Coupling of Redox and Structural States in Cytochrome P450 Reductase Studied by Molecular Dynamics Simulation

Iijima, Mikuru; Ohnuki, Jun; Sato, Takato; Sugishima, Masakazu; Takano, Mitsunori

doi:10.1038/s41598-019-45690-2

Cited by 13 publications

(13 citation statements)

References 47 publications

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“…HMBS was immersed in a truncated octahedral unit cell containing 10843 water molecules, 29 potassium ions, and 21 chloride ions. The whole system was initially equilibrated in the same way as in our previous study [ 39 ]. In the production run, multiple 0.7-μs-long simulations (16 independent simulations of total 11.2 μs) were conducted at isothermal (310 K) and isobaric (0.1 MPa) conditions.…”

Section: Methodsmentioning

confidence: 99%

Crystal structures of hydroxymethylbilane synthase complexed with a substrate analog: a single substrate-binding site for four consecutive condensation steps

Satō

Sugishima

Tsukaguchi

et al. 2021

Biochemical Journal

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Hydroxymethylbilane synthase (HMBS), which is involved in the heme biosynthesis pathway, has a dipyrromethane cofactor and combines four porphobilinogen (PBG) molecules to form a linear tetrapyrrole, hydroxymethylbilane. Enzyme kinetic study of human HMBS using a PBG-derivative, 2-iodoporphobilinogen (2-I-PBG), exhibited noncompetitive inhibition with the inhibition constant being 5.4 ± 0.3 µM. To elucidate the reaction mechanism of HMBS in detail, crystal structure analysis of 2-I-PBG-bound holo-HMBS and its reaction intermediate possessing two PBG molecules (ES2), and inhibitor-free ES2 was performed at 2.40, 2.31, and 1.79 Å resolution, respectively. Their overall structures are similar to that of inhibitor-free holo-HMBS, and the differences are limited near the active site. In both 2-I-PBG-bound structures, 2-I-PBG is located near the terminus of the cofactor or the tetrapyrrole chain. The propionate group of 2-I-PBG interacts with the side chain of Arg173, and its acetate group is associated with the side chains of Arg26 and Ser28. Furthermore, the aminomethyl group and pyrrole nitrogen of 2-I-PBG form hydrogen bonds with the side chains of Gln34 and Asp99, respectively. These amino acid residues form a single substrate-binding site, where each of the four PBG molecules covalently binds to the cofactor (or oligopyrrole chain) consecutively, ultimately forming a hexapyrrole chain. Molecular dynamics simulation of the ES2 intermediate suggested that the thermal fluctuation of the lid and cofactor-binding loops causes substrate recruitment and oligopyrrole chain shift needed for consecutive condensation. Finally, the hexapyrrole chain is hydrolyzed self-catalytically to produce hydroxymethylbilane.

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Section: Methodsmentioning

confidence: 99%

Crystal structures of hydroxymethylbilane synthase complexed with a substrate analog: a single substrate-binding site for four consecutive condensation steps

Satō

Sugishima

Tsukaguchi

et al. 2021

Biochemical Journal

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“…Hence, the complete conformational transition likely takes place beyond the timescale of our simulations. Further, a recent study implied the closed conformation to be energetically favored [11], rendering it unlikely to observe a transition in the microsecond timescale despite our efforts promoting it.…”

Section: Open Conformation Of the Cprmentioning

confidence: 96%

“…In the aforementioned conformational conversion, it is thought that the FMN domain undergoes a rotational or stretching motion caused by adaptations of the hinge region [6,9,10]. Further, the redox state of the cofactors and the salt concentration of the surrounding environment were proposed to be involved in the conformational transition [7,9,11]. As the CPR is the main redox partner serving electrons to all microsomal CYPs [6], conformational adaptations to fine-tune these individual interactions are necessary.…”

Section: Introductionmentioning

confidence: 99%

Conformational Landscape of Cytochrome P450 Reductase Interactions

Sellner

Fischer

Don

et al. 2021

IJMS

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Oxidative reactions catalyzed by Cytochrome P450 enzymes (CYPs), which constitute the most relevant group of drug-metabolizing enzymes, are enabled by their redox partner Cytochrome P450 reductase (CPR). Both proteins are anchored to the membrane of the endoplasmic reticulum and the CPR undergoes a conformational change in order to interact with the respective CYP and transfer electrons. Here, we conducted over 22 microseconds of molecular dynamics (MD) simulations in combination with protein–protein docking to investigate the conformational changes necessary for the formation of the CPR–CYP complex. While some structural features of the CPR and the CPR–CYP2D6 complex that we highlighted confirmed previous observations, our simulations revealed additional mechanisms for the conformational transition of the CPR. Unbiased simulations exposed a movement of the whole protein relative to the membrane, potentially to facilitate interactions with its diverse set of redox partners. Further, we present a structural mechanism for the susceptibility of the CPR to different redox states based on the flip of a glycine residue disrupting the local interaction network that maintains inter-domain proximity. Simulations of the CPR–CYP2D6 complex pointed toward an additional interaction surface of the FAD domain and the proximal side of CYP2D6. Altogether, this study provides novel structural insight into the mechanism of CPR–CYP interactions and underlying conformational changes, improving our understanding of this complex machinery relevant for drug metabolism.

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“…These data suggest that the “open–close” transition of CPR is necessary for a smooth electron transfer. Computational simulation provided evidence of the “open–close” transition of CPR with the redox change in its coenzymes [ 28 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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Coupling of Redox and Structural States in Cytochrome P450 Reductase Studied by Molecular Dynamics Simulation

Cited by 13 publications

References 47 publications

Crystal structures of hydroxymethylbilane synthase complexed with a substrate analog: a single substrate-binding site for four consecutive condensation steps

Crystal structures of hydroxymethylbilane synthase complexed with a substrate analog: a single substrate-binding site for four consecutive condensation steps

Conformational Landscape of Cytochrome P450 Reductase Interactions

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

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