Involvement of NADP(H) in the interaction between heme oxygenase-1 and cytochrome P450 reductase
Involvement of NADP(H) in the interaction between heme oxygenase-1 and cytochrome P450 reductase
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DOI：
10.1074/jbc.m406203200
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发表时间：
2005-01-07
影响因子：
4.8
通讯作者：
Noguchi, M
中科院分区：
文献类型：
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作者：
Higashimoto, Y;Sakamoto, H;Noguchi, M
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。本研究利用表面等离子体共振技术研究了NADP（H）对HO-1与CPR相互作用的影响。我们发现HO-1在NADP（+）存在时（K-D = 0.5 μ M）比在NADP（+）不存在时（K-D = 2.4 μ M）更紧密地与CPR相关。HO-1突变体K149 A、K149 A/K153 A和R185 A在NADPH-CPR下几乎没有血红素降解活性，而在使用抗坏血酸钠时，它们表现出与野生型相当的活性。与野生型相比，R185 A显示出对CPR的亲和力降低了100倍，即使在NADP（+）存在下（K-D = 36.3 μ M）。K149 A和K149 A/K153 A对CPR的亲和力分别降低了7倍和9倍（K-D = 16.8和21.8 μ M）。与R185 A相反，K149 A和K149 A/K153 A的亲和力通过加入NADP（+）而提高（K-D分别为5.2和9.6 μ M），与野生型的情况一样。HO-1/CPR复合物的计算机模拟显示，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而有助于与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，以定向氧化还原配偶体，以实现从CPR的FMN到HO-1的血红素的最佳电子转移。
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). In this study, we investigated the effect of NADP( H) on the interaction of HO-1 with CPR by surface plasmon resonance. We found that HO-1 associated with CPR more tightly in the presence of NADP(+) (K-D = 0.5 muM) than in its absence (K-D = 2.4 muM). The HO-1 mutants, K149A, K149A/K153A, and R185A, showed almost no heme degradation activity with NADPH-CPR, whereas they exhibited activity comparable to that of the wild type when sodium ascorbate was used. R185A showed a 100-fold decreased affinity for CPR compared with wild type, even in the presence of NADP(+) (K-D = 36.3 muM). The affinities of K149A and K149A/K153A for CPR were decreased 7- and 9-fold (K-D = 16.8 and 21.8 muM), respectively. In contrast to R185A, the affinities of K149A and K149A/K153A were improved by the addition of NADP(+) (K-D = 5.2 and 9.6 muM, respectively), as was the case with wild type. Computer modeling of the HO-1/CPR complex showed that the guanidino group of Arg(185) is located within the hydrogen bonding distance of 2'-phosphate of NADPH, suggesting that Arg185 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.