PMID- 10757991
OWN - NLM
STAT- MEDLINE
DCOM- 20000616
LR  - 20190613
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 39
IP  - 15
DP  - 2000 Apr 18
TI  - Nature of the inhibition of horseradish peroxidase and mitochondrial cytochrome c 
      oxidase by cyanyl radical.
PG  - 4415-22
AB  - Previous studies established that the cyanyl radical ((*)CN), detected as 
      5,5-dimethyl-1-pyrroline N-oxide (DMPO)/(*)CN by the electron spin resonance 
      (ESR) spin-trapping technique, can be generated by horseradish peroxidase (HRP) 
      in the presence of hydrogen peroxide (H(2)O(2)) and by mitochondrial cytochrome c 
      oxidase (CcO) in the absence of H(2)O(2). To investigate the mechanism of 
      inhibition by cyanyl radical, we isolated and characterized the iron 
      protoporphyrin IX and heme a from the reactions of CN(-) with HRP and CcO, 
      respectively. The purified heme from the reaction mixture of HRP/H(2)O(2)/KCN was 
      unambiguously identified as cyanoheme by the observation of the protonated 
      molecule, (M + H)(+), of m/z = 642.9 in the matrix-assisted laser 
      desorption/ionization (MALDI) mass spectrum. The proton NMR spectrum of the 
      bipyridyl ferrous cyanoheme complex revealed that one of the four meso protons 
      was missing and had been replaced with a cyanyl group, indicating that the 
      single, heme-derived product was meso-cyanoheme. The holoenzyme of HRP from the 
      reconstitution of meso-cyanoheme with the apoenzyme of HRP (apoHRP) showed no 
      detectable catalytic activity. The Soret peak of cyanoheme-reconstituted apoHRP 
      was shifted to 411 nm from the 403 nm peak of native HRP. In contrast, the heme a 
      isolated from partially or fully inhibited CcO did not show any change in the 
      structure of the protoporphyrin IX as indicated by its MALDI mass spectrum, which 
      showed an (M + H)(+) of m/z = 853.6, and by its pyridine hemochromogen spectrum. 
      However, a protein-centered radical on the CcO can be detected in the reaction of 
      CcO with cyanide and was identified as the thiyl radical(s) based on inhibition 
      of its formation by N-ethylmaleimide pretreatment, suggesting that the protein 
      matrix rather than protoporphyrin IX was attacked by the cyanyl radical. In 
      addition to the difference in heme structures between HRP and CcO, the available 
      crystallographic data also suggested that the distinct heme environments may 
      contribute to the different inhibition mechanisms of HRP and CcO by cyanyl 
      radical.
FAU - Chen, Y R
AU  - Chen YR
AD  - Laboratories of Pharmacology and Chemistry and of Structural Biology, National 
      Institute of Environmental Health Sciences, National Institutes of Health, 
      Research Triangle Park, North Carolina 27709, USA. chen6@niehs.nih.gov
FAU - Deterding, L J
AU  - Deterding LJ
FAU - Tomer, K B
AU  - Tomer KB
FAU - Mason, R P
AU  - Mason RP
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Apoenzymes)
RN  - 0 (Cyanides)
RN  - 0 (Cyclic N-Oxides)
RN  - 0 (Holoenzymes)
RN  - 0 (Protons)
RN  - 42VZT0U6YR (Heme)
RN  - 6JKA7MAH9C (Guaiacol)
RN  - 7170JZ1QF3 (5,5-dimethyl-1-pyrroline-1-oxide)
RN  - BBX060AN9V (Hydrogen Peroxide)
RN  - EC 1.11.1.- (Horseradish Peroxidase)
RN  - EC 1.9.3.1 (Electron Transport Complex IV)
RN  - MQD255M2ZO (Potassium Cyanide)
RN  - O3C74ACM9V (Ethylmaleimide)
SB  - IM
MH  - Apoenzymes/metabolism
MH  - Catalysis/drug effects
MH  - Chromatography, High Pressure Liquid
MH  - Cyanides/metabolism/*pharmacology
MH  - Cyclic N-Oxides/metabolism
MH  - Electron Spin Resonance Spectroscopy
MH  - Electron Transport Complex IV/*antagonists & inhibitors/metabolism
MH  - Ethylmaleimide/pharmacology
MH  - Guaiacol/metabolism
MH  - Heme/analogs & derivatives/chemistry/isolation & purification/metabolism
MH  - Holoenzymes/metabolism
MH  - Horseradish Peroxidase/*antagonists & inhibitors/metabolism
MH  - Hydrogen Peroxide/metabolism/pharmacology
MH  - Magnetic Resonance Spectroscopy
MH  - Mitochondria/*enzymology
MH  - Models, Chemical
MH  - Molecular Weight
MH  - Oxidative Stress
MH  - Potassium Cyanide/metabolism/*pharmacology
MH  - Protons
MH  - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
MH  - Spin Trapping
EDAT- 2000/04/12 09:00
MHDA- 2000/06/24 11:00
CRDT- 2000/04/12 09:00
PHST- 2000/04/12 09:00 [pubmed]
PHST- 2000/06/24 11:00 [medline]
PHST- 2000/04/12 09:00 [entrez]
AID - bi992652+ [pii]
AID - 10.1021/bi992652+ [doi]
PST - ppublish
SO  - Biochemistry. 2000 Apr 18;39(15):4415-22. doi: 10.1021/bi992652+.