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。我们在本项目中得到了（1）-（3）的结果。(1)we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复合物的计算机模拟进行了分析。Arg-185的胍基位于NADPH的2 '-磷酸的氢键距离内，表明Arg-185通过与磷酸基团的静电相互作用有助于与CPR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，从而定向氧化还原伴侣，以实现从CPR的FMN到HO-1的血红素的最佳电子转移。(2)To为了研究CPR的电子传递途径，我们用大鼠FMN-de检测了大鼠HO-1（rHO-1）对血红素的降解， 关于我们 做了心肺复苏通过用2 M KBr透析CPR突变体Y140 A/Y178 A来制备耗尽了FMN的CPR。还原亚铁血红素铁到亚铁态的第一个电子必须由CPR的FMN提供。用于将绿血红素转化为铁胆绿素-铁螯合物的电子可以由FAD或NADPH通过血红素丙酸盐提供，并且还原铁胆绿素的最终电子，导致亚铁和胆绿素的释放，应该由CPR的FMN提供。(3)O用快速扫描停流法研究了α-羟基血红素的铁和亚铁形式与水溶性大鼠血红素加氧酶-1络合的反应。在pH 7.4和9.0时，α-羟基血红素铁与O_2反应生成绿血红素铁，其依赖于O_2的速率常数为4 × 10^5 M^<-1>s^<-1>。在pH6.5时，反应速率常数下降到2.8 × 10 ~（^5）M ~<-1>（^8<-1>），表明反应是通过O_2直接攻击α-羟基血红素的p-中性自由基形式进行的，而α-羟基血红素是由α-羟基去质子化产生的。亚铁α-羟基血红素与O_2反应生成亚铁绿血红素，并在415和815 nm处有一个新的吸收峰。该两步反应的速率常数为68和145 s-1<-1>。这些结果表明，在生理条件下，α-羟基血红素向绿血红素的转化比配位铁的还原快得多（&lt;1 s +1<-1>），表明在体内，铁α-羟基血红素向铁绿血红素的转化先于铁α-羟基血红素的还原。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