The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels

The enzyme NADPH oxidase in phagocytes is important in the body's defence against microbes: it produces superoxide anions (O-2(-), precursors to bactericidal reactive oxygen species(1)). Electrons move from intracellular NADPH, across a chain comprising FAD ( flavin adenine dinucleotide) and two haems, to reduce extracellular O-2 to O-2(-). NADPH oxidase is electrogenic(2), generating electron current (I-e) that is measurable under voltage-clamp conditions(3,4). Here we report the complete current-voltage relationship of NADPH oxidase, the first such measurement of a plasma membrane electron transporter. We find that Ie is voltage-independent from -100 mV to >0 mV, but is steeply inhibited by further depolarization, and is abolished at about +190 mV. It was proposed that H+ efflux(2) mediated by voltage-gated proton channels(5,6) compensates I-e, because Zn2+ and Cd2+ inhibit both H+ currents(7-9) and O-2(-) production(10). Here we show that COS-7 cells transfected with four NADPH oxidase components(11), but lacking H+ channels(12), produce O-2(-) in the presence of Zn2+ concentrations that inhibit O-2(-) production in neutrophils and eosinophils. Zn2+ does not inhibit NADPH oxidase directly, but through effects on H+ channels. H+ channels optimize NADPH oxidase function by preventing membrane depolarization to inhibitory voltages.