Inhibition of plant and animal cytochrome oxidases by nitrous oxide as a function of cytochrome c concentration.

Cytochrome oxidase from both pea leaves and bovine heart shows lower activity under a mixture of 79% N2O/21% O2 than under ambient air. This inhibition is not detectable below 5 microM cytochrome c but appears with increasing concentrations of cytochrome c. These results suggest that the N2O-induced inhibition of cytochrome c oxidase is modulated by cytochrome c concentration. This seems to concern only the lowest affinity site of the oxidase. Apparently, N2O and cytochrome c do not share the same site of fixation on the oxidase.     

Mitochondrial respiration at low levels of oxygen and cytochrome c

In the intracellular microenvironment of active muscle tissue, high rates of respiration are maintained at near-limiting oxygen concentrations. The respiration of isolated heart mitochondria is a hyperbolic function of oxygen concentration and half-maximal rates were obtained at 0.4 and 0.7 microM O(2) with substrates for the respiratory chain (succinate) and cytochrome c oxidase [N,N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)+ascorbate] respectively at 30 degrees C and with maximum ADP stimulation (State 3). The respiratory response of cytochrome c-depleted mitoplasts to external cytochrome c was biphasic with TMPD, but showed a monophasic hyperbolic function with succinate. Half-maximal stimulation of respiration was obtained at 0.4 microM cytochrome c, which was nearly identical to the high-affinity K(')(m) for cytochrome c of cytochrome c oxidase supplied with TMPD. The capacity of cytochrome c oxidase in the presence of TMPD was 2-fold higher than the capacity of the respiratory chain with succinate, measured at environmental normoxic levels. This apparent excess capacity, however, is significantly decreased under physiological intracellular oxygen conditions and declines steeply under hypoxic conditions. Similarly, the excess capacity of cytochrome c oxidase declines with progressive cytochrome c depletion. The flux control coefficient of cytochrome c oxidase, therefore, increases as a function of substrate limitation of oxygen and cytochrome c, which suggests a direct functional role for the apparent excess capacity of cytochrome c oxidase in hypoxia and under conditions of intracellular accumulation of cytochrome c after its release from mitochondria.     

The superoxide-generating oxidase of leucocytes. NADPH-dependent reduction of flavin and cytochrome b in solubilized preparations.

An NADPH-dependent O2.- -generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence.     

Cytochrome oxidase-catalyzed superoxide generation from hydrogen peroxide.

Superoxide dismutase is shown to affect spectral changes observed upon cytochrome c oxidase reaction with H2O2, which indicates a possibility of O2- radicals being formed in the reaction. Using DMPO as a spin trap, generation of superoxide radicals from H2O2 in the presence of cytochrome oxidase is directly demonstrated. The process is inhibited by cyanide and is not observed with a heat-denatured enzyme pointing to a specific reaction in the oxygen-reducing centre of cytochrome c oxidase. The data support a hypothesis on a catalase cycle catalyzed by cytochrome c oxidase in the presence of excess H2O2 (Vygodina and Konstantinov (1988) Ann. NY Acad. Sci., 550, 124-138): (formula: see text)     

Copper-dependent inhibition of cytochrome c oxidase by Abeta(1-42) requires reduced methionine at residue 35 of the Abeta peptide

By altering key amino acid residues of the Alzheimer's disease-associated amyloid-beta peptide, we investigated the mechanism through which amyloid-beta inhibits cytochrome c oxidase (EC 1.9.3.1). Native amyloid-beta inhibited cytochrome oxidase by up to 65%, and the level of inhibition was determined by the period of amyloid-beta ageing before the cytochrome oxidase assay. Substituting tyrosine-10 with alanine did not affect maximal enzyme inhibition, but the altered peptide required a longer period of ageing. By contrast, oxidizing the sulfur of methionine-35 to a sulfoxide, or substituting methionine-35 with valine, completely abrogated the peptide's inhibitory potential towards cytochrome oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the loss of inhibitory potential towards cytochrome oxidase with the methionine-35-altered peptides did not correlate with a substantially different distribution of amyloid-beta oligomeric species. Although the amyloid-beta-mediated inhibition of cytochrome oxidase was completely dependent on the presence of divalent Cu2+, it was not supported by monovalent Cu+, and experiments with catalase and H2O2 indicated that the mechanism of cytochrome oxidase inhibition does not involve amyloid-beta-mediated H2O2 production. We propose that amyloid-beta-mediated inhibition of cytochrome oxidase is dependent on the peptide's capacity to bind, then reduce Cu2+, and that it may involve the formation of a redox active amyloid-beta-methionine radical.     

Respiratory electron transfer activity in an asolectin-isooctane reverse micellar system.

Bovine heart submitochondrial particles (SMP) were solubilized in an asolectin isooctane reverse micellar system and the functionality of the respiratory chain was tested by spectroscopic and amperometric techniques. Electron transfer rate supported by NADH was very slow as evidenced by the low cytochrome reduction levels attained over long incubation periods. In the presence of KCN, NADH caused 34% and 12.5% reduction of the cytochromes aa3 and c, respectively, and negligible reduction of cytochrome b. Supplementation of the system with menadione rose the NADH-dependent reduction of all the cytochromes to levels that were close to the total content. However, no measurable O2 uptake activity took place in the presence of NADH plus menadione, or with ascorbate (or NADH) plus TMPD reducing systems. Therefore, it is suggested that in the organic medium, electron transfer from NADH to O2 is arrested at the terminal oxidase step. Cytochrome oxidase reduced by ascorbate (or NADH) plus TMPD seems to be trapped in its half reduced state (ie, a2+ a3(3+)). Although it is poorly reactive with O2, it can transfer electrons back to cytochrome c and TMPD. The electron transfer block to O2 was overcome when PMS was used instead of TMPD. This seems to be due to the recognized capacity of PMSH2 to carry out simultaneous reduction of both a CuA and a3 CuB redox centers of cytochrome oxidase. The cytochrome oxidase reaction in the organic solvent was highly sensitive to KCN (Ki 1.9 microM) and showed bell-shaped kinetics towards the PMS concentration and a sigmoidal response to water concentration, reaching its maximal turnover number (18 s-1) at 4 mM PMS and 1.1% (v/v) water.(ABSTRACT TRUNCATED AT 250 WORDS)     

The membrane-associated component of the amphiphile-activated, cytosol-dependent superoxide-forming NADPH oxidase of macrophages is identical to cytochrome b559.

The superoxide (O2-) forming NADPH oxidase complex of resting phagocytes can be activated in a cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). For O2- production to occur, the participation of both membrane-associated and cytosol-derived components is required. The purpose of this investigation was to isolate and characterize the membrane component of NADPH oxidase. For this purpose, guinea pig macrophage membranes were extracted with 1 M NaCl, solubilized by 40 mM octyl glucoside, and subjected to a purification sequence consisting of absorption with DEAE-Sepharose, affinity chromatography on heparin-agarose, and chromatography on hydroxylapatite. At each purification step, fractions were assayed for their ability to support SDS-elicited, cytosol-dependent O2- production, following incorporation in liposomes of phosphatidylcholine. We found that membrane oxidase activity copurified strictly with cytochrome b559. Peak hydroxylapatite fractions exhibited specific O2(-)-forming activity in the range of 81-115 mumol of O2-/mg protein/min and a specific cytochrome b559 content of 7-14 nmol of cytochrome b559/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the peak oxidase activity fractions, derived by hydroxylapatite chromatography, revealed essentially two bands that were identified as the beta (54-60 kDa) and alpha (21/22 kDa) subunits of guinea pig cytochrome b559. The relation of the two polypeptides to cytochrome b559 was established by correlation with a spectral signal characteristic of cytochrome b559, immunoblotting with antibodies against defined human cytochrome b559 beta and alpha chain peptides, cross-linking studies, and deglycosylation experiments. Hydroxylapatite-purified membrane oxidase preparations did not contain FAD and were free of cytochrome c reductase activity. Purified membrane oxidase preparations were also capable of cooperating with purified cytosolic components in SDS-elicited cell-free O2- production. We conclude that the membrane-associated component of the O2- generating NADPH oxidase is identical to cytochrome b559.     

Composition of partially purified NADPH oxidase from pig neutrophils.

The superoxide (O2.-)-forming enzyme NADPH oxidase from pig neutrophils was solubilized and partially purified by gel-filtration chromatography. The purification procedure allowed the separation of NADPH oxidase activity from NADH-dependent cytochrome c reductase and 2,6-dichlorophenol-indophenol reductase activities. O2.-forming activity was co-purified with cytochrome b-245 and was associated with phospholipids. However, active fractions endowed with cytochrome b were devoid of ubiquinone and contained only little FAD. The cytochrome b/FAD ratio was 1.13:1 in the crude solubilized extract and increased to 18.95:1 in the partially purified preparations. Most of FAD was associated with fractions containing NADH-dependent oxidoreductases. These results are consistent with the postulated role of cytochrome b in O2.-formation by neutrophil NADPH oxidase, but raise doubts about the participation of flavoproteins in this enzyme activity.     

Effect of oxygen on the synthesis and assembly of mitochondrial encoded subunits of cytochrome oxidase and cytochrome b.c1 in mouse embryo fibroblasts.

Mouse embryo fibroblasts were grown in low and control O2 for 24 h (average medium oxygen tensions, 7 torr and 143 torr, respectively). Relative to controls, there was a reduction in radiolabeled subunits in immunoprecipitates of cytochrome oxidase and cytochrome b.c1 prepared from low O2 cells. Incorporation of radiolabeled amino acids into subunit I of cytochrome oxidase and the apocytochrome b protein of the b.c1 complex ranged from 51-100% of control, whereas the appearance of these pulse-labeled subunits into holoenzymes immunoprecipitated from low O2 cells was in the range of 6-39% of control. The synthesis of subunit II of cytochrome oxidase by low O2 cells ranged from 63-100% of control, and assembly of this protein into the low O2 immunoprecipitated enzyme ranged from 15-61% of control. Thus, the data suggest that O2 had an effect on the assembly of these mitochondrially translated proteins that was independent of any effect on their synthesis.     

Mechanism of the superoxide-producing oxidase of neutrophils. O2 is necessary for the fast reduction of cytochrome b-245 by NADPH.

A soluble oxidase from phorbol-stimulated pig neutrophils contained FAD and cytochrome b-245. A typical preparation produced 13.03 mol of superoxide (O2-.) X S-1 X mol of cytochrome b-1 (348 nmol X min-1 X mg of protein-1). In the aerobic steady state, cytochrome b was 8.9% reduced. Steady-state cytochrome b reduction was absent from extracts of unstimulated cells; Km values for NADPH, for O2-. production and cytochrome b reduction were similar. The calculated aerobic rate of cytochrome b reduction was equal to the measured rate of O2-. production in a variety of preparations and in the presence of a range of inhibitors. Under anaerobic conditions the rate was slow: O2 is apparently required for rapid electron flow into the oxidase complex. Cytochrome b is shown to be kinetically competent to act as part of the O2-.-generating complex.     

The reduction of cytochrome b558 and the activity of the respiratory burst oxidase from human neutrophils.

It is known that in respiratory burst oxidase preparations engaged in O2- production, cytochrome b558, a characteristic oxidase component, is partly reduced. This result has been interpreted in terms of a mechanism in which cytochrome b558 functions as an electron-carrying component of the respiratory burst oxidase, its level of reduction reflecting a steady-state partitioning of the cytochrome between reduced and oxidized forms as it ferries electrons from NADPH to oxygen. Kinetic arguments based on this interpretation have supported the proposal that the cytochrome is reduced at a rate sufficient to account for the rate of O2- production by activated neutrophils. We have confirmed the partial reduction of cytochrome b558 in neutrophil cytoplasts and in oxidase preparations exposed to NADPH, but have found that the reduction of the cytochrome bears no apparent relation to the activity of the oxidase, and can occur when NADPH is added to neutrophil membrane preparations that are unable to manufacture O2-. We therefore conclude that the NADPH-dependent reduction of cytochrome b558 seen in these preparations is unlikely to be a reflection of a catalysis-related steady state and that inferences drawn from such observations regarding the kinetic competence of the cytochrome may need to be reconsidered.     

Interactions of the anesthetic nitrous oxide with bovine heart cytochrome c oxidase. Effects on protein structure, oxidase activity, and other properties

The interactions of nitrous oxide with cytochrome c oxidase isolated from bovine heart muscle have been investigated in search of an explanation for the inhibition of mitochondrial respiration by the inhalation anesthetic. Oxidase activity of the isolated enzyme is partially and reversibly reduced by nitrous oxide. N2O molecules are shown by infrared spectroscopy to occupy sites within the oxidase. Occupancy of sites within the protein by N2O has no observed effects on visible Soret spectra or on the O2 reaction site; no evidence is found for N2O serving as a ligand to a metal. The anesthetic does not substitute for O2 as an oxygen atom donor in either the cytochrome c oxidase or carbon monoxide dioxygenase reactions catalyzed by the enzyme. N2O appears to affect oxidase activity by reducing the rate of electron transfer from cytochrome c to the O2 reaction site rather than by interfering directly with the reduction of O2 to water. Cytochrome c oxidase represents a target site for nitrous oxide and possibly other anesthetics, and the inhibition of oxidase activity may contribute significantly to the anesthetic and/or toxic effects of these substances.     

Cytochrome c oxidase is not a proton pump.

We conclude that the reduction of O2 to 2 H2O by cytochrome c oxidase of rat liver mitochondria involves the translocation of 4-from cytochrome c at the outer surface of the cristae membrane per O2 reduced and protonated by 4 H+ ions that enter the reaction domain from the inner aqueous phase. This net electron-translocating function of cytochrome c oxidase plugged through the mitochondrial cristae membrane is not linked to a proton-pumping function, such as that proposed by Wikstr?m [7,8].     

Tryptic cleavage of rat liver sulfite oxidase. Isolation and characterization of molybdenum and heme domains.

Treatment of rat liver sulfite oxidase with trypsin leads to loss of ability to oxidize sulfite in the presence of cytochrome c as electron acceptor. Ability to oxidize sulfite with ferricyanide as acceptor is undiminished, while sulfite leads to O2 activity is partially retained. Gel filtration of the proteolytic products has led to the isolation of two major fragments of dissimilar size derived from sulfite oxidase. The smaller fragment has a molecular weight of 9500 and appears to be monomeric when detached from sulfite oxidase. It contains the heme in its cytochrome b5 structure, has no sulfite oxidase activity, and is reducible with dithionite but not with sulfite. The heme fragment can mediate electron transfer between pig liver microsomal NADH cytochrome b5 reductase and cytochrome c. The larger fragment has a molecular weight of 47,400 under denaturing conditions but elutes from Sephadex G-200 as a dimer. It contains no heme but retains all of the molybdenum and the modified sulfite-oxidizing capacity present in the proteolytic mixture. All of the EPR properties of the molybdenum center of native sulfite oxidase are retained in the molybdenum fragment. The molybdenum center is a weak chromophore with an absorption sectrum suggestive of coordination with sulfur ligands. Reduction by sulfite generates a spectrum attributable to molybdenum (V). Spectra of oxidized and sulfite-reduced preparations are sensitive to anions and pH. NH2-terminal analysis of native sulfite oxidase and the two tryptic fragments has permitted the conclusion that the sequence represented by the heme fragment is the NH2 terminus of native enzyme. These studies have demonstrated that the two cofactor moieties of sulfite oxidase are contained in distinct domains which are covalently held in contiguity by means of an exposed hinge region. Isolation of functional heme and molybdenum domains of sulfite oxidase after tryptic cleavage has demonstrated conclusively that the cytochrome b5 region of the molecule is required for electron transfer to the physiological acceptor, cytochrome c.     

Properties of enzymes. V. Catalase inhibition of glucose oxidase reaction.

The radical produced aerobically in the glucose oxidase reaction reduces the oxidated cytochrome c. The extent of reduction depends on the concentrations of substrate (glucose) and enzyme. Superoxide dismutase purified from various sources does not inhibit the cytochrome c reduction, but catalase does, in proportion to its concentration. This inhibition, which might be utilized for quantitative catalase determinations, provides further evidence for the formation of H2O2 in the glucose oxidase reaction.     

Kinetic evidence for the re-definition of electron transfer pathways from cytochrome c to O2 within cytochrome oxidase

The reaction with O2 of equimolar mixtures of cytochrome c and cytochrome c oxidase in high and low ionic strength buffers has been examined by flow-flash spectrophotometry at room temperature. In low ionic strength media where cytochrome c and the oxidase are bound in an electrostatic, 1:1 complex some of the cytochrome c is oxidised at a faster rate than a metal centre of the oxidase. In contrast, when cytochrome c and cytochrome c oxidase are predominantly dissociated at high ionic strength cytochrome c oxidation occurs only slowly (t1/2 = 5 s) following the complete oxidation of the oxidase. These results demonstrate that maximal rates of electron transfer from cytochrome c to O2 occur when both substrates are present on the enzyme. The heterogeneous oxidation of cytochrome c observed in the complex implies more than one route for electron transfer within the enzyme. Possibilities for new electron transfer pathways from cytochrome c to O2 are proposed.     

Formation and reduction of a ''peroxy'' intermediate of cytochrome c oxidase by hydrogen peroxide.

In the presence of micromolar concentrations of H2O2, ferric cytochrome c oxidase forms a stable complex characterized by an increased absorption intensity at 606-607 nm with a weaker absorption band in the 560-580 nm region. Higher (millimolar) concentrations of H2O2 result in an enzyme exhibiting a Soret band at 427 nm and an alpha-band of increased intensity in the 589-610 nm region. Addition of H2O2 to ferric cytochrome c oxidase in the presence of cyanide results in absorbance increases at 444nm and 605nm. These changes are not seen if H2O2 is added to the cyanide complex of the ferric enzyme. The results support the idea that direct reaction of H2O2 with ferric cytochrome a 3 produces a 'peroxy' intermediate that is susceptible to further reduction by H2O2 at higher peroxide concentrations. Electron flow through cytochrome a is not involved, and the final product of the reaction is the so-called 'pulsed' or 'oxygenated' ferric form of the enzyme.     

Effects of thyroid state on H2O2 production by rat heart mitochondria: sites of production with complex I- and complex II-linked substrates.

This work was designed to determine possible effects of altered thyroid states on rates and sites of H 2 O 2 production by rat heart mitochondria. Rates of O 2 consumption and H 2 O 2 release, capacities to remove the peroxide, lipid peroxidation, cytochrome oxidase activities and ubiquinone levels were determined in heart mitochondria from euthyroid, hypothyroid, and hyperthyroid rats. Hypothyroidism decreased, whereas hyperthyroidism increased the rates of O 2 consumption and H 2 O 2 release during both state 4 and state 3 respiration with Complex I- or Complex II-linked substrates. The percentage of O 2 released as H 2 O 2 was not significantly affected by thyroid state. However, the mitochondrial capacity to remove H 2 O 2 increased in the transition from hypothyroid to hyperthyroid state, which indicates that H 2 O 2 production did not modify in proportion to the rate of O 2 consumption. The thyroid-state-linked changes in H 2 O 2 production were well correlated with the levels of hydroperoxides. Rates of H 2 O 2 release in the presence of respiratory inhibitors indicated that changes in the H 2 O 2 production occurred at both sites at which H 2 O 2 was generated in euthyroid state. This result and the observation that ubiquinol levels and cytochrome oxidase activities increase in the transition from hypothyroid to hyperthyroid state suggest that the modifications of H 2 O 2 production are due to a modulation by thyroid hormone of mitochondrial content of autoxidisable electron carriers.     

Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation.

Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of the production of O2- by the activated NADPH oxidase of bovine neutrophils. In a cell-free system of NADPH oxidase activation consisting of neutrophil membranes and cytosol from resting cells, supplemented with guanosine 5'-[gamma-thio]triphosphate, MgCl2 and arachidonic acid, or in membranes isolated from neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate, addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to contain the Ph2I-sensitive component(s). In the presence of a concentration of Ph2I sufficient to fully inhibit O2- production (around 10 nmol/mg membrane protein), addition of catalytic amounts of the redox mediator dichloroindophenol (Cl2Ind) resulted in a by-pass of the electron flow to cytochrome c, the rate of which was about half of that determined in non-inhibited oxidase. A marked increase in the efficiency of this by-pass was achieved by addition of sodium deoxycholate. The Cl2-Ind-mediated cytochrome c reduction was negligible in membranes isolated from resting neutrophils. At a higher concentration of Ph2I (100 nmol/mg membrane protein), the Cl2Ind-mediated cytochrome c reductase activity was only half inhibited, which indicated that, in the NADPH oxidase complex, there are at least two Ph2I sensitive components, differing by their sensitivity to the inhibitor. At low concentrations of Ph2I (less than 10 nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil membranes was modified, suggesting that the component sensitive to low concentrations of Ph2I is the heme binding component of cytochrome b558. Higher concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase component of the oxidase complex. A number of membrane and cytosolic proteins were labeled by [125I]Ph2I. However, the radiolabeling of a membrane-bound 24-kDa protein, which might be the small subunit of cytochrome b558, responded more specifically to the conditions of activation and reduction which are required for inhibition of O2- production by Ph2I. The O2(-)-generating form of xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a non-heme iron flavoprotein, by Ph2I had a number of features in common with that of the neutrophil NADPH oxidase, namely the requirement of reducing conditions for inhibition of O2- production by Ph2I and the induction of a by-pass of electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some similarity in the molecular organization of the two enzymes.     

Studies on the electron-transfer mechanism of the human neutrophil NADPH oxidase.

A superoxide-generating NADPH oxidase was solubilized from phorbol 12-myristate 13-acetate-activated human neutrophils with a mixture of sodium deoxycholate (0.125%, w/v) and Lubrol-PX (0.125%, v/v). The solubilized preparation contained FAD (577 pmol/mg of protein) and cytochrome b-245 (479 pmol/mg of protein) and produced 11.61 mol of O2-./s per mol of cytochrome b (340 nmol of O2-./min per mg of protein). On addition of NADPH, the cytochrome b-245 was reduced by 7.9% and the FAD by 38% in the aerobic steady state; NADH addition caused little steady-state reduction of cytochrome b and FAD. In this preparation, and several others, the measured rate of O2-. production correlated with the turnover of cytochrome b calculated from the extent of cytochrome b-245 reduction under aerobic conditions. Addition of diphenyleneiodonium abolished the reduction of both the FAD and cytochrome b-245 components and inhibited O2-. production. The haem ligand imidazole inhibited O2-. generation and cytochrome b reduction while permitting FAD reduction. These results support the suggestion that the human neutrophil NADPH oxidase has the electron-transport sequence: NADPH----FAD----cytochrome b-245----O2.