RESUMO
Genetic variations in POR, encoding NADPH-cytochrome P450 oxidoreductase (CYPOR), can diminish the function of numerous cytochromes P450, and also have the potential to block degradation of heme by heme oxygenase-1 (HO-1). Purified full-length human CYPOR, HO-1, and biliverdin reductase were reconstituted in lipid vesicles and assayed for NADPH-dependent conversion of heme to bilirubin. Naturally-occurring human CYPOR variants queried were: WT, A115V, Y181D, P228L, M263V, A287P, R457H, Y459H, and V492E. All CYPOR variants exhibited decreased bilirubin production relative to WT, with a lower apparent affinity of the CYPOR-HO-1 complex than WT. Addition of FMN or FAD partially restored the activities of Y181D, Y459H, and V492E. When mixed with WT CYPOR, only the Y181D CYPOR variant inhibited heme degradation by sequestering HO-1, whereas Y459H and V492E were unable to inhibit HO-1 activity suggesting that CYPOR variants might have differential binding affinities with redox partners. Titrating the CYPOR-HO-1 complex revealed that the optimal CYPOR:HO-1 ratio for activity was 1:2, lending evidence in support of productive HO-1 oligomerization, with higher ratios of CYPOR:HO-1 showing decreased activity. In conclusion, human POR mutations, shown to impact P450 activities, also result in varying degrees of diminished HO-1 activity, which may further complicate CYPOR deficiency.
Assuntos
Heme Oxigenase-1/química , Complexos Multienzimáticos/química , Mutação de Sentido Incorreto , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , Multimerização Proteica , Mononucleotídeo de Flavina/química , Mononucleotídeo de Flavina/genética , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Heme/química , Heme/genética , Heme/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Humanos , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismoRESUMO
Heme oxygenase-1 (HO-1) catalyzes the oxidative degradation of heme to biliverdin, carbon monoxide, and free iron in a reaction requiring the interaction of HO-1 with NADPH-cytochrome P450 reductase (CPR). HO-1 is bound to the endoplasmic reticulum by 23 C-terminal amino acids; however, a soluble HO-1 (sHO-1) lacking this membrane spanning region has been extensively studied. The goal of this project was to characterize the effect of the C-terminal hydrophobic domain on formation of the HO-1/CPR complex. Full-length HO-1 was shown to exhibit higher reaction rates than sHO-1, particularly at subsaturating CPR, indicating that the C-terminal region influences HO-1 binding to CPR. The increased activity of HO-1 was attributable to a time-dependent formation of a low K(m) HO-1/CPR complex that was not seen with sHO1. Gel filtration analysis confirmed the formation of multiple high molecular weight complexes in the presence and absence of the synthetic lipid dilauroylphosphatidylcholine (DLPC). However, the largest complex appeared following a 2 h incubation of HO-1 and CPR in DLPC, suggesting that the C-terminal region was required for the high-affinity HO-1/CPR complex formation and membrane incorporation. These data demonstrate that the C-terminal region of HO-1 influenced complex formation and ultimately its affinity for CPR.