Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction
Heme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH–cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium betwee...
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doaj-c3addd8353f943f18e8dedeab21cb5542020-11-25T03:27:43ZengMDPI AGAntioxidants2076-39212020-07-01967367310.3390/antiox9080673Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase ReactionMasakazu Sugishima0Junichi Taira1Tatsuya Sagara2Ryota Nakao3Hideaki Sato4Masato Noguchi5Keiichi Fukuyama6Ken Yamamoto7Takuo Yasunaga8Hiroshi Sakamoto9Department of Medical Biochemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, JapanDepartment of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka 820-8502, JapanDepartment of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka 820-8502, JapanDepartment of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka 820-8502, JapanDepartment of Medical Biochemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, JapanDepartment of Medical Biochemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, JapanDepartment of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka 560-0043, JapanDepartment of Medical Biochemistry, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, JapanDepartment of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka 820-8502, JapanDepartment of Bioscience and Bioinformatics, Graduate School of Computer Science and Systems Engineering, Kyushu Institute of Technology, 680-4 Kawazu, Iizuka 820-8502, JapanHeme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH–cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium between the open and the closed forms of CPR. We previously demonstrated that the open-form stabilized CPR (ΔTGEE) is tightly bound to heme–HO-1, whereas the reduction in heme–HO-1 coupled with ΔTGEE is considerably slow because the distance between FAD and FMN in ΔTGEE is inappropriate for electron transfer from FAD to FMN. Here, we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514). Additionally, we analyzed the interaction between 147CC514 and heme–HO-1 by analytical ultracentrifugation. The results indicate that the interaction between 147CC514 and heme–HO-1 is considerably weak, and the enzymatic activity of 147CC514 is markedly weaker than that of CPR. Further, using cryo-electron microscopy, we confirmed that the crystal structure of ΔTGEE in complex with heme–HO-1 is similar to the relatively low-resolution structure of CPR complexed with heme–HO-1 in solution. We conclude that the “open–close” transition of CPR is indispensable for electron transfer from CPR to heme–HO-1.https://www.mdpi.com/2076-3921/9/8/673analytical ultracentrifugecryo-electron microscopyelectron transferprotein–protein interaction |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Masakazu Sugishima Junichi Taira Tatsuya Sagara Ryota Nakao Hideaki Sato Masato Noguchi Keiichi Fukuyama Ken Yamamoto Takuo Yasunaga Hiroshi Sakamoto |
spellingShingle |
Masakazu Sugishima Junichi Taira Tatsuya Sagara Ryota Nakao Hideaki Sato Masato Noguchi Keiichi Fukuyama Ken Yamamoto Takuo Yasunaga Hiroshi Sakamoto Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction Antioxidants analytical ultracentrifuge cryo-electron microscopy electron transfer protein–protein interaction |
author_facet |
Masakazu Sugishima Junichi Taira Tatsuya Sagara Ryota Nakao Hideaki Sato Masato Noguchi Keiichi Fukuyama Ken Yamamoto Takuo Yasunaga Hiroshi Sakamoto |
author_sort |
Masakazu Sugishima |
title |
Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction |
title_short |
Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction |
title_full |
Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction |
title_fullStr |
Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction |
title_full_unstemmed |
Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction |
title_sort |
conformational equilibrium of nadph–cytochrome p450 oxidoreductase is essential for heme oxygenase reaction |
publisher |
MDPI AG |
series |
Antioxidants |
issn |
2076-3921 |
publishDate |
2020-07-01 |
description |
Heme oxygenase (HO) catalyzes heme degradation using electrons supplied by NADPH–cytochrome P450 oxidoreductase (CPR). Electrons from NADPH flow first to FAD, then to FMN, and finally to the heme in the redox partner. Previous biophysical analyses suggest the presence of a dynamic equilibrium between the open and the closed forms of CPR. We previously demonstrated that the open-form stabilized CPR (ΔTGEE) is tightly bound to heme–HO-1, whereas the reduction in heme–HO-1 coupled with ΔTGEE is considerably slow because the distance between FAD and FMN in ΔTGEE is inappropriate for electron transfer from FAD to FMN. Here, we characterized the enzymatic activity and the reduction kinetics of HO-1 using the closed-form stabilized CPR (147CC514). Additionally, we analyzed the interaction between 147CC514 and heme–HO-1 by analytical ultracentrifugation. The results indicate that the interaction between 147CC514 and heme–HO-1 is considerably weak, and the enzymatic activity of 147CC514 is markedly weaker than that of CPR. Further, using cryo-electron microscopy, we confirmed that the crystal structure of ΔTGEE in complex with heme–HO-1 is similar to the relatively low-resolution structure of CPR complexed with heme–HO-1 in solution. We conclude that the “open–close” transition of CPR is indispensable for electron transfer from CPR to heme–HO-1. |
topic |
analytical ultracentrifuge cryo-electron microscopy electron transfer protein–protein interaction |
url |
https://www.mdpi.com/2076-3921/9/8/673 |
work_keys_str_mv |
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