Studies of heme degradation mechanism by heme oxygenase based on the 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，以牺牲分子氧为代价催化血红素的生理降解。在这个项目中我们得到了结果(1)-(3)。(1)通过表面等离子体共振研究了NADP(H)对HO-1与CPR相互作用的影响，并通过HO-1/CPR配合物的计算机模拟进行了分析。Arg-185的胍基位于NADPH 2'-磷酸的氢键距离内，表明Arg-185通过与磷酸基的静电相互作用参与了CPR的结合。另一方面，Lys-149靠近CPR的FMN结合位点附近的酸性氨基酸簇。因此，Lys-149和Lys-153似乎以这样的方式与CPR相互作用，从而定向氧化还原伙伴，以实现从CPR的FMN到HO-1的血红素的最佳电子转移。(2)为了探讨心肺复苏术的电子传递途径，我们利用大鼠FMN-de - More complete CPR检测了大鼠HO-1（rHO-1）对血红素的降解。通过对CPR突变体Y140A/Y178A进行2 M KBr透析，制备了fmn缺失的CPR。将铁血红素还原为亚铁态的第一个电子必须由CPR的FMN提供。将胆绿素转化为铁-胆绿素-铁螯合物所需的电子可由FAD或NADPH通过丙酸血红素提供，还原铁-胆绿素并释放亚铁和胆绿素的最终电子应由CPR的FMN提供。(3)采用快速扫描停流法检测α-羟基血红素与水溶性大鼠血红素加氧酶-1的铁态和铁态o_2依赖性反应。铁α-羟基血红素与O_2反应生成铁α-羟基血红素，反应速率常数为4x10^5 M^<-1>s^<-1>。当pH值为6.5时，反应速率常数降至2.8x10^5 M^<-1>s^<-1>，表明O_2直接攻击α-羟基血红素的p中性自由基形式（α-羟基去质子化）。α-羟基血红素亚铁与O_2反应生成了双相氧化亚铁，该反应涉及一种新的中间体，在415和815 nm处具有最大吸收。两步反应的速率常数分别为68和145 s^<-1>。这些结果表明，在生理条件下α-羟血红素转化为铁素的速度远快于配位铁的还原（<1 s^<-1>），这表明在体内，铁α-羟血红素转化为铁铁的铁素先于铁α-羟血红素的还原。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 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'-phosphate 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. (2)To investigate the electron transfer pathway from CPR, we examined the heme degradation by rat HO-1(rHO-1)using rat FMN-de … More pleted CPR. The FMN-depleted CPR was prepared by dialyzing the CPR mutant, Y140A/Y178A, against 2 M KBr. The first electron to reduce the ferric heme iron to the ferrous state must be supplied from FMN of CPR. The electrons for conversion of verdoheme to ferric biliverdin-iron chelate can be supplied from FAD or NADPH via heme propionates, and that the final electron to reduce ferric biliverdin, leading to the release of ferrous iron and biliverdin, should be provided from FMN of CPR. (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. Ferric α-hydroxyheme reacted with O_2 to form ferric verdoheme with an O_2-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 O_2 on the p-neutral radical form of α-hydroxyheme, which is generated by deprotonation of the α-hydroxy group. The reaction of ferrous α-hydroxyheme with O_2 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 α-hydroxyheme to verdoheme is much faster than the reduction of coordinated iron(<1 s^<-1>)under physiological conditions suggesting that, in vivo, the conversion of ferric α-hydroxyheme to ferric verdoheme precedes the reduction of ferric α-hydroxyheme. Less