PMID- 11931642
OWN - NLM
STAT- MEDLINE
DCOM- 20020905
LR  - 20181113
IS  - 0264-6021 (Print)
IS  - 1470-8728 (Electronic)
IS  - 0264-6021 (Linking)
VI  - 365
IP  - Pt 2
DP  - 2002 Jul 15
TI  - Mechanism in the reaction of cytochrome c oxidase with organic hydroperoxides: an 
      ESR spin-trapping investigation.
PG  - 461-9
AB  - Organic hydroperoxides are of great utility in probing the reaction mechanism and 
      the toxicological consequences of lipid peroxidation. In the present study, ESR 
      spin-trapping was employed to investigate the peroxidation of mitochondrial 
      cytochrome c oxidase (CcO) with t-butyl hydroperoxide (t-BuOOH) and cumene 
      hydroperoxide (CumOOH). The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was 
      used to detect the radical species formed from the reaction of CcO with t-BuOOH. 
      The presence of t-BuOOH-derived alkoxyl radical (t-BuO*) as the primary radical 
      indicates reductive scission of the O-O bond by CcO. The ESR signal of 
      DMPO/*Ot-Bu can be partially abolished by cyanide, implying that the reductive 
      cleavage involved the haem a(3)Cu(B) binuclear site of CcO. A nitroso spin trap, 
      2-methyl-2-nitrosopropane (MNP), was used to detect and identify radical species 
      from the reaction of CcO with CumOOH. In addition to the t-BuOOH-derived methyl, 
      hydroxylmethyl and tertiary carbon-centred radicals, a protein-derived radical 
      was detected. The intensity of the ESR signal from the protein radical increased 
      with the CumOOH concentration at low CumOOH/CcO ratios, with maximal intensity at 
      a ratio of 100 mol of CumOOH/mol of CcO. The immobilized protein radical adduct 
      of MNP was stable and persistent after dialysis; it was also resistant to 
      proteolytic digestion, suggesting that it was formed in the transmembrane region, 
      a region that is not accessible to proteases. Its signal was greatly enhanced 
      when CcO cysteine residues were chemically modified by N-ethylmaleimide, when the 
      tryptophan residues in CcO were oxidized by N-bromosuccimide, and when tyrosine 
      residues on the surface of CcO were iodinated, showing that a radical equilibrium 
      was established among the cysteine, tryptophan and tyrosine residues of the 
      protein-centred radical. Pre-treatment of CcO with cyanide prevented detectable 
      MNP adduct formation, confirming that the haem a(3)-Cu(B) binuclear centre was 
      the initial reaction site. When the CcO was pre-treated with 10 mM (100 
      equivalents) of CumOOH, the enzyme activity decreased by more than 20%. This 
      inhibition was persistent after dialysis, suggesting that the detected 
      protein-centred radical was, in part, involved in the irreversible inactivation 
      by CumOOH. Visible spectroscopic analysis revealed that the haem a of CcO was not 
      affected during the reaction. However, the addition of pyridine to the reaction 
      mixture under alkaline conditions resulted in the destruction of the haem centre 
      of CcO, suggesting that its protein matrix rather than its haem a is the target 
      of oxidative damage by the organic hydroperoxide.
FAU - Chen, Yeong-Renn
AU  - Chen YR
AD  - The Laboratory of Pharmacology and Chemistry, National Institute of Environmental 
      Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, 
      USA. chen6@niehs.nih.gov
FAU - Mason, Ronald P
AU  - Mason RP
LA  - eng
PT  - Journal Article
PL  - England
TA  - Biochem J
JT  - The Biochemical journal
JID - 2984726R
RN  - 0 (Spin Labels)
RN  - 42HK56048U (Tyrosine)
RN  - 8DUH1N11BX (Tryptophan)
RN  - 955VYL842B (tert-Butylhydroperoxide)
RN  - EC 1.9.3.1 (Electron Transport Complex IV)
RN  - K848JZ4886 (Cysteine)
SB  - IM
MH  - Cysteine/metabolism
MH  - Electron Spin Resonance Spectroscopy
MH  - Electron Transport Complex IV/chemistry/*metabolism
MH  - Oxidative Stress
MH  - Spin Labels
MH  - Tryptophan/metabolism
MH  - Tyrosine/metabolism
MH  - tert-Butylhydroperoxide/*metabolism
PMC - PMC1222682
EDAT- 2002/04/05 10:00
MHDA- 2002/09/06 10:01
PMCR- 2003/01/15
CRDT- 2002/04/05 10:00
PHST- 2002/04/02 00:00 [accepted]
PHST- 2002/03/25 00:00 [revised]
PHST- 2002/01/29 00:00 [received]
PHST- 2002/04/05 10:00 [pubmed]
PHST- 2002/09/06 10:01 [medline]
PHST- 2002/04/05 10:00 [entrez]
PHST- 2003/01/15 00:00 [pmc-release]
AID - BJ20020170 [pii]
AID - 10.1042/BJ20020170 [doi]
PST - ppublish
SO  - Biochem J. 2002 Jul 15;365(Pt 2):461-9. doi: 10.1042/BJ20020170.