Conformational Equilibrium of NADPH–Cytochrome P450 Oxidoreductase Is Essential for Heme Oxygenase Reaction

Abstract

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.

Inhibition of MAGL activates the Keap1/Nrf2 pathway to attenuate glucocorticoid‐induced osteonecrosis of the femoral head

“…[50][51][52][53] The NADPH/NADP ratio is downregulated by a significant upregulation of NQO1 and HO1, which then leads to a reduction in NOX activity. 54,55 In addition, products of HO1 metabolism, namely, biliverdin and CO, are potent antioxidants. 56 GC suppresses Nrf2 transcription, whereas Nrf2 activation can significantly reduce GC-induced oxidative stress in osteoblasts.…”

“…Nrf2, a major regulator of intracellular antioxidants, can directly reduce ROS generation by increasing the levels of ROS‐scavenging enzymes, or by indirectly inhibiting NOX activation by increasing the expression of downstream targets, such as NQO1 and HO1 50–53 . The NADPH/NADP ratio is downregulated by a significant upregulation of NQO1 and HO1, which then leads to a reduction in NOX activity 54,55 . In addition, products of HO1 metabolism, namely, biliverdin and CO, are potent antioxidants 56 .…”

Complex Formation of Heme Oxygenase-2 with Heme Is Competitively Inhibited by the Cytosolic Domain of Caveolin-1

“…To determine the affinity in heterocomplex formation of CAV(1−101) and HO samples, sedimentation equilibrium experiments were performed. 38 Comparison of the sedimentation behaviors of CAV(1−101) with tHO-2 or F226A/Y228A/F233A is shown in Figure 3. A complex model was well fitted to the data with small and symmetrically distributed residuals (Figure 3, top panels).…”

“…To determine the affinity in heterocomplex formation of CAV(1–101) and HO samples, sedimentation equilibrium experiments were performed . Comparison of the sedimentation behaviors of CAV(1–101) with tHO-2 or F226A/Y228A/F233A is shown in Figure .…”

“…Sedimentation equilibrium analytical ultracentrifugation was performed as previously reported. 38 Data were collected at 25 °C in 0.1 M potassium phosphate buffer (pH 7.4) using an Optima XL-A analytical ultracentrifuge (Beckman Coulter, Brea, CA) with an An-60 Ti rotor; data were processed using ProteomeLab XL-A software (Beckman Coulter). Mixtures of the CAV(1−101) and equimolar HO-2 samples, i.e., tHO-2 or F226A/Y228A/ F233A, were subjected to sedimentation equilibrium analysis.…”