Specific modification of two tryptophans within the nuclear-encoded subunits of bovine cytochrome c oxidase by hydrogen peroxide.
Hydrogen peroxide does more than react with the binuclear center of oxidized bovine cytochrome c oxidase and generate the well-characterized "peroxy" and "ferryl" forms. Hydrogen peroxide also inactivates detergent-solubilized cytochrome c oxidase in a time- and concentration-dependent manner. There is a 70-80% decrease of electron-transport activity, peroxidation of bound cardiolipin, modification of two nuclear-encoded subunits (IV and VIIc), and dissociation of approximately 60% of subunits VIa and VIIa. Modification of subunit VIIc and dissociation of subunit VIIa are coupled events that probably are responsible for the inactivation of cytochrome c oxidase. When cytochrome c oxidase is exposed to 500 microM hydrogen peroxide for 30 min at pH 7.4 and room temperature, subunits IV (modified up to 20%) and VIIc (modified up to 70%) each have an increased mass of 16 Da as detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and electrospray ionization mass spectrometry. In each case, the increased mass is caused by oxidation of a tryptophan (Trp19 within subunit VIIc and Trp48 within subunit IV), almost certainly due to formation of hydroxytryptophan. We conclude that hydrogen peroxide-induced oxidation of tryptophan and cardiolipin proceeds via the binuclear center since both modifications are prevented if the binuclear center is first blocked with cyanide. Bound cardiolipin and oxidized tryptophans are localized relatively far from the binuclear center (30-60 A); therefore, oxidation probably occurs by migration of a free radical generated at the binuclear center to these distal reaction sites.
Musatov
Andrej
A
Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas 78229-3900, USA. musatov@uthscsa.edu
Hebert
Emmy
E
Carroll
Christopher A
CA
Weintraub
Susan T
ST
Robinson
Neal C
NC
D000818:Animals; D002308:Cardiolipins / Q000737:chemistry; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003486:Cyanides / Q000737:chemistry; D003902:Detergents / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry*; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000737:chemistry; D005960:Glucosides / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry*; D015227:Lipid Peroxidation; D008353:Mannitol / Q000737:chemistry; D009687:Nuclear Proteins / Q000737:chemistry*; D021122:Protein Subunits / Q000737:chemistry*; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013482:Superoxide Dismutase / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*
D002308:Cardiolipins; D003486:Cyanides; D003902:Detergents; D004791:Enzyme Inhibitors; D005960:Glucosides; D009687:Nuclear Proteins; D021122:Protein Subunits; D008353:Mannitol; C040358:dodecyl maltoside; D014364:Tryptophan; D006861:Hydrogen Peroxide; D013482:Superoxide Dismutase; D003576:Electron Transport Complex IV