title: Involvement of NADPH in the interaction between heme oxygenase-1 and cytochrome P450 reductase.
abstract: 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 microm) than in its absence (K(D) = 2.4 microm). 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 microm). The affinities of K149A and K149A/K153A for CPR were decreased 7- and 9-fold (K(D) = 16.8 and 21.8 microm), 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 microm, 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 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.
authors.affiliation: Department of Medical Biochemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Higashimoto
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakamoto
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Hayashi
authors.forename: Masakazu
authors.initials: M
authors.lastname: Sugishima
authors.forename: Keiichi
authors.initials: K
authors.lastname: Fukuyama
authors.forename: Graham
authors.initials: G
authors.lastname: Palmer
authors.forename: Masato
authors.initials: M
authors.lastname: Noguchi
mesh_terms: D000818:Animals; D001665:Binding Sites; D006419:Heme Oxygenase (Decyclizing) / Q000737:chemistry / Q000378:metabolism*; D051547:Heme Oxygenase-1; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D055672:Static Electricity; D020349:Surface Plasmon Resonance
chemical_list: D009249:NADP; D006419:Heme Oxygenase (Decyclizing); D051547:Heme Oxygenase-1; D009251:NADPH-Ferrihemoprotein Reductase