The NADPH oxidase is the molecular machinery responsible for making O2- in the phagocytic vacuole. It is the prototype of a number of NADPH Oxidase or NOX enzymes that have been identified in a wide variety of tissues. It consists of a flavocytochrome that spans the membrane and passes electrons from the substrate, NADPH, in the cytoplasm, across a short electron transport chain consisting of a FAD and two haems, to O2 in the vacuole. Activation of this oxidase requires the participation of a number of cytosolic proteins including p47phox, p67phox, and the small GTP binding protein, p21rac. These proteins seem to activate electron transport, possibly by modifying the conformation of the flavocytochrome, and providing NADPH access to the active site of the enzyme. Function of oxidase Microbial killing – Neutrophils consume large amounts of oxygen when they phagocytose bacteria and fungi. This oxygen consumption, known as the “respiratory burst” is required for the efficient killing and digestion of the engulfed microbe which are killed poorly under anaerobic conditions or in Chronic Granulomatous Disease (CGD), in which the oxidase is dysfunctional. The oxidase produces superoxide, O2-, in the vacuole, and this dismutates to form hydrogen peroxide. The neutrophil granules contain myeloperoxidase which can use H2O2 as substrate to oxidise halides to hypohalous acids. The discovery that neutrophils produce oxygen free radicals and H2O2, coupled with the fact that the absence of this production results in impaired microbial killing, led to the belief that the oxygen radicals themselves killed the microbes. This concept that the products of the oxidase were themselves toxic and directly responsible for killing the bacteria was attractive and gained almost universal support. However it was then shown that mice in whom the major neutrophil neutral proteases cathepsin G and elastase had been knocked out were susceptible to bacterial infection, and that their neutrophils demonstrated a profound defect of microbial killing, despite the production of normal amounts of ROS and normal activity of myeloperoxidase. The oxidase had to be doing something else! Charge compensation – The oxidase is electrogenic, which means that the passage of electrons across the vacuolar membrane produces a charge across this membrane that will curtail further electron transport unless compensated. Electrons carry one negative charge so this charge compensation requires the passage of negatively charged ions in the opposite direction or positively charge ions in the same direction. We have found that combinations of the two occur. The neutrophil granules contain a number of digestive enzymes and other proteins and high concentrations of ions, particularly Cl- (320mM), K+ (240mM) and Na+ (80mM). The main compensating ion is Cl- which passes from the vacuole to the cytoplasm. In addition K+ passes through BKCa channels into the vacuole. The efflux of Cl- from, and influx of K+ into, the vacuole activate the granule enzymes to kill and digest the microbes. pH regulation – Electrons are accepted in the vacuole by O2 to produce O2- which dismutates to form O22-(peroxide) which becomes protonated to form H2O2: 4NADPH + 4O2 → 4NADP + 4H+ + 4O2- 4O2- → 2O22- +2O2 2O22- + 4H+ → 2H2O2 2H2O2 → 2H2O + O2 The H+s released from NADPH are left in the cytoplasm, which is acidified, whereas the consumption of H+s in the vacuole causes the pH to rise in this compartment. It is important that the pH in the vacuole is maintained at that optimal for the activity of the neutral proteases that kill and digest the microbes. This is accomplished by the buffering capacity of the granule contents, exchange of H+ for Na+ by NHE1 and compensation of some of the charge by the passage of H+s and K+ from the cytoplasm to the vacuole. Implications for the role of free radicals in the pathogenesis of disease ROS production by neutrophils provided the paradigm for the toxic effect of these molecules in a biological system, and by extrapolation, to their potentially toxic role in the pathogenesis of human diseases, including oncogenesis. Now that the true mechanism by which the NADPH oxidase promotes microbial killing has been established, the involvement of ROS as causal agents in disease processes must be reassessed.