Uncompetitive inhibition of superoxide generation by a synthetic peptide corresponding to a predicted NADPH binding site in gp91-phox, a component ofthe phagocyte respiratory oxidase

The large subunit of cytochrome b558, gp91-phox, is believed to play a hey role in superoxide generation in neutrophils by accepting electrons from NA DPH and donating them to molecular oxygen. We found that a peptide correspo nding to a predicted NADPH binding site in gp91-phox inhibited superoxide g eneration in a cell-fi ee system consisting of neutrophil membrane and cyto sol. Minimum essential sequence for the inhibition was KSVWYK, which corres ponded to residues 420-425 (IC50 = 30 mu M). Unlike other peptides known to inhibit the reaction, this peptide was effective even when added to the sy stem after activation or to activated membrane from stimulated neutrophils. Furthermore, the peptide inhibited superoxide generation in a membrane sys tem activated without cytosol. Kinetic analysis revealed that the peptide i nhibited. the reaction uncompetitively. These results suggest that the pept ide combines with the activated cytochrome b558-NADPH complex and thereby i nhibits electron transfer from. NADPH to molecular oxygen. (C) 1999 Academi c Press.