O.T.G. Jones

ENZYMIC MECHANISMS OF SUPEROXIDE PRODUCTION

The realization that superoxide, and other reactive oxygen species, is produced in significant quantities in biological systems is a relatively recent one and the nature and purpose of the radical-generating systems is in most cases, only beginning to be understood. It is the purpose of this review to describe the known biological sources of superoxide, the enzymic machinery used to generate it, and the possible functions to which it is put

Absence of cytochrome b reduction in stimulated neutrophils from both female and male patients with chronic granulomatous disease

We have recently described a cytochrome b in human neutrophils [ 11. It is located predominantly within the plasma membrane [2,3] and is rapidly incorporated into the phagocytic vacuole [ 1,3]. We believe that the microbicidal oxidase system of neutrophils contains an electron transport chain in which this cytochrome b is a central component. The evidence for the involvement of this cytochrome b in the oxidase system is 2-fold. Stimulation of neutrophils with phorbol myristate acetate (PMA) activates the oxidase system [4] and results in the reduction and subsequent oxidation of this cytochrome b [5]. This cytochrome b was not found in reducedoxidised spectra of neutrophils from a group of patients with Chronic Granulomatous Disease (CGD) [6], a syndrome in which a predisposition to bacterial infection is associated with the absence of demonstrable oxidase activity in their neutrophils upon stimulation [7]. However, this cytochrome b was later found in some patients with CGD, particularly those with an autosomal mode of inheritance [8,9]. This study was conducted to investigate whether neutrophils from patients with CGD, both those with and those without spectroscopic evidence of the cytochrome b, demonstrate the normal pattern of reduction of this cytochrome after stimulation of the cells with PMA. It was found that the cytochrome b was not reduced in any of these patients. This strongly implicates this electron transport chain in the microbicidal oxidase system. It also confirms that the syndrome of CGD is the result of at least two different molecular aberrations; in some cases the cytochrome b is missing, in others it fails to become reduced upon stimulation of the cell, either because of the absence

REDUCTION AND SUBSEQUENT OXIDATION OF A CYTOCHROME b OF HUMAN NEUTROPHILS AFTER STIMULATION WITH PHORBOL MYRISTATE ACETATE

Abstract Difference spectroscopy of human neutrophils stimulated with phorbol myristate acetate as compared with unstimulated cells produced the characteristic spectral changes of the reduced cytochrome that has recently been described in these cells. The reduction of this cytochrome b is enhanced under anaerobic conditions and is partially reversed upon the reintroduction of air. This reversible oxidation and reduction after stimulation with phorbol myristate provides additional evidence for the role of this cytochrome b as a component of the microbicidal oxidase system.

Cytochrome c2 — an electron carrier shared by the respiratory and photosynthetic electron transport chain of Rhodopseudomonas capsulata

The function of cytochrome c2 as the primary electron donor to the reaction center in photosynthetic electron transport of Rhodospirillaceae has been clearly established [ 1,2]. However, its role in the respiratory pathways on which these bacteria depend under heterotrophic growth conditions has been a matter of debate since many years [3-81. The study of respiration in these organisms is often complicated by the presence of branched electron transport chains [7,9,19] and by possible regulatory phenomena in the activity of the terminal oxidases dependent upon physical parameters of growth, such as oxygen concentration and light intensity [ 11 ,121. The Rhodopseudomonas capsulata cytochrome c2 is thought to function in only one of the branches as electron donor for a cytochrome b-type oxidase (cyt. b& [6,13]. This suggestion rests o,n experimental evidence, such as the observed redox changes of cytochrome c2 upon addition of antimycin A or during transition to anaerobiosis, and the lack of reoxidation of cytochrome c2 in membranes from a respiration-deficient mutant (M7) in which cytochrome beI is absent [6]. In this communication we present evidence for a role of cytochrome c2 in respiration of Rps. capsulata; definite proof has been obtained by an immunological ahproach taking into account both the vectorial orientation of the membrane and the complexity of the respiratory chain. These two difficulties have been overcome using spheroplast preparations of a respiratory mutant (M6) endowed with a linear respiratory chain where cytochrome c oxidase (cyt. b,& is the only terminal enzyme present [6,10]. The data demonstrate conclusively the role of this cytochrome in the respiratory as well as in the photosynthetic chain of Rps. capsulata.

Energy-linked electron transfer reactions in Rhodopseudomonas viridis.

The particulate fraction of Rhodopseudomonas viridis when supplied with succinate catalyses the reduction of NAD+ by light; this reaction is inhibited by uncouplers of oxidative phosphorylation but not by oligomycin. Formation of NADH takes place in the dark when ATP or PPi is supplied. Both light and dark reactions are inhibited by valinomycin and nigericin, when added together, but not by either separately. NADH formation in R. viridis appears to take place by an energy-dependent reversal of electron flow and energy may be conserved in the form of a membrane potential. The addition of ATP caused the oxidation of both C553 and C558 in chromatophores; carbonylcyanide p-trifluoromethoxyphenylhydrazone and oligomycin abolished this oxidation. The NAD+ and NADH concentrations at equilibrium in the light-dependent reaction were determined and the oxidation-reduction potential of this couple calculated. From this value it was calculated that under these experimental conditions the energy requirement to form NADH from the succinate/fumarate couple at Eh = o V was 9.4 kcal. Particles of R. viridis contained an active transhydrogenase, driven by either light or ATP, that was sensitive to uncouplers of oxidative phosphorylation; the light-driven reaction was insensitive to oligomycin and was inhibited by antimycin A and 2-heptyl-4-hydroxyquinone-N-oxide. R. viridis did not grow aerobically but particles contained NADH oxidase activity that was cyanide sensitive. There was no spectroscopic evidence for cytochromes of the b-type in reduced-minus-oxidised spectra of particles or in pyridine haemochrome spectra of whole cells.

The cytochrome b and flavin content and properties of the O2−-forming NADPH oxidase solubilized from activated neutrophils

NADPH-dependent O2- -generating activity was extracted and partially purified from guinea pig polymorphonuclear leukocytes. The most active preparation generated 202.8 nmol O2- min/min per mg protein. This activity was 30-fold higher than that of extracts from resting cells, indicating that the activated state of the oxidase was retained after solubilization. The solubilization and purification of the enzyme activity were followed by a parallel solubilization and purification of cytochrome b. Spectroscopic studies showed that solubilized cytochrome b has an Em of -245 mV and binds CO to about 30%. Cytochrome b was reduced by NADPH in anaerobiosis at a low rate and was rapidly reoxidized by air. A correlation was found between the inhibition of O2- formation caused by the SH reagent p-chloromercuribenzoate and the alterations induced by this compound on the Em of cytochrome b. These observations strongly support the participation of cytochrome b in the catalytic activity of the solubilized NADPH oxidase. The enzyme preparations contained FAD, which was found to be associated both with NADPH oxidase and with diaphorase activities. The fraction with the highest O2- forming activity contained FAD and cytochrome b in a ratio of about 0.5:1. The participation of FAD in the electron transport from NADPH to O2 is supported also by the inhibitory effect exerted by quinacrine on O2- formation.

Keratinocyte superoxide generation.

We have demonstrated using the reduction of cytochrome c, that the keratinocyte cell line H357 generates superoxide at significant rates (8.36 nmol/h/10[6] cells). The rate of superoxide release decreased as the cells reached confluence. Superoxide production was increased more than twofold following preincubation with IL-1beta, or by the addition of the Ca2+ ionophore, Ionomycin. Other stimuli known to activate the NADPH oxidase of phagocytes were ineffective, but the regulatory cytokine IFNgamma lowered the rate of release. Inhibitors of lipoxygenase function decreased the rate of superoxide production, whereas inhibitors of cyclo-oxygenase, xanthine oxidase, or NADPH oxidase failed to inhibit. The addition of NADH or NADPH to whole cells increased the rate threefold.

A variant form of X-linked chronic granulomatous disease with normal nitroblue tetrazolium slide test and cytochrome b

Abstract. Chronic granulomatous disease was diagnosed in a boy who suffered from severe generalized infections. Family investigations revealed the inheritance of the disease to be X‐linked. However, unlike other cases of X‐linked chronic granulomatous disease, the membrane oxidase of the neutrophils from this patient was not totally defective and sufficient activity was left to result in a normal phorbol myristate acetate‐stimulated nitroblue tetrazolium slide test. Also, unlike the usual findings in X‐linked chronic granulomatous disease, cytochrome b was present in normal amounts in the neutrophils from this patient. The cytochrome was normal, judged from its midpoint potential of —245 mV and its ability to bind CO. It is thus apparent that X‐linked chronic granulomatous disease may result from at least two different defects and that the phorbol myristate acetate stimulated nitroblue tetrazolium slide test fails to detect some cases.

A nitrite reductase from Neurospora crassa

Nitrite reductase has been purified, over 50-fold from ordinary felts of Neurospora crassa, macroconidial wild type Em 5297a. The enzyme is a DPNH-dependent flavoprotein containing FAD, Fe and Cu and -SH groups. Copper may act by coupling the flavin component of the enzyme to nitrite since an external supply of Cu1+ reduced NO2 non-enzymically; this reduction increased in the presence of the enzyme. The role of iron in the enzyme is not known since neither Fe2+ nor reduced cytochrome c will reduce nitrite. The enzyme is also dependent on Mg and pyridoxine for maximal activity. Although the addition of pyridoxine or pyridoxal or pyridoxal phosphate reactivated the enzyme in crude extracts of the pyridoxine-requiring mutant deficient in the vitamin, there was no similar effect with the purified enzyme. A possible explanation for this phenomenon is presented. The reduction product inhibited the enzyme and it is shown that the addition of hyponitrite but not of hydroxylamine depressed nitrite reductase activity.

Rapid incorporation of the human neutrophil plasma membrane cytochrome b into phagocytic vacuoles

Abstract Phagocytic vacuoles containing lgG coated latex particles were isolated from human neutrophils by floatation. The absorbance spectrum of the cytochrome b was associated with the vacuoles within 10 sec of particle uptake and the vacuolar concentration increased little thereafter. In contrast, the cytoplasmic granule proteins myeloperoxidase and vitamin B 12 binding protein associate with the vacuoles more slowly. The addition of dithionite to intact cells rapidly reduces most of the cytochrome b , whereas only a small proportion of the myeloperoxidase, which is located intracellularly, is reduced in the absence of detergent. Most of the cytochrome b appears to be localised in the neutrophil plasma membrane.