Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction
Abstract
:1. Introduction
2. Materials and Methods
2.1. Protein Expression and Purification
2.2. Enzymatic Assay
2.3. Analytical Ultracentrifugation
2.4. Cryo-Electron Microscopy (EM), Image Processing, and Molecular Modeling of the rCPR–Heme–rHO-1 Complex
3. Results
3.1. Enzymatic Assay
3.2. Reduction Kinetics
3.3. Complex Formation between CPRs and Heme–rHO-1 Evaluated by Ultracentrifugation
3.4. Cryo-EM Structure of the rCPR–Heme–rHO-1 Complex
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Reduction System | HO Activity | Apparent Reduction Rate Constant | ||
---|---|---|---|---|
(min−1) | (%) | (min−1) | (%) | |
rCPR | 1.96 ± 0.35 | 100 | 122 ± 3.8 | 100 |
147CC514 | 0.229 ± 0.0037 | 11.7 ± 0.19 | 23.9 ± 5.1 | 19.6 ± 4.2 |
DTT-treated rCPR | ND | ND | 130 ± 10.8 | 107 ± 8.9 |
DTT-treated 147CC514 | ND | ND | 112 ± 23.6 | 91.8 ± 19 |
IAM/DTT-treated rCPR | 1.17 ± 0.0024 | 59.7 ± 0.12 | ND | ND |
IAM/DTT-treated 147CC514 | 1.25 ± 0.197 | 63.8 ± 10 | ND | ND |
ΔTGEE [24] | ND | ND | 0.250 | 0.205 |
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Sugishima, M.; Taira, J.; Sagara, T.; Nakao, R.; Sato, H.; Noguchi, M.; Fukuyama, K.; Yamamoto, K.; Yasunaga, T.; Sakamoto, H. Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction. Antioxidants 2020, 9, 673. https://doi.org/10.3390/antiox9080673
Sugishima M, Taira J, Sagara T, Nakao R, Sato H, Noguchi M, Fukuyama K, Yamamoto K, Yasunaga T, Sakamoto H. Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction. Antioxidants. 2020; 9(8):673. https://doi.org/10.3390/antiox9080673
Chicago/Turabian StyleSugishima, Masakazu, Junichi Taira, Tatsuya Sagara, Ryota Nakao, Hideaki Sato, Masato Noguchi, Keiichi Fukuyama, Ken Yamamoto, Takuo Yasunaga, and Hiroshi Sakamoto. 2020. "Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction" Antioxidants 9, no. 8: 673. https://doi.org/10.3390/antiox9080673