Interactions between NADPH oxidase and voltage-gated proton channels: why electron transport depends on proton transport

Leukocytes kill microbes by producing reactive oxygen species, using a multi-component enzyme complex, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Electrons pass from intracellular NADPH through a redox chain within the enzyme, to reduce extracellular O2 to O2-. Electron flux is electrogenic, and rapidly depolarizes the membrane potential. Excessive depolarization can turn off electron transport by self-inhibition, but this is prevented by proton flux that balances the electron flux. Although the membrane potential depolarizes by approximately 100 mV during the respiratory burst (NADPH oxidase activity), NADPH oxidase activity is independent of voltage in this range, which permits optimal function and prevents self-inhibition.     

Redox-driven proton pumping by heme-copper oxidases

One of the key problems of molecular bioenergetics is the understanding of the function of redox-driven proton pumps on a molecular level. One such class of proton pumps are the heme-copper oxidases. These enzymes are integral membrane proteins in which proton translocation across the membrane is driven by electron transfer from a low-potential donor, such as, e.g. cytochrome c, to a high-potential acceptor, O(2). Proton pumping is associated with distinct exergonic reaction steps that involve gradual reduction of oxygen to water. During the process of O(2) reduction, unprotonated high pK(a) proton acceptors are created at the catalytic site. Initially, these proton acceptors become protonated as a result of intramolecular proton transfer from a residue(s) located in the membrane-spanning part of the enzyme, but removed from the catalytic site. This residue is then reprotonated from the bulk solution. In cytochrome c oxidase from Rhodobacter sphaeroides, the proton is initially transferred from a glutamate, E(I-286), which has an apparent pK(a) of 9.4. According to a recently published structure of the enzyme, the deprotonation of E(I-286) is likely to result in minor structural changes that propagate to protonatable groups on the proton output (positive) side of the protein. We propose that in this way, the free energy available from the O(2) reduction is conserved during the proton transfer. On the basis of the observation of these structural changes, a possible proton-pumping model is presented in this paper. Initially, the structural changes associated with deprotonation of E(I-286) result in the transfer of a proton to an acceptor for pumped protons from the input (negative) side of the membrane. After reprotonation of E(I-286) this acceptor releases a proton to the output side of the membrane.     

Electron and proton transport across the plasma membrane

Transplasma membrane electron transport in both plant and animal cells activates proton release. The nature and components of the electron transport system and the mechanism by which proton release is activated remains to be discovered. Reduced pyridine nucleotides are substrates for the plasma membrane dehydrogenases. Both plant and animal membranes have unusual cyanide-insensitive oxidases so oxygen can be the natural electron acceptor. Natural ferric chelates or ferric transferrin can also act as electron acceptors. Artificial, impermeable oxidants such as ferricyanide are used to probe the activity. Since plasma membranes containb cytochromes, flavin, iron, and quinones, components for electron transport are present but their participation, except for quinone, has not been demonstrated. Stimulation of electron transport with impermeable oxidants and hormones activates proton release from cells. In plants the electron transport and proton release is stimulated by red or blue light. Inhibitors of electron transport, such as certain antitumor drugs, inhibit proton release. With animal cells the high ratio of protons released to electrons transferred, stimulation of proton release by sodium ions, and inhibition by amilorides indicates that electron transport activates the Na⁺/H⁺ antiport. In plants part of the proton release can be achieved by activation of the H⁺ ATPase. A contribution to proton transfer by protonated electron carriers in the membrane has not been eliminated. In some cells transmembrane electron transport has been shown to cause cytoplasmic pH changes or to stimulate protein kinases which may be the basis for activation of proton channels in the membrane. The redox-induced proton release causes internal and external pH changes which can be related to stimulation of animal and plant cell growth by external, impermeable oxidants or by oxygen.     

Cross talk between mitochondria and NADPH oxidases

Reactive oxygen species (ROS) play an important role in physiological and pathological processes. In recent years, a feed-forward regulation of the ROS sources has been reported. The interactions between the main cellular sources of ROS, such as mitochondria and NADPH oxidases, however, remain obscure. This work summarizes the latest findings on the role of cross talk between mitochondria and NADPH oxidases in pathophysiological processes. Mitochondria have the highest levels of antioxidants in the cell and play an important role in the maintenance of cellular redox status, thereby acting as an ROS and redox sink and limiting NADPH oxidase activity. Mitochondria, however, are not only a target for ROS produced by NADPH oxidase but also a significant source of ROS, which under certain conditions may stimulate NADPH oxidases. This cross talk between mitochondria and NADPH oxidases, therefore, may represent a feed-forward vicious cycle of ROS production, which can be pharmacologically targeted under conditions of oxidative stress. It has been demonstrated that mitochondria-targeted antioxidants break this vicious cycle, inhibiting ROS production by mitochondria and reducing NADPH oxidase activity. This may provide a novel strategy for treatment of many pathological conditions including aging, atherosclerosis, diabetes, hypertension, and degenerative neurological disorders in which mitochondrial oxidative stress seems to play a role. It is conceivable that the use of mitochondria-targeted treatments would be effective in these conditions.     

A burst of plant NADPH oxidases

Reactive oxygen species (ROS) are highly reactive molecules able to damage cellular components but they also act as cell signalling elements. ROS are produced by many different enzymatic systems. Plant NADPH oxidases, also known as respiratory burst oxidase homologues (RBOHs), are the most thoroughly studied enzymatic ROS-generating systems and our understanding of their involvement in various plant processes has increased considerably in recent years. In this review we discuss their roles as ROS producers during cell growth, plant development and plant response to abiotic environmental constraints and biotic interactions, both pathogenic and symbiotic. This broad range of functions suggests that RBOHs may serve as important molecular 'hubs' during ROS-mediated signalling in plants.     

NADPH oxidases contribute to autophagy regulation

Reactive oxygen species (ROS) are emerging as regulators of autophagy in various cellular contexts. There are many cellular sources of ROS in eukaryotic cells. In phagocytes, the critical immune cells for host defense, the Nox2 NADPH oxidase generates ROS during phagocytosis and plays a central role in microbial killing. Toll-like receptors (TLRs) are important membrane microbial sensing receptors, which can activate Nox2,¹ and were recently demonstrated to signal autophagy targeting of phagosomes to promote their maturation.² Our recent study reveals that Nox2 activity and its generated ROS are key signals that induce TLR-activated autophagy of phagosomes. Our results provide the first evidence that ROS from the Nox2 NADPH oxidase can contribute to regulating autophagy in host defense against bacteria. The association of TLR, Nox2 and autophagy with inflammatory bowel disease (IBD) suggests a significant role of this antibacterial pathway in these diseases.     

Identification and characterization of Sclerotinia sclerotiorum NADPH oxidases

Numerous studies have shown both the detrimental and beneficial effects of reactive oxygen species (ROS) in animals, plants, and fungi. These organisms utilize controlled generation of ROS for signaling, pathogenicity, and development. Here, we show that ROS are essential for the pathogenic development of Sclerotinia sclerotiorum, an economically important fungal pathogen with a broad host range. Based on the organism's completed genome sequence, we identified two S. sclerotiorum NADPH oxidases (SsNox1 and SsNox2), which presumably are involved in ROS generation. RNA interference (RNAi) was used to examine the function of SsNox1 and SsNox2. Silencing of SsNox1 expression indicated a central role for this enzyme in both virulence and pathogenic (sclerotial) development, while inactivation of the SsNox2 gene resulted in limited sclerotial development, but the organism remained fully pathogenic. ΔSsnox1 strains had reduced ROS levels, were unable to develop sclerotia, and unexpectedly correlated with significantly reduced oxalate production. These results are in accordance with previous observations indicating that fungal NADPH oxidases are required for pathogenic development and are consistent with the importance of ROS regulation in the successful pathogenesis of S. sclerotiorum.     

The microbicidal and cytoregulatory roles of NADPH oxidases

Despite their prominent microbicidal roles, NADPH oxidases (NOXs) have been recently found to regulate a wide variety of physiological activities. Through generation of reactive oxygen species (ROS), NOXs actively participate in cellular activities, including NET formation, inflammasome activation and wound sensing. The microbicidal and cytoregulatory roles of NOXs are contrasted in this review.     

Versatile roles of plant NADPH oxidases and emerging concepts

NADPH oxidase (NOX) is a key player in the network of reactive oxygen species (ROS) producing enzymes. It catalyzes the production of superoxide (O₂⁻), that in turn regulates a wide range of biological functions in a broad range of organisms. Plant Noxes are known as respiratory burst oxidase homologs (Rbohs) and are homologs of catalytic subunit of mammalian phagocyte gp91^phox. They are unique among other ROS producing mechanisms in plants as they integrate different signal transduction pathways in plants. In recent years, there has been addition of knowledge on various aspects related to its structure, regulatory components and associated mechanisms, and its plethora of biological functions. This update highlights some of the recent developments in the field with particular reference to important members of the plant kingdom.     

Molecular mechanism for activation of superoxide-producing NADPH oxidases

The membrane-integrated protein gp91phox, existing as a heterodimer with p22phox, functions as the catalytic core of the phagocyte NADPH oxidase, which plays a crucial role in host defence. The oxidase, dormant in resting cells, becomes activated to produce superoxide, a precursor of microbicidal oxidants, by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. In the past few years, several proteins homologous to gp91phox were discovered as superoxide-producing NAD(P)H oxidases (Nox's) in non-phagocytic cells; however, regulatory mechanisms for the novel oxidases have been largely unknown. Current identification of proteins highly related to p47phox and p67phox, designated Noxol (Nox organizer 1) and Noxal (Nox activator 1), respectively, has shed lights on common and distinct mechanisms underlying activations of Nox family oxidases.     

Mechanism and energetics of proton translocation by the respiratory heme-copper oxidases

Recent time-resolved optical and electrometric experiments have provided a sequence of events for the proton-translocating mechanism of cytochrome c oxidase. These data also set limits for the mechanistic, kinetic, and thermodynamic parameters of the proton pump, which are analysed here in some detail. The analysis yields limit values for the pK of the "pump site", its modulation during the proton-pumping process, and suggests its identity in the structure. Special emphasis is made on side-reactions that may short-circuit the pump, and the means by which these may be avoided. We will also discuss the most prominent proton pumping mechanisms proposed to date in relation to these data.     

The role of NADPH oxidases in diabetic cardiomyopathy

Systemic changes during diabetes such as high glucose, dyslipidemia, hormonal changes and low grade inflammation, are believed to induce structural and functional changes in the cardiomyocyte associated with the development of diabetic cardiomyopathy. One of the hallmarks of the diabetic heart is increased oxidative stress. NADPH-oxidases (NOXs) are important ROS-producing enzymes in the cardiomyocyte mediating both adaptive and maladaptive changes in the heart. NOXs have been suggested as a therapeutic target for several diabetic complications, but their role in diabetic cardiomyopathy is far from elucidated. In this review we aim to provide an overview of the current knowledge regarding the understanding of how NOXs influences cardiac adaptive and maladaptive processes in a “diabetic milieu”. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

Differential vascular functions of Nox family NADPH oxidases

PURPOSE OF REVIEW: Reactive oxygen species have been implicated in the initiation and progression of atherosclerosis. Reactive oxygen species can oxidize lipoproteins, limit the vascular availability of antiatherosclerotic nitric oxide and promote vascular expression of cytokines and adhesion molecules. Nox proteins of the NADPH oxidase family are prominent sources of vascular reactive oxygen species, and Nox protein-dependent reactive oxygen species production has been linked to atherogenesis. Recently, significant progress has been made in the understanding of differences among the Nox proteins. RECENT FINDINGS: Nox proteins exhibit cell-specific expression patterns and divergent molecular mechanisms controlling activity have been identified for individual Nox proteins. These aspects may relate to cellular activation, differentiation, proliferation, angiogenesis and gene expression, and may also be modulated by the functional states of the vessel such as endothelial dysfunction: in quiescent vessels, Nox proteins contribute to signal transduction and to the physiological responses to growth factors such as vascular endothelial growth factor or thrombin. Excessive Nox-dependent reactive oxygen species formation in vascular disease such as hyperlipidemia or diabetes, however, largely contributes to vascular dysfunction resulting in defective angiogenesis and inflammatory activation. SUMMARY: Reactive oxygen species, specifically generated by individual Nox proteins, act as secondary messengers. Selective inhibition of Nox proteins might be a novel approach to prevent and treat cardiovascular diseases.     

NADPH oxidases participate to doxorubicin-induced cardiac myocyte apoptosis

Cumulative doses of doxorubicin, a potent anticancer drug, lead to serious myocardial dysfunction. Numerous mechanisms including apoptosis have been proposed to account for its cardiotoxicity. Cardiac apoptosis induced by doxorubicin has been related to excessive reactive oxygen species production by the mitochondrial NADH dehydrogenase. Here, we explored whether doxorubicin treatment activates other superoxide anion generating systems such as the NADPH oxidases, membrane-embedded flavin-containing enzymes, and whether the subsequent oxidative stress contributes to apoptosis. We showed that doxorubicin treatment of rat cardiomyoblasts H9c2 triggers increases in caspase-3 like activity and hypoploid cells, both common features of apoptosis. Doxorubicin exposure also leads to a rapid superoxide production through NADPH oxidase activation. Inhibition of these enzymes using diphenyliodonium and apocynin reduces doxorubicin-induced reactive oxygen species production, caspase-3 like activity and sub-G1 cell population. In conclusion, NADPH oxidases participate to doxorubicin-induced cardiac apoptosis.     

Mechanism and energetics of proton translocation by the respiratory heme-copper oxidases

Recent time-resolved optical and electrometric experiments have provided a sequence of events for the proton-translocating mechanism of cytochrome c oxidase. These data also set limits for the mechanistic, kinetic, and thermodynamic parameters of the proton pump, which are analysed here in some detail. The analysis yields limit values for the pK of the “pump site”, its modulation during the proton-pumping process, and suggests its identity in the structure. Special emphasis is made on side-reactions that may short-circuit the pump, and the means by which these may be avoided. We will also discuss the most prominent proton pumping mechanisms proposed to date in relation to these data.     

Towards the mechanism of proton pumping by the haem-copper oxidases

The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a3/CuB centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the "prepumped" proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different Em values of the electron acceptor in the binuclear site.