Mutations of human cytochrome P450 reductase differentially modulate heme oxygenase-1 activity and oligomerization |
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Authors: | Marohnic Christopher C Huber Iii Warren J Patrick Connick J Reed James R McCammon Karen Panda Satya P Martásek Pavel Backes Wayne L Masters Bettie Sue S |
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Institution: | aThe University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229, USA;bThe Louisiana State University Health Science Center, Department of Pharmacology and Stanley S. Scott Cancer Center, New Orleans, LA 70112, USA;cCharles University, 1st School of Medicine, Department of Pediatrics, 121 09 Prague, Czech Republic |
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Abstract: | 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. |
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Keywords: | CYPOR HO-1 P450 Reductase Heme Oxygenase POR deficiency |
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