Involvement of NADP(H) in the interaction between heme oxygenase-1 and cytochrome P450 reductase

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.