A kinetic study of the mechanism of conversion of alpha-hydroxyheme to verdoheme while bound to heme oxygenase.
O2-dependent reactions of the ferric and ferrous forms of alpha-hydroxyheme complexed with water-soluble rat heme oxygenase-1 were examined by rapid-scan stopped-flow measurements. Ferric alpha-hydroxyheme reacted with O2 to form ferric verdoheme with an O2-dependent rate constant of 4x10(5) M(-1) s(-1) at pH 7.4 and 9.0. A decrease of the rate constant to 2.8x10(5) M(-1) s(-1) at pH 6.5 indicates that the reaction proceeds by direct attack of O2 on the pi-neutral radical form of alpha-hydroxyheme, which is generated by deprotonation of the alpha-hydroxy group. The reaction of ferrous alpha-hydroxyheme with O2 yielded ferrous verdoheme in a biphasic fashion involving a new intermediate having absorption maxima at 415 and 815 nm. The rate constants for this two-step reaction were 68 and 145 s(-1). These results show that conversion of alpha-hydroxyheme to verdoheme is much faster than the reduction of coordinated iron (<1 s(-1)) under physiological conditions [Y. Liu, P.R. Ortiz de Montellano, Reaction intermediates and single turnover rate constants for the oxidation of heme by human heme oxygenase-1, J. Biol. Chem. 275 (2000) 5297-5307], suggesting that, in vivo, the conversion of ferric alpha-hydroxyheme to ferric verdoheme precedes the reduction of ferric alpha-hydroxyheme.
Sakamoto
Hiroshi
H
Department of Medical Biochemistry, Kurume University School of Medicine, Kurume, Japan. sakakan@bio.kyutech.ac.jp
Takahashi
Kenichi
K
Higashimoto
Yuichiro
Y
Harada
Saori
S
Palmer
Graham
G
Noguchi
Masato
M
D000818:Animals; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D051381:Rats; D013053:Spectrophotometry
C099546:alpha-hydroxyheme; C099547:verdoheme; D006418:Heme; D007501:Iron; D006419:Heme Oxygenase (Decyclizing); C491523:Hmox1 protein, rat