Identification of bovine heart cytochrome c oxidase subunits modified by the lipid peroxidation product 4-hydroxy-2-nonenal.
Bovine heart cytochrome c oxidase (CcO) was inactivated by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) in a time- and concentration-dependent manner with pseudo-first-order kinetics. Cytochrome c oxidase electron transport activity decreased by as much as 50% when the enzyme was incubated for 2 h at room temperature with excess HNE (300-500 microM). HNE-modified CcO subunits were identified by two mass spectrometric methods: electrospray ionization mass spectrometry (ESI/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). All of the experimentally determined molecular masses were in excellent agreement with published sequence values with an accuracy of approximately 1 part per 10000 mass units for subunits smaller than 20 kDa and approximately 1 part per 1000 mass units for the three subunits larger than 20 kDa. Both MS methods detected six CcO subunits with an increased mass of 156 Da after reaction with HNE (subunits II, IV, Vb, VIIa, VIIc, and VIII); this result indicates a single Michael-type reaction site on either a lysine or histidine residue within each subunit. Reaction of HNE with either subunit VIIc or subunit VIII (modified approximately 30% and 50-75%, respectively) must be responsible for CcO inhibition. None of the other subunits were modified more than 5% and could not account for the observed loss of activity. Reaction of HNE with His-36 of subunit VIII is most consistent with the approximately 50% inhibition of CcO: (1) subunit VIII is modified more than any other subunit by HNE; (2) the time dependence of subunit VIII modification is consistent with the percent inhibition of CcO; (3) His-36 was identified as the HNE-modified amino acid residue within subunit VIII by tandem MS analysis.
Musatov
Andrej
A
Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA. musatov@uthscsa.edu
Carroll
Christopher A
CA
Liu
Yuan-Chao
YC
Henderson
George I
GI
Weintraub
Susan T
ST
Robinson
Neal C
NC
D000447:Aldehydes / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003576:Electron Transport Complex IV / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D015227:Lipid Peroxidation / Q000187:drug effects; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology* / Q000378:metabolism; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000447:Aldehydes; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D003576:Electron Transport Complex IV; C027576:4-hydroxy-2-nonenal