title: Heme histidine ligands within gp91(phox) modulate proton conduction by the phagocyte NADPH oxidase.
abstract: The membrane subunit of the phagocyte NADPH oxidase, gp91(phox), possesses a H(+) channel motif formed by membrane-spanning histidines postulated to coordinate the two heme groups forming the redox center of the flavocytochrome. To study the role of heme-binding histidines on proton conduction, we stably expressed the gp91(phox) cytochrome in human embryonic kidney 293 cells and measured proton currents with the patch clamp technique. Similar to its shorter homologue, NADPH oxidase homologue 1, which is predicted not to bind heme, gp91(phox) generated voltage-activated, pH-dependent, H(+)-selective currents that were reversibly blocked by Zn(2+). The gp91(phox) currents, however, activated faster, deactivated more slowly, and were markedly affected by the inhibition of heme synthesis. Upon heme removal, the currents had larger amplitude, activated faster and at lower voltages, and became sensitive to the histidine reagent diethylpyrocarbonate. Mutation of the His-115 residue to leucine abolished both the gp91(phox) characteristic 558-nm absorbance peak and voltage-activated currents, indicating that His-115 is involved in both heme ligation and proton conduction. These results indicate that the gp91(phox) proton channel is activated upon release of heme from its His-115 ligand. During activation of the oxidase complex, changes in heme coordination within the cytochrome might increase the mobility of histidine ligands, thereby coupling electron and proton transport.
authors.affiliation: Department of Physiology, University of Geneva Medical Center, 1211 Geneva 4, Fondation pour Recherches Médicales, University of Geneva, Switzerland.
authors.forename: A
authors.initials: A
authors.lastname: Maturana
authors.forename: S
authors.initials: S
authors.lastname: Arnaudeau
authors.forename: S
authors.initials: S
authors.lastname: Ryser
authors.forename: B
authors.initials: B
authors.lastname: Banfi
authors.forename: J P
authors.initials: JP
authors.lastname: Hossle
authors.forename: W
authors.initials: W
authors.lastname: Schlegel
authors.forename: K H
authors.initials: KH
authors.lastname: Krause
authors.forename: N
authors.initials: N
authors.lastname: Demaurex
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D004583:Electrons; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007477:Ions; D007700:Kinetics; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010586:Phagocytes / Q000201:enzymology; D010587:Phagocytosis; D011485:Protein Binding; D011522:Protons; D013053:Spectrophotometry; D013997:Time Factors; D014162:Transfection; D015032:Zinc / Q000494:pharmacology
chemical_list: D007477:Ions; D008024:Ligands; D008562:Membrane Glycoproteins; D011522:Protons; D006418:Heme; D006639:Histidine; D006859:Hydrogen; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D015032:Zinc