今天推送的文章发表在ACS Catalysis的“Enhancing Local Electric Fields Drives the Proton-Coupled Electron Transfer within Cytochrome P450 ReductasePrecursor”，通讯作者为厦门大学化学化工学院王斌举教授。
Cytochrome P450 enzymes play a key role in biosynthesis and metabolic transformation. Cytochrome P450 reductase (CPR) is a key electron donor for P450 monooxygenase to activate oxygen, but the mechanism of electron transport in CPR is largely unknown. The authors elucidated the electron transport mechanism of CPR through molecular dynamics (MD) and quantum mechanical/molecular mechanics (QM/MM) calculations. First, the electron transfer from FADH− to FMN occurs through the proton-coupled electron transfer (PCET) mechanism. Glu142 transfers one proton to FMN through a two-aqueous molecular chain, and simultaneously transfers electrons from FADH− to FMN. The electron transfer of FADH• to FMNH• is an Asp675-mediated PCET process: ser457 assists in the transfer of protons from FADH to Asp675, coupling the electron transport from FADH• to FMNH. Among them, the local electric field of the double-protonated His180 significantly enhanced the PCET reaction in terms of kinetics and thermodynamics. The authors' research highlights the important role of local electric fields in facilitating bioelectron transport in enzymatic reactions.
A typical p450 catalytic cycle activates oxygen to form an active iron(iv)porphyrin π-radical cation known as compound I (CpdI). Oxygen activation typically requires two electrons, provided by NAD(P)H through continuous electron transfer by redox partners. CPR is the main redox partner of microsomal cytochromes and plays a role in the P450 electron transport process through the nadph-cytochrome P450 reductase (CPR) pathway. CPR is the initial member of the diflavin reductase family and contains two cofactors, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), in a single polypeptide chain. NADPH enters the FAD domain of CPR to facilitate hydrogen transfer. Subsequently, the FAD accepts and sequentially supplies two electrons to the FMN. Finally, the fully reduced FMN (FMNH–) in turn contributes two electrons to P450 to complete the catalytic cycle.
Figure 1: Mechanism of reaction of electron transfer process between NADPH and FMN in cytochrome P450 reductase
There have been a number of experimental studies on electron transfer in CPR, but the specifics of the excitation response, particularly the effects of proton transfer channels and the protein environment on electron transfer, remain unclear. The authors investigated these issues by combining molecular dynamics (MD) simulations and hybrid quantum mechanical/molecular mechanics (QM/MM) simulations. Based on the crystal structure of the brown rat (PDB code: 5urd), the authors studied the initial structure of the transfer of hydrogen ions from NADPH to FAD in P450 reductase. MD simulations showed that the nicotinamide ring of NADPH was superimposed on the isoalloxazine ring of FAD, and based on the MD results, the authors performed a single-point energy calculation by QM/MM. In the QM/mm optimized reactant complex (RC), one hydrogen atom of the NADPH nicotinamide functional group is directed towards the N5 atom of the FAD at a distance of 2.14 Å, which is the ideal bond length for hydride transfer and the calculated activation energy barrier is consistent with the experimental value, and the charge on NADP+ increases to 0.74 after the hydride transfer is completed. The transfer of hydrogen ions from NADPH to FAD may involve hydrogen atom transfer and electron transfer.
FIGURE 2 RELATIVE ENERGY DISTRIBUTION OF HYDROGEN IONS FROM NADPH TO FAD IN QM/MM
Kinetic experiments36,37 show that in the first electron transfer (ET) of FADH−toFMN, the FMN accepts 1 electron to form FMNH•. Therefore, this ET process may be coupled to proton transfer, possibly through the proton-coupled electron transfer (PCET) mechanism. To study the PCET process from FADH− to FMN, the authors constructed a system using a QM/mm optimized product structure for hydrogen ion transfer between NADPH and FAD. Since Glu142 is the closest base residue to the FMN N5 site, it was chosen to be protonated to donate protons during PCET. PROPKA analysis showed that the ratio of His180 to the CPR reduced state was 6.55, which means that His180 may exist in neutral or double deproton form.
FIGURE 3 QM/MM ENERGY PROFILE OF THE PCET PROCESS FROM FADH− TO FADH− IS PROPOSED
Starting from IC2, protons are transferred from protonated Glu142 to N5 of FMN, facilitated by two water molecules (WAT1 and WAT2), simultaneously driving electron transfer from FADH−toFMN, resulting in FADH• and FMNH• in IC3. Mulliken population analysis showed that the spin density of FADH in IC2 was ~0. In IC3, the spin density of FADH increases to 0.99, indicating a successful transfer of electrons from FADH−toFMN. This PCET process leads to the formation of FMNH• and FADH•. The authors showed that the pure electron transfer of FADH−toFMN was endothermic with a heat absorption of 3.88 kcal/mol, indicating that the process was very thermodynamically unfavorable. In the PCET process, proton input can increase the redox potential of FMN, thereby promoting the electron transfer of FADH−toFMN.
To evaluate the effect of His180 protonation on the PCET process, the authors deprotonated His180 in IC2 (resulting in HID) and performed additional QM/MM calculations. After removing a proton from HIP and converting it to HID, the energy barrier of the PCET process increased significantly from 12.4 kcal/mol (Figure 4A) to 19.9 kcal/mol (Figure 5A), a finding consistent with the experimental results. In addition, the energy of the product IC3' is 11.6 kcal/mol higher than that of the intermediate IC2', compared to only 0.46 kcal/mol in Figure 4A. This suggests that the removal of the positive charge of His180 will make the PCET reaction from FADH− to FMN unfavorable both kinetically and thermodynamically. Therefore, the authors propose that positively charged HIP180 significantly promotes the electron transfer from FADH− to FMN.
Fig. 4 Proton-coupled electron transfer for the CPR reaction initiated by FADH− to FMN and HID180 residues
A large number of experiments and theories have shown that the external electric field, or the internal electric field present in some catalysts, can be used to effectively control chemical activity and selectivity. By protonation, a positive charge is added to the imidazole ring of its side chain, which significantly changes the electrostatic environment in which the PCET reaction occurs. The authors detected the electrostatic effects of positively charged HIP180 by Mulliken charge in QM/MM calculations to evaluate the interaction energies between the charged side chains of His180 and PCET reaction complexes, including FADH−, FMN, Glu142, and bridging water molecules.
Fig.5 Electrostatic interaction energy between the charged side chain in HIP180 and the PCET reaction complex in (A) IC2, (B) TS3 and (C) IC3.
The static interaction of positively charged imidazole rings in TS3 and IC3 with PCET-reactive complexes is more favorable compared to IC2. In the PCET reaction, charge redistribution transfers the negative charge from FADH−toGlu142 and from IC2 to IC3. Protonation of His180 during PCET brings the reaction complex negatively charged closer to the positively charged imidazole ring, resulting in electrostatic stabilization of TS3 and IC3 (as shown in Figures 6A and 6C). The electrostatic interaction analysis was consistent with the QM/MM results, and the protonation of His180 reduced the PCET energy barrier by 7.5 kcal/mol and the endothermic properties of the reaction by 11.1 kcal/mol. The results show that the electric field of the charged imidazole ring in protonated HIP180 is favorable for the reaction, and the electrostatic stability in IC3 is better than that of TS3. This explains why the protonation of His180 significantly reduces the PCET reaction energy more than the reaction barrier. After the formation of FMNH• in IC3, our MD simulations showed that the CO group of the backbone of Gly141 was close to FMN, forming hydrogen bonds with FMNH•. In view of the fact that the two-electron reduced CPR structure with FADH•/FMNH• may have similar active site structure and protein conformation to the four-electron reduced CPR structure, we subsequently performed a QM/MM study on the PCET process from FADH• to FMNH• based on the crystal structure of the four-electron reduced CPR of Brown mouse (PDB ID: 5ure).
FIGURE 6 QM/MM ENERGY DISTRIBUTION OF THE PCET PROCESS FROM FADH• TO FMNH•
In the QM/mm optimized IC4, there is a proton channel between the H1 atom of FADH • and the O atom of Asp675, which is bridged by Ser457 (Figure 7B). Starting from IC4, protons are transferred from FADH• to Asp675, which, with the assistance of Ser457, facilitates the transfer of electrons to FMNH•, resulting in FAD and FMNH−inIC5 (Figure 7A). This process encountered an energy barrier of about 11.7 kcal/mol. The predicted value pKa of His180 in the fully reduced CPR state was 7.4, indicating that His180 was still in a diprotonated state.
For comparison, the PCET process from FADH• to FMNH• was analyzed using QM/MM calculations, where His180 was the neutral form (HID180). Under these conditions, the calculated energy barrier rises to 15.1 kcal/mol, and the product IC6′ is 6.7 kcal/mol higher than that of the intermediate product IC4′.
Summary:
THE AUTHORS INVESTIGATED THE PRIMING RESPONSE IN CPR THROUGH EXTENSIVE MD SIMULATIONS AND QM/MM CALCULATIONS. The author's simulation results are as follows:
(1) The transfer of NADPH to FAD occurs through hydrogen atom transfer reactions and electron transfer.
(II) ELECTRON TRANSFER FROM FADH− TO FMN IS CARRIED OUT BY THE PCET MECHANISM, IN WHICH Glu142 TRANSFERS ONE PROTON TO FMN, THROUGH A WATER CHAIN OF TWO MOLECULES, AND FADH− TRANSFERS ONE ELECTRON AT THE SAME TIME, FORMING FADH•/FMNH• RADICALS.
(III)随后从FADH•到FMNH•的ET涉及Asp675介导的PCET, ser457辅助质子从FADH•转移到Asp675，再加上电子转移，导致FAD和FMNH−。
(IV) THE PROTONATION STATE OF His180 SIGNIFICANTLY AFFECTS THE PROTON-COUPLED ELECTRON TRANSFER OF FADH−toFMN.