Heme histidine ligands within gp91(phox) modulate proton conduction by thephagocyte NADPH oxidase

The membrane subunit of the phagocyte NADPH oxidase, gp91(phox), possesses a H+ channel motif formed by membrane-spanning histidines postulated to coo rdinate the two heme groups forming the redox center of the flavocytochrome . To study the role of heme-binding histidines on proton conduction, we sta bly expressed the gp91(phox) cytochrome in human embryonic kidney 293 cells and measured proton currents with the patch clamp technique. Similar to it s shorter homologue, NADPH oxidase homologue 1, which is predicted not to b ind heme, gp91(phox) generated voltage-activated, pH-dependent, H+-selectiv e currents that were reversibly blocked by Zn2+. The gp91(phox) currents, h owever, activated faster, deactivated more slowly, and were markedly affect ed by the inhibition of heme synthesis. Upon heme removal, the currents had larger amplitude, activated faster and at lower voltages, and became sensi tive to the histidine reagent diethylpyrocarbonate. Mutation of the His-115 residue to leucine abolished both the gp91(phox) characteristic 558-nm abs orbance peak and voltage-activated currents, indicating that His-115 is inv olved in both heme ligation and proton conduction. These results indicate t hat the gp91(phox) proton channel is activated upon release of heme from it s His-115 ligand. During activation of the oxidase complex, changes in heme coordination within the cytochrome might increase the mobility of histidin e ligands, thereby coupling electron and proton transport.