NAD(P)H oxidase

Biological Function

Superoxide produced from NADPH oxidase is used to kill bacteria and fungi by mechanisms that are not yet fully understood. Thus far, in E. coli it has been shown to undergo spontaneous dismutation in a pH and concentration-dependent reaction to yield hydrogen peroxide which together with superoxide anion damage a variety of biomolecules, most importantly damaging iron clusters. Release of iron can undergo Fenton reaction with hydrogen peroxide to yield hydroxyl radicals, which damage any biological molecule, including DNA. The enzymatic activity for producing this type of Reactive Oxygen Species (ROS) exceeds those of other oxidase enzymes including Xanathine oxidase and mitochondrial oxidase. In most studies, NADH is believed to be the preferred electron donor, although reports have shown that NAPDH is equally used as the electron donor.

Chemical Reaction

The overall chemical reaction for the superoxide anion formation from NADPH is shown in Figure 1.

Enzyme Structure

The membrane-bound vascular enzyme is composed of five parts: two cytosolic subunits (p47phox and p67phox), a cytochrome b558 which consists of gp91phox (shown as Nox 1 or 4 in Figure 2), p22phox and a small G protein Rac. Generation of the superoxide in vascular NADPH occurs by a one-electron reduction of oxygen via the gp91phox subunit, using reduced NADPH as the electron donor (Figure 2). The small G protein carries an essential role in the activation of the oxidase by switching between a GDP-bound (inactive) and GTP-linked (active) forms.

Biological Diversity

NAD(P)H oxidase is found in two types: one in white blood cells (neutrophilic) and the other in vascular cells, differing in biochemical structure and functions. Both types of enzymes are inhibited by diphenylene iodonium (DPI) and stimulated by agonists and arachidonic acid. Apocynin is also known to inhibit NAD(P)H oxidase by preventing the assembly of the NADPH oxidase subunits. In one study, apocynin showed clinical benefits in the treatment of influenza as it decreased influenza-induced lung inflammation in mice in vivo. Vascular NAD(P)H oxidase produces superoxide species in minutes to hours, while the neutrophilic enzyme releases the radical dioxygen almost instantaneously. Moreover, in white blood cells superoxide have been found to transfer electrons across the membrane to extracellular oxygen, while in vascular cells the radical anion appears to be released mainly intracellularly.

Regulation

Since NAD(P)H oxidase supplies the major source of the highly reactive superoxide anions, careful regulation of the enzyme is crucial to maintain a healthy level of ROS in the body. NADPH oxidase is dormant in resting cells but becomes rapidly activated by several stimuli, including bacterial products and cytokines. Vascular NADPH oxidases are regulated by a variety of hormones and factors known to be important players in vascular remodeling and disease. These include thrombin, platelet-derived growth factor (PDGF), tumor necrosis factor (TNFa), lactosylceramide, interleukin-1, and oxidized LDL.

Pathology and Disease Relevance

Superoxides are crucial in killing foreign bacteria in the human body. A known mutation in the gene coding for the NAD(P)H oxidase causes an immunodeficiency syndrome called chronic granulomatous disease, characterized by extreme susceptibility to infection. An excessive production of ROS in vascular cells— referred to as oxidative stress— has been shown to lead to many forms of cardiovascular disease including hypertension, atherosclerosis, myocardial infarction, and ischemic stroke. Nitric oxide (NO) inhibits the NADPH enzyme, blocking the source of oxidative stress in the vasculature. NO donor drugs (also known as nitrovasodilators) have been used for more than a century to treat coronary artery disease, hypertension, and heart failure by preventing excess superoxide anion from deteriorating healthy vascular cells.