Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of 
the production of O2- by the activated NADPH oxidase of bovine neutrophils. In a 
cell-free system of NADPH oxidase activation consisting of neutrophil membranes 
and cytosol from resting cells, supplemented with guanosine 
5'-[gamma-thio]triphosphate, MgCl2 and arachidonic acid, or in membranes 
isolated from neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate, 
addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced 
inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to 
contain the Ph2I-sensitive component(s). In the presence of a concentration of 
Ph2I sufficient to fully inhibit O2- production (around 10 nmol/mg membrane 
protein), addition of catalytic amounts of the redox mediator dichloroindophenol 
(Cl2Ind) resulted in a by-pass of the electron flow to cytochrome c, the rate of 
which was about half of that determined in non-inhibited oxidase. A marked 
increase in the efficiency of this by-pass was achieved by addition of sodium 
deoxycholate. The Cl2-Ind-mediated cytochrome c reduction was negligible in 
membranes isolated from resting neutrophils. At a higher concentration of Ph2I 
(100 nmol/mg membrane protein), the Cl2Ind-mediated cytochrome c reductase 
activity was only half inhibited, which indicated that, in the NADPH oxidase 
complex, there are at least two Ph2I sensitive components, differing by their 
sensitivity to the inhibitor. At low concentrations of Ph2I (less than 10 
nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil 
membranes was modified, suggesting that the component sensitive to low 
concentrations of Ph2I is the heme binding component of cytochrome b558. Higher 
concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase 
component of the oxidase complex. A number of membrane and cytosolic proteins 
were labeled by [125I]Ph2I. However, the radiolabeling of a membrane-bound 
24-kDa protein, which might be the small subunit of cytochrome b558, responded 
more specifically to the conditions of activation and reduction which are 
required for inhibition of O2- production by Ph2I. The O2(-)-generating form of 
xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a 
non-heme iron flavoprotein, by Ph2I had a number of features in common with that 
of the neutrophil NADPH oxidase, namely the requirement of reducing conditions 
for inhibition of O2- production by Ph2I and the induction of a by-pass of 
electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some 
similarity in the molecular organization of the two enzymes.