Activation of human neutrophil NADPH oxidase results in coupling of electron carrier function between ubiquinone-10 and cytochrome b559 (original) (raw)

The enzymatic activity underlying the respiratory burst in human neutrophils was examined in a subcellular fraction with high specific activity and shown to be a membrane-associated complex of a flavoprotein, ubiquinone-10, and cytochrome baas in an approximate 1.3:1:2 molar ratio. Study of the redox poise of these electron carriers indicated that electron flow in the intact complex from unstimulated cells proceeded: NADPH + E-FAD -P ubiquinone-10. Similar studies on the complex prepared from stimulated neutrophils indicated that electron flow proceeded NADPH + E-FAD + ubiquinone-10 + cytochrome bass + oxygen. The active enzyme complex was inhibited by p-chloromercuribenzoate. Inhibition persisted after removal of excess inhibitor, was reversed by dithiothreitol, and could be blocked by prior addition of substrate (NADPH). Inhibition of the active oxidase complex by p-chloromercuribenzoate also inhibited electron flow from NADPH to all purported electron carriers in the chain (Le. E-FAD, ubiquinone-10, and cytochrome bass). We conclude that activation of the oxidase enzyme complex in the intact neutrophil resulted in linkage of electron carrier function between endogenous ubiquinone-10 and cytochrome bass and was without demonstrable effect on proximal electron flow. The pchloromercuribenzoate sensitive site(s) proximal to the initial electron acceptor (E-FAD) did not appear to be altered by the cellular activation process.