythmic scores in the allopurinol-treated infarcted rats were significantly lower than those in vehicle. For similar levels of ventricular remodeling, apocynin had no beneficial effects on oxidative stress, sympathetic hyperinnervation, or arrhythmia vulnerability. Allopurinol-treated hearts had significantly decreased nerve growth factor expression, which was substantially increased after coadministration of 3-morpholinosydnonimine. These results indicate that xanthine oxidase but not NADPH oxidase largely mediates superoxide production after myocardial infarction. Xanthine oxidase inhibition ameliorates sympathetic innervation and arrhythmias possibly via inhibition of the peroxynitrite-mediated nerve growth factor pathway.     

A 1.1-kb duplication in the p67-phox gene causes chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency that is caused by a functional defect of the NADPH oxidase of phagocytes, and that leads to severe recurrent infections. CGD results from the absence or the dysfunction of various components of NADPH oxidase, and autosomal recessive CGD with the lack of p67-phox (A67 CGD) is the rarest form of the disease. Identifying familiar mutations in subjects with A67 CGD provides the most reliable method of detecting carriers and is the basis for prenatal diagnosis. In the present study, we report the detailed characterization of the first duplication in the p67-phox gene identified in a 30-year-old patient affected by systemic aspergillosis attributable to p67-phox deficiency. We show that this new mutation involving exons 9 and 10 is the result of a tandem duplication of approximately 1.1 kb, which resulted from the juxtaposition of intron 8 to intron 10. We have sequenced both the junction fragment of this duplication and the corresponding wild-type regions and have found that the breakpoint regions in intron 8 and in intron 10 show limited homology. Our result suggests that this interchange arose as an illegitimate recombination event. As in other non-homologous rearrangements previously reported, the duplication breakpoints are located within the sequence motif 5'-CCAG-3' and its complement 5'-CTGG-3'.     

Race-specific elicitors of Cladosporium fulvum promote translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells.

The effect of race-specific elicitors on NADPH oxidase was examined in vivo by treating tomato cells with elicitor-containing intercellular fluids prepared from infected tomato leaves inoculated with specific Cladosporium fulvum races. Treatment of Cf-4 or Cf-5 cells with intercellular fluids from incompatible but not from compatible races of C. fulvum increased oxidase activity and the amount of p67-phox, p47-phox, and rac2 in the plasma membrane. Comparison of these three components in the cytosol and plasma membrane indicated that elicitors promoted the translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells carrying the appropriate resistance gene. Protein kinase C activators and inhibitors did not affect enzyme activity or the binding of these three components to the plasma membrane. In contrast, staurosporine, calmodulin antagonists, and EGTA inhibited elicitor-induced oxidase activity and the translocation of the cytosolic components. The assembly process involves a Ca(2+)-dependent protein kinase that catalyzes the phosphorylation of p67-phox and p47-phox, facilitating their translocation to the plasma membrane. Our data suggest that although both plants and animals share common elements in eukaryotic signal transduction, the involvement of different protein kinases mediating the activation of phosphorylation of p67-phox and p47-phox may reflect the unique spatial and temporal distribution of signal transduction pathways in plants.     

Statistical and mutational analysis of chronic granulomatous disease in Japan with special reference to gp91-phox and p22-phox deficiency

Chronic granulomatous disease (CGD) is a group of inherited disorders of host defense caused by a mutation in any of the four components of phagocyte NADPH oxidase, namely gp91-, p22-, p47-, and p67-phox. We have made a precise statistical analysis of 229 registered patients from 195 families in Japan and mutation analysis of 28 and 5 independent patients, respectively, with gp91- and p22-phox deficiency. The gp91- and p22-phox proteins form the membrane cytochrome b558, which plays important roles in the assembly of the active oxidase and electron-transfer reaction, and the lesions in either subunit account for more than 80% of cases. The ratio of male to female patients was 6.6/1, the incidence was calculated to be about 1 out of 220,000 birth, and the life expectancy of the patients born in the 1970s was estimated to be 25-30 years old. For the X-linked gp91-phox deficiency, we found five missense and nine nonsense mutations, seven deletions, three insertions, and four splice site mutations, which included the following novel mutations: four missense, five nonsense, six deletions, one insertion, and two splice site abnormalities. With regard to p22-phox deficiency, two homozygous nonsense mutations and one homozygous deletion, a missense mutation together with a splice site mutation, and two different missense mutations were found. These mutations have not been reported before. Based on the present and reported data from Japan, we discuss the molecular defects of the disease and the difference in statistics between western countries and Japan.     

Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide

We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2*-) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rapla) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2*-. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2*- is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2*- is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2*- is transported to the plasma membrane to be released and to ensure host defense against infection.     

Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide

Abstract

We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rap1a) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2 is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2 is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2 is transported to the plasma membrane to be released and to ensure host defense against infection.     

P67-phox-mediated NADPH oxidase assembly: imaging of cytochrome b558 liposomes by atomic force microscopy

NADPH oxidase activity depends on the assembly of the cytosolic activating factors, p67-phox, p47-phox, p40-phox, and Rac with cytochrome b(558). The transition from an inactive to an active oxidase complex induces the transfer of electrons from NADPH to oxygen through cytochrome b(558). The assembly of oxidase complex was studied in vitro after reconstitution in a heterologous cell-free assay by using true noncontact mode atomic force microscopy. Cytochrome b(558) was purified from neutrophils and Epstein-Barr virus-immortalized B lymphocytes and incorporated into liposomes. The effect of protein glycosylation on liposome size and oxidase activity was investigated. The liposomes containing the native hemoprotein purified from neutrophils had a diameter of 146 nm, whereas after deglycosylation, the diameter was reduced to 68 nm, although oxidase activity was similar in both cases. Native cytochrome b(558) was used after purification in reconstitution experiments to investigate the topography of NADPH oxidase once it was assembled. For the first time, atomic force microscopy illustrated conformational changes of cytochrome b(558) during the transition from the inactive to the active state of oxidase; height measurements allow the determination of a size of 4 nm for the assembled complex. In the processes that were studied, p67-phox displayed a critical function; it was shown to be involved in both assembly and activation of oxidase complex while p47-phox proceeded as a positive effector and increased the affinity of p67-phox with cytochrome b(558), and p40-phox stabilizes the resting state. The results suggest that although an oligomeric structure of oxidase machinery has not been demonstrated, allosteric regulation mechanisms may be proposed.     

Surfactant protein D regulates NF-kappa B and matrix metalloproteinase production in alveolar macrophages via oxidant-sensitive pathways

Targeted ablation of the surfactant protein D (SP-D) gene caused progressive pulmonary emphysema associated with pulmonary infiltration by foamy alveolar macrophages (AMs), increased hydrogen peroxide production, and matrix metalloproteinase (MMP)-2, -9, and -12 expression. In the present study, the mechanisms by which SP-D influences macrophage MMP activity were assessed in AMs from SP-D(-/-) mice. Tissue lipid peroxides and reactive carbonyls were increased in lungs of SP-D(-/-) mice, indicating oxidative stress. Immunohistochemical staining of AMs from SP-D(-/-) mice demonstrated that NF-kappaB was highly expressed and translocated to the nucleus. Increased NF-kappaB binding was detected by EMSA in nuclear extracts of AMs isolated from SP-D(-/-) mice. Antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate inhibited MMP production by AMs from SP-D(-/-) mice. To assess whether increased oxidant production influenced NF-kappaB activation and production of MMP-2 and -9, AMs from SP-D(-/-) mice were treated with the NADPH oxidase inhibitors diphenylene iodonium chloride and apocynin. Inhibition of NADPH oxidase suppressed NF-kappaB binding by nuclear extracts and decreased production of MMP-2 and 9 in AMs from SP-D(-/-) mice. SN-50, a synthetic NF-kappaB-inhibitory peptide, decreased MMP production by AMs from SP-D(-/-) mice. Oxidant production and reactive oxygen species were increased in lungs of SP-D(-/-) mice, in turn activating NF-kappaB and MMP expression. SP-D plays an unexpected inhibitory role in the regulation of NF-kappaB in AMs.     

Involvement of Na/H exchanger and respiratory burst enzymes NADPH oxidase and NO synthase,in Cd-induced lipid peroxidation and DNA damage in haemocytes of mussels

This study investigated cadmium-induced oxidative and genotoxic effects, such as lipid peroxidation and disturbance of DNA integrity (DNA damage) in haemocytes of mussel Mytilus galloprovincialis and the possible involvement of Na⁺/H⁺ exchanger (NHE), and/or the main enzymes of respiratory burst, NADPH oxidase and nitric oxide (NO) synthase, in the induction of Cd toxic effects. In order to verify the role of either NHE, or NADPH oxidase and NO synthase in Cd-mediated toxicity, inhibitors such as ethyl-N-isopropyl-amiloride (EIPA), diphenyleneiodonium chloride (DPI) and N^G-nitro-l-arginine methyl ester (L-NAME) were used in each case. Moreover, phorbol-myristate acetate (PMA), a well-known protein kinase C (PKC)-mediated NADPH oxidase and NO synthase stimulator, as well as hydrogen peroxide (H₂O₂), a well-known genotoxic agent, was also used for elucidating the modulation of signaling molecules within cells, thus leading to the induction of lipid peroxidation and DNA damage. The results of the present study showed that micromolar concentrations of Cd (0.05–50 μΜ) could enhance both lipid peroxidation and DNA damage, possible via a PKC-mediated signaling pathway with the involvement of NHE, thus leading to the induction of NADPH oxidase and NO synthase activity, since inhibition of either NHE, or NADPH oxidase and NO synthase activity, significantly attenuates Cd-induced toxic effects in each case.     

Role of gp91phox-containing NADPH oxidase in left ventricular remodeling induced by intermittent hypoxic stress

Intermittent hypoxia due to sleep apnea syndrome is associated with cardiovascular diseases. However, the precise mechanisms by which intermittent hypoxic stress accelerates cardiovascular diseases are largely unclear. The aim of this study was to investigate the role of gp91(phox)-containing NADPH oxidase in the development of left ventricular (LV) remodeling induced by intermittent hypoxic stress in mice. Male gp91(phox)-deficient (gp91(-/-)) mice (n = 26) and wild-type (n = 39) mice at 7-12 wk of age were exposed to intermittent hypoxia (30 s of 4.5-5.5% O(2) followed by 30 s of 21% O(2) for 8 h/day during daytime) or normoxia for 10 days. Mean blood pressure and LV systolic and diastolic function were not changed by intermittent hypoxia in wild-type or gp91(-/-) mice, although right ventricular systolic pressure tended to be increased. In wild-type mice, intermittent hypoxic stress significantly increased the diameter of cardiomyocytes and interstitial fibrosis in LV myocardium. Furthermore, intermittent hypoxic stress increased superoxide production, 4-hydroxy-2-nonenal protein, TNF-alpha and transforming growth factor-beta mRNA, and NF-kappaB binding activity in wild-type, but not gp91(-/-), mice. These results suggest that gp91(phox)-containing NADPH oxidase plays a crucial role in the pathophysiology of intermittent hypoxia-induced LV remodeling through an increase of oxidative stress.     

The effects of hyperoxia, hypoxia, and ischemia/reperfusion on the activity of cytochrome oxidase from the rat retina

Cytochrome oxidase activity from the retina can be enhanced or depressed by free radical-mediated reactions both in positive and negative aspect. The greatest effect was exerted by ischemia/reperfusion, which significantly increased the fluorescent products of lipid peroxidation (358 %, P < 0.01) and inhibited the enzyme activity (14%, P < 0.001). After hyperoxia the fluorescent products slightly increased (192%, P < 0.05) as well as the enzyme activity (133 %, P < 0.05). Hypoxia had no effect on any of these parameters. Specific changes in the composition of fluorophores after ischemia/reperfusion were revealed in the fluorescence spectra. The fact that increased lipid peroxidation after hyperoxia and after ischemia/reperfusion does not produce the same effect upon cytochrome oxidase activity might be explained by changes in the kinetic behavior of cytochrome oxidase. In the control enzyme preparation, two binding sites for cytochrome c were observed. One was of the low-affinity (Km = 60 microM) and the other of the high-affinity (Km = 1.12 microM). After in vitro-initiated lipid peroxidation, the low-affinity binding site was lost and the activity measured under "optimum" conditions at a single cytochrome concentration was higher than in the controls. This implies that oxidative damage to cytochrome oxidase in vivo can be site-specific and its extent should be estimated by performing detailed kinetic analysis as otherwise the results might be misleading.