How does heme degrade ? -Studies of the catalytic mechanism of heme oxygenase based on its crystal structures-

点击翻译按钮获取中文摘要

Heme oxygenase-1 (HO-1) catalyzes the physiological degradation of heme at the expense of molecular oxygen using electrons donated by NADPH-cytochrome P450 reductase (CPR). We obtained the results (1)-(3) in this project.(1) We investigated whether or not hydroxylamine (HA) and hydrazine (HZ) interact with heme bound to heme oxygenase-1. Anaerobic addition of either HA or HZ to the ferric heme-enzyme complex produced a low-spin heme species. Titration studies at different pHs revealed that the neutral form of each of HA and HZ selectively binds to the heme with dissociation constants of 9.8 and 1.8 mM, respectively. Electron spin resonance analysis suggested that the nitrogen atom of each amine is coordinated to the ferric heme iron.(2) We investigated the effect of NADP(H) on the interaction of HO-1 with CPR by surface plasmon resonance and analyzed by computer modeling of the HO-1/CPR complex. The guanidino group of Arg-185 is located within the hydrogen bonding distance of 2'-phosph … More ate of NADPH, suggesting that Arg-185 contributes to the binding to CPR through an electrostatic interaction with the phosphate group. On the other hand, Lys-149 is close to a cluster of acidic amino acids near the FMN binding site of CPR. Thus, Lys-149 and Lys-153 appear to interact with CPR in such a way as to orient the redox partners for optimal electron transfer from FMN of CPR to heme of HO-1.(3) O_2-dependent reactions of the ferric and ferrous forms of α-hydroxyheme complexed with water-soluble rat heme oxygenase-1 were examined by rapid-scan stopped-flow measurements. We demonstrate that either the ferric or ferrous form of α-hydroxyheme can be converted to verdoheme in O_2-depending manner in vitro. Ferric α-hydroxyheme is converted to ferric verdoheme by direct attack of 0_2 on the π-neutral radical form and ferrous α-hydroxyheme is converted to ferrous verdoheme via the 815-nm species. In view of the ambient oxygen concentration and efficiency of NADPH-cytochrome P450 reductase system in tissues, we consider that the ferric α-hydroxyheme to ferric verdoheme pathway prevails in vivo. Less