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

血红素加氧酶-1(HO-1)利用NADPH-细胞色素P450还原酶(CPR)提供的电子，以分子氧为代价催化血红素的生理降解。(1)研究了羟胺(HA)和肼(HZ)与血红素加氧酶-1结合后是否与血红素相互作用。将HA或HZ厌氧添加到铁血红素-酶复合体中，可产生低自旋的血红素物种。在不同pH下的滴定研究表明，HA和HZ的中性形式分别选择性地与血红素结合，解离常数分别为9.8和1.8 mm。电子自旋共振分析表明，每个胺的氮原子都与铁血红素铁配位。(2)利用表面等离子体共振法研究了NaDP(H)对HO-1与CPR相互作用的影响，并对HO-1/CPR复合体进行了计算机模拟分析。精氨酸185中的胍基位于2‘-磷酸…的氢键距离内更多的NADPH，这表明Arg-185通过与磷酸基团的静电相互作用参与了与CPR的结合。另一方面，Lys-149靠近CPR的FMN结合部位附近的一簇酸性氨基酸。因此，Lys-149和Lys-153似乎以这样一种方式与CPR相互作用，使氧化还原伙伴的电子从CPR的FMN最佳地转移到HO-1的血红素。(3)通过快速扫描停流测量研究了铁和铁形式的α-羟基血红素与水溶性大鼠血红素加氧酶-1的络合反应。我们证明了在体外，无论是铁形式还是铁形式的α-羟基血红素都可以通过氧依赖的方式转化为马鞭铁。铁的α-羟基血红素通过0_2对π-中性自由基形式的直接攻击转化为铁的马鞭铁，亚铁的α-羟基血红素通过815 nm的物种转化为亚铁的马鞭铁。鉴于NADPH-细胞色素P450还原酶系统在组织中的环境氧浓度和效率，我们认为在体内存在α铁-羟基血红素到马鞭铁的途径。较少

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