Full-length structure of CPR-containing self-sufficient cytochrome P450

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Rey-Ting Guo doi: https://doi.org/10.1101/2025.11.29.689641 Zhenzhen Xie 1 Zhejiang Key Laboratory of Medical Epigenetics, Hubei Hongshan Laboratory, Department of Immunology and Pathogen Biology, School of Basic Medical Sciences, Hangzhou Normal University , Hangzhou, 311121, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ziwei Liu 1 Zhejiang Key Laboratory of Medical Epigenetics, Hubei Hongshan Laboratory, Department of Immunology and Pathogen Biology, School of Basic Medical Sciences, Hangzhou Normal University , Hangzhou, 311121, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Siyu Li 1 Zhejiang Key Laboratory of Medical Epigenetics, Hubei Hongshan Laboratory, Department of Immunology and Pathogen Biology, School of Basic Medical Sciences, Hangzhou Normal University , Hangzhou, 311121, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kangwei Xu 2 School of Pharmaceutical Sciences, Sun Yat-sen University , Guangzhou 510006, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jian-Wen Huang 1 Zhejiang Key Laboratory of Medical Epigenetics, Hubei Hongshan Laboratory, Department of Immunology and Pathogen Biology, School of Basic Medical Sciences, Hangzhou Normal University , Hangzhou, 311121, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jian Min 3 School of Life Sciences, Hubei University , Wuhan, 430062, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Qiru Li 3 School of Life Sciences, Hubei University , Wuhan, 430062, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jingxue Zhai 3 School of Life Sciences, Hubei University , Wuhan, 430062, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Te Wang 3 School of Life Sciences, Hubei University , Wuhan, 430062, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yutong Wang 1 Zhejiang Key Laboratory of Medical Epigenetics, Hubei Hongshan Laboratory, Department of Immunology and Pathogen Biology, School of Basic Medical Sciences, Hangzhou Normal University , Hangzhou, 311121, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lu Yang 1 Zhejiang Key Laboratory of Medical Epigenetics, Hubei Hongshan Laboratory, Department of Immunology and Pathogen Biology, School of Basic Medical Sciences, Hangzhou Normal University , Hangzhou, 311121, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Junjie Duan 1 Zhejiang Key Laboratory of Medical Epigenetics, Hubei Hongshan Laboratory, Department of Immunology and Pathogen Biology, School of Basic Medical Sciences, Hangzhou Normal University , Hangzhou, 311121, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Junjie Chen 1 Zhejiang Key Laboratory of Medical Epigenetics, Hubei Hongshan Laboratory, Department of Immunology and Pathogen Biology, School of Basic Medical Sciences, Hangzhou Normal University , Hangzhou, 311121, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ruibo Wu 2 School of Pharmaceutical Sciences, Sun Yat-sen University , Guangzhou 510006, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: wurb3{at}mail.sysu.edu.cn cckate0722{at}hznu.edu.cn guoreyting{at}hznu.edu.cn Chun-Chi Chen 1 Zhejiang Key Laboratory of Medical Epigenetics, Hubei Hongshan Laboratory, Department of Immunology and Pathogen Biology, School of Basic Medical Sciences, Hangzhou Normal University , Hangzhou, 311121, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: wurb3{at}mail.sysu.edu.cn cckate0722{at}hznu.edu.cn guoreyting{at}hznu.edu.cn Rey-Ting Guo 1 Zhejiang Key Laboratory of Medical Epigenetics, Hubei Hongshan Laboratory, Department of Immunology and Pathogen Biology, School of Basic Medical Sciences, Hangzhou Normal University , Hangzhou, 311121, PR China Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: wurb3{at}mail.sysu.edu.cn cckate0722{at}hznu.edu.cn guoreyting{at}hznu.edu.cn Abstract Full Text Info/History Metrics Supplementary material Preview PDF Summary Cytochrome P450 (P450s) are heme-thiolate monooxygenases that exploit electrons sourced from pyridine nucleotide to reduce the oxygen and heme iron to catalyze hydroxylation of inactivated C-H bond 1 – 4 . Self-sufficient P450s that contain the substrate-binding heme-domain and the electron-donating NADPH:cytochrome P450 reductase (CPR) domain in the same polypeptide chain are highly effective and have been engineered to catalyze various challenging reactions. The high efficacy attributes to the effective electron transfer rate, but how the electrons travel among the redox centers remains elusive owing to the lack of structural information of the full-length protein. Here, we report the structure of a homologue of the most extensively studied P450BM3 from Shimazuella soli ( So P450) resolved by single particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. So P450 primarily exists as a homodimer formed via the intertwined CPR-domains. The spatial alignment of the heme-domain that is linked via an extensive loop was also determined. Notably, a class of structure that lacks one heme-domain was identified from the cryo-EM analyses, indicating that the heme-domain is mobile. We suspected that the heme-domain could move to reach the CPR-domain for electron acquisition and built a model of putative catalytic state to reveal how the electrons are relayed from NADPH to heme. These results are of fundamental importance to understand the catalytic reaction of CPR-containing self-sufficient P450s, which shall provide critical information to the engineering and applications of these enzymes. Main Cytochrome P450s have attracted much attentions in organic chemistry, pharmaceutical industry and synthetic biology owing to their capability to catalyze many challenging reactions and can be engineered to accept myriad types of substrates 5 – 9 . The action of P450s is to catalyze the reductive scission of molecular oxygen, leading to the insertion of one atom of the dioxygen to the substrate and reduction of the other to water. The activation of the dioxygen is mediated by the highly reactive oxo iron porphyrin known as compound I, which is formed by two electrons that successively flow to the heme 10 , 11 . In majority of P450s, the electrons are sourced by reduced pyridine nucleotide (NADPH or NADH) and transferred to the heme prosthetic by two types of redox partners. One is NADPH:cytochrome P450 reductase (CPR) that contains a FAD and an FMN, and the other is phthalate dioxygen reductase (PFOR) that contains an iron-sulfur cluster and an FMN. P450s are classified as multi-component or self-sufficient systems depending on whether the heme-domain and the redox partners are located on the same gene 12 , 13 . For multi-component P450s, the heme-domain and the ancillary redox proteins are encoded independently such that the addition of cognate or suitable exogenous reductase is generally required 14 , 15 . For self-sufficient P450s, the heme-domain and the redox partner are fused in a single polypeptide thus the searching for a matching reductase is omitted. Compared with multi-component P450s, self-sufficient P450s show higher coupling efficiency and catalytic activity 16 . This superior performance of self-sufficient P450s relies on the integrity of the protein, as reconstituting individual domains of these enzymes drastically reduces the enzyme activity 17 – 20 . These features also make self-sufficient P450s useful frameworks for the construction of artificial chimeras to improve the catalytic activity of multi-component P450s 21 – 26 . There are two types of self-sufficient P450s, one fuses with a CPR-domain and the other a PFOR-domain, which exploit electron transfer route of NADPH→FAD→FMN→heme and NAD(P)H→FMN→[2Fe-2S]→heme, respectively. In 2020, we solved the crystal structure of a PFRO-containing self-sufficient P450 termed CYP116B46 27 . This structure demonstrates that the direct electron transfer can occur between FMN and [2Fe-2S] as the distance between these two redox centers is around 8 Å. But the edge-to-edge distance between [2Fe-2S] and heme is measured to approximately 25 Å, much longer than that is demanded for efficient direct electron transfer. We thus proposed that protein residues that line the connecting tunnel between FMN and heme could serve as media for the tunneling of the long-range electron transfer and applied mutagenesis experiments to validate the role of these residues 27 , 28 . The number of CPR-containing self-sufficient P450s has rapidly expanded since the first member of this superfamily termed CYP102A1 from Bacillus megaterium was identified ( https://cyped.biocatnet.de ) 29 – 31 . This enzyme, also known as P450BM3, is the most extensively investigated P450, which exhibits high coupling rate and turnover rate, and its heme-domain has been engineered to conduct various reactions 32 – 35 . A number of structures of heme-domain, FMN-domain and FAD-domain of P450BM3 and other homologous P450s have been reported 36 , but the steric organization of each domain of the full-length protein so as the mechanism that contributes to the efficient electron transfer remain elusive. It has been proposed that the FMN-domain should undergo conformational change such that the electrons from the reduced FMN could be transferred to the heme 37 . This is mainly based on the structural studies of mammalian CPRs 38 , 39 and a crystal structure of partial P450BM3 fragment that contains the heme- and FMN-domain 40 . It is worth noting that 24 amino acids between the heme- and FMN-domain of P450BM3 are missing in the latter structure, complicating the determination of the domain alignment. It has been demonstrated that P450BM3, as well as its homologs, can dimerize in solution, and the dimer configuration is essential for the enzyme to exert catalytic activity 41 – 45 . Biochemical and mutagenesis experiments indicate that the electrons are transferred from the FAD-domain on one polypeptide to the FMN-domain of the other polypeptide and to the heme-domain that is on the same polypeptide chain that houses the FMN-domain 44 . The dimeric configuration of P450BM3 has been revealed through negative stain electron microscopy images and single particle cryogenic electron (cryo-EM) microscopic analyses 46 , 47 . However, the domain alignment remains uncertain owing to low resolution. In this study, we report the cryo-EM structure and crystal structure of a P450BM3 homologous protein to elucidate the three-dimensional structure of a CPR-containing self-sufficient P450 at atomic level. Results and Discussion Full-length structure of a CPR-containing P450 Our attempt to resolve the structure of P450BM3 failed as no crystal or cryo-EM images that can be used for structure determination was obtained. This is suspected a result of high protein flexibility as mentioned in previous reports 46 , 47 . Since the goal is to explore the full-length structure of CPR-containing self-sufficient P450s, we examined a panel of P450BM3 homologous proteins ( Extended Data Table 1 ) and eventually obtained cryo-EM images of So P450 from Shimazuella soli (60.4 % protein sequence identity to P450BM3) that can be reconstituted for structure determination ( Extended Data Fig. 1 and Extended Data Table 2 ). A main class of the cryo-EM structure of So P450 that was resolved to 2.72 Å displays a dimeric configuration as the cryo-EM maps can be modeled with two heme-domains, two FMN-domains and two FAD-domains, tentatively termed as heme-A, heme-B, FAD-A, FAD-B, FMN-A and FMN-B, respectively ( Fig. 1a ). This is consistent with the size-exclusion chromatographic analyses, which indicate that So P450, resembling P450BM3, exists as a dimer in solution ( Supplementary Fig. 1 ). The spatial location of all domains can be unambiguously determined because the electron densities of heme, FMN and FAD prosthetics were clearly identified ( Fig. 1b ). However, assigning each domain to two individual polypeptide chains is complicated as these domains that appear to intertwine to each other are linked via extensive connecting loops. The connecting regions that link heme- and FMN-domain (N456-N496) and FMN- and FAD-domain (S650-G669) are designated as CR I and CR II , respectively ( Fig. 1a ). For CR II , the electron densities of the first seven amino acids are missing whereas those from D657 to G669 are sufficiently clear for the modeling ( Extended Data Fig. 2a ). Judged from the orientation of CR II , FMN-A and FAD-A (and FMB-B and FAD-B) are on the same polypeptide chain ( Extended Data Fig. 2b ). As such, FMN-A and FAD-B that belong to different polypeptide chains comprise a functional CPR-domain ( Extended Data Fig. 2c and 2d ), with the FMN and FAD juxtapositioned as those in other known CPR structures (see below). Download figure Open in new tab Fig. 1 The domain assignment of cryo-EM structure of So P450. a , Top: scheme of domain distribution of So P450. Residues on both ends of each domain are indicated on the scheme. Linker CR I and CR II are indicated by asterisks. Bottom: cryo-EM structure of So P450 with each domain labeled and colored as the scheme shown above. b , The electron density maps of prosthetic groups (sticks) bound in the structure contoured at 5.0 σ are shown in mesh. c , Two views of So P450 in homodimeric configuration, with two chains colored in green and magenta. Compared with the flavin-containing domains, the assignment of heme-domain is more challenging since the electron density of the entire CR I segment is missing. Taking heme-A as an instance, it could be connected to the adjacent FMN-A via path 1 or the distal FMN-B via path 2 ( Extended Data Fig. 3a ). Despite path 1 that is shorter a more possible route, the modeling study indicates that linking heme-A to FMN-B through path 2 is also viable since CR I that contains 41 amino acids (N456-N496) is sufficiently long to traverse across such a distance ( Extended Data Fig. 3b ). To resolve this issue, we constructed variant So P450-CR I -10 that contains ten amino acids in the CR I region and solved its cryo-EM structure to 2.34 Å ( Extended Data Table 2 and Extended Data Fig. 4a ). As a result, So P450-CR I -10 forms a dimer the same as the full-length protein ( Extended Data Fig. 4b ). Given that a CR I that contains ten amino acids is too short to afford a dimer configured via path 2, the homodimer should be assembled through path 1 ( Supplementary Fig. 2 ). As such, heme-A and FMN-A should belong to a polypeptide chain, and the structure of So P450 was determined ( Fig. 1c ). In addition, we successfully grew crystals of So P450 and the structure was resolved at a resolution of 3.38 Å by using the template obtained in the cryo-EM analysis ( Fig. 2 ). The crystal structure of So P450 contains two polypeptide chains that are organized as those in the cryo-EM structure (Cα root mean square deviation, 0.919 Å) ( Fig. 2a ). The electron density maps of prosthetics bound in each domain ( Fig. 2b ) and K655 to G669 and D656 to G669 in the CR II of chain A and chain B, respectively, were clearly seen ( Fig. 2c ). As the case in the cryo-EM structure, no electron density map in the CR I region can be seen. Download figure Open in new tab Fig. 2 Crystal structure of So P450. a , The overall structure of crystal structure of So P450 and its superimposition to the cryo-EM structure of So P450. b , The 2 F o - F c electron density maps of prosthetic groups (sticks) bound in the structure contoured at 2.0 σ are shown in mesh. Each domain and the bound cofactors are displayed in a color scheme that is applied in Fig. 1c . c , The overall structure and 2 F o - F c electron density maps of CR II segment in the crystal structure of So P450 contoured at 2.0 σ are shown in mesh. Cofactor-binding modes in So P450 The heme-domain located on the most N-terminus that mainly comprises α-helices adopts a canonical triangular fold of P450s ( Fig. 3a ) 12 , 48 . The heme is thiol-ligated to an invariant cysteine (C404 in So P450) on the proximal side as in other P450s 12 , 49 , and an elongated tunnel that should house the substrate formed on the distal side ( Fig. 3a ). The FMN-domain consists of a parallel β-sheet flanked by five α-helices with a FMN bound to the apex of the cone-shaped domain ( Fig. 3b ). The FAD-domain consists of an anti-parallel β-barrel and the putative NADPH-binding motif in another parallel five-stranded β-sheet sandwiched by several α-helices ( Fig. 3b ). We also solved the cryo-EM structure of NADPH-bound complex of So P450 to probe the binding mode of the nicotinamide pyridine ( Extended Data Table 2 and Extended Data Fig. 5 ). The cryo-EM structure of So P450/NADPH is highly identical to the apo-form structure ( Extended Data Fig. 5b ) and contains electron density maps that can be easily modeled with NADPH molecules ( Fig. 3b and Extended Data Fig. 5c ). The ribityl-nicotinamide moiety of the NADPH is missing, probably owing to the disordered structure of this portion as the case in other complex structures of CPR ( Fig. 3b ) 50 – 52 . Download figure Open in new tab Fig. 3 The domain structure of So P450. a , Left: two views of the heme-domain of So P450 with main α-helices labeled alphabetically. Right: the putative substrate-binding cavity above the prosthetic heme (cyan stick) displayed as yellow bubble. The residues lining the cavity are shown in lines. 1, A331; 2, A267; 3, I266; 4, L183; 5, T442. b , The di-flavin CPR domain consisting of FMN-A and FAD-B domains from different dimeric counterparts in the cryo-EM structures of So P450 and SoP450/NADPH are shown in green and magenta, respectively. The bound cofactors are displayed as sticks. The electron density maps of prosthetic groups bound in the complex of So P450/NADPH are contoured at 5.0 σ are shown in mesh and displayed in the zoom-in view. c , The structures of rat CPR and the functional CPR-domain of So P450 with hinge in the former and CR II in the latter highlighted. The FMN, FAD and NADPH bind to the protein via a number of polar interactions and the isoalloxazine and adenine are packed against several aromatic residues ( Extended Data Fig. 6a ). The complex structures of these cofactors and P450BM3 have been revealed by the crystal structures of the individual FAD-domain 42 and a FMN-domain in complex with the heme-domain 40 , which indicate that the location and interaction networks of FAD and FMN of So P450 and P450BM3 are highly identical ( Extended Data Fig. 6b ). The isoalloxazine rings of two flavins are juxtaposed by their C7- and C8-methyl moieties with a distance of approximately 4 Å ( Fig. 3b ), a pose that facilitates the direct electron transfer 53 . The nicotinamide of NADPH that should pack against the isoalloxazine ring of FAD to allow the electron transfer lacks electron density maps while the indole side group of W1064 remains stacks to the isoalloxazine ring of FAD ( Fig. 3b and Extended Data Fig. 6a ). This suggests a competition between the nicotinamide and W1064, a phenomenon consistently seen in other complex structures of flavin-containing NAD(P)H reductase, and the nicotinamide portion could be revealed by replacing the FAD-stacking aromatic residue with small amino acids 54 , 55 . The relative locations of these cofactors resemble those in CPR from other species ( Fig. 3c and Supplementary Fig. 3 ), though the FAD- and FMN-domain in the latter group are on the same polypeptide chain 50 – 52 . The CR II segment of So P450 stretches to the other CPR-domain of the dimer, whereas the equivalent region in other CPR structures forms a hinge ( Fig. 3c ). It has been suggested that the hinge region accounts for the back-and-forth movement of the FMN-domain 39 , 52 , 55 . However, such movements were not observed in the CPR-domain in all So P450 structures presented in this study. The heme-domain of So P450 is mobile These structures can clearly show the electron flowing path from NADPH to FAD to FMN but that from FMN to heme would be very inefficient, if ever occurs, in the observed homodimer configuration. This is because the FMN is located distantly from either heme prosthetic with straight-line edge-to-edge distances of longer than 40 Å ( Supplementary Fig. 4 ), much longer than the distance that allows efficient electron transfer 12 , 27 . In this context, domain rearrangement that brings FMN and heme closer is expected to take place. Structural investigations of CPRs from the multi-component P450s suggest a mobile FMN-domain that can stretch out to approach the proximal side of the heme-domain 39 , 56 , 57 . As such, FMN-A should dissociate from FAD-B, or FMN-B from FAD-A, to reach for the heme-domain. However, we did not observe movement of FMN-domain. The interaction pose of FMN-domain and heme-domain revealed in the crystal structure of partial P450BM3 40 is also unlikely to take place in the configuration of the dimeric structure as CR II segment is too short to allow the relocation of the FMN-domain ( Supplementary Fig. 5 ). Instead, we identified a class of structure that lacks one heme-domain during processing the cryo-EM images of So P450 ( Fig. 4a , Extended Data Figs. 1b and 1c ). The one-heme-missing structure, termed class 2 conformation, contains all cofactors, except for one heme, that bind to the same positions as those in the dimeric structure ( Fig. 4b ). The class 2 conformation superimposes well to the dimer though FAD-B slightly deviates from the position in the dimeric form, which could owe to the removal of the heme-A domain located beneath ( Supplementary Fig. 6 ). Download figure Open in new tab Fig. 4 The heme-domain of So P450 in mobile. a , The cryo-EM structure of class 2 conformation of So P450 displayed in a cartoon model with two chains colored in green and magenta. b , The cofactors bound in the structure of the class 2 conformation with the cognate electron density maps displayed as described in Fig. 1b . c , The areas of the contact interface between each domain in the dimeric form So P450. nd., not detectable. d , The proposed electron transfer mechanism of a heme-moving model of So P450. The presence of class 2 conformation implies that the heme-domain is mobile, which could be, at least partially, supported by the fact that the heme-domain forms fewer interactions to the adjacent domains than the FMN- and FAD-domain that form a CPR-domain. The buried interface of FMN-A and FAD-B is estimated to 1,838 and 1,627 Å 2 , respectively, accounting for approximately 18.5% and 7.2% of the solvent accessible surface area of each domain. The interactions between FMN-A and FAD-B, so as FMN-B and FAD-A, represent the most extensive contact among all in the dimeric configuration ( Fig. 4c ). In comparison, the heme-domain forms fewer contact to others. Accordingly, we proposed that the heme-domain is mobile and could relocate to reach the FMN-domain to acquire the electrons ( Fig. 4d ). The correlation of CR I length and the activity of So P450 Based on the heme-domain moving mechanism, we built a model to simulate the catalytic state displayed in Fig. 4d . As shown in Fig. 5a , the heme-domain should flip up to allow the proximal side to approach the CPR-domain with the CR I -anchoring residues on either end spatially close to each other. The bulge between helix H and I of the heme-domain docks into the concave side of the CPR-domain ( Supplementary Fig. 7a ) and the contact interfaces on the heme- and CPR-domain are electrostatic complementary ( Supplementary Fig. 7b ). The edge-to-edge distance between heme-1 and FMN-1 is measured to 17.8 Å ( Fig. 5b ). The methyl moieties of FMN remains directing toward the FAD instead of being exposed to orient to the heme-domain. As previous studies reported, W574 that stacks the isoalloxazine ring of FMN assists the electron flow between FMN and heme in P450BM3 40 , 58 . Accordingly, the equivalent residue W591 in So P450 that packs against the isoalloxazine ring of FMN may serve to relay the electron from FMN ( Fig. 5b ). Download figure Open in new tab Fig. 5 The putative electron transfer route of So P450. a, The model of a catalytic state of So P450, in which the mobile heme-domain (green) is relocated to approach the CPR-domain (magenta). The contact interface of heme- and FMN-domain is framed. The model construction processes are described in Methods. b, The zoom-in view of the framed area shown in a , with the edge-to-edge distance between heme and FMN prosthetics indicated on the right. Two possible electron traveling paths from FMN to heme are depicted by curved arrows, with nonbond jumps on the path connected by dashed lines. c, 1 mL reactions containing 5 µM enzyme ( So P450/G84F and variants), 4 mM indole, and 0.2 mM NADPH were incubated at 37 °C for 20 min. The indigo production was quantified based on a standard curve established with indigo of known concentration. The average and individual values of each group are shown by bars and circles. d, 1 mL reactions containing So P450/G84F or P450BM3/A83F that possess varying length of CR I assembled as described in c were incubated at 37 °C. The indigo formation during indicated time period was monitored via optical density at 670 nm (OD670) and the average values of a triplicate assay are displayed. The sequences of CR I -truncated variants are shown in Supplementary Fig. 10 . FL, full-length; Ctrl, no enzyme. The results demonstrated in c and d are representatives of three independent experiments. Since no prosthetic exists between FMN and heme, we suspected that the electrons shall be tunneling to the heme center through amino acids en route and hired HARLEM program 59 to identify candidate residues ( Supplementary Fig. 8 ). The predicted path that begins with the indole of W591 passes through covalent bonds that connect residues A592, S593, Q401, R402, A403, C404 along with a non-bonded jump between S593 O and Q401 NE2 and eventually to the heme iron ( Fig. 5b ). Mutagenesis experiment was then applied to investigate the role of these residues play in the activity of So P450. Because the natural substrate of So P450 remains unrevealed, variant G84F was constructed, based on the analogy to the variant A83F of P450BM3 60 , to confer the protein capability of synthesizing indigo by using indole as a substrate ( Supplementary Fig. 9 ). Residue A592, S593, R402 and A403 contribute the main chain to the path, and C404 and W591 are indispensable to the binding of cofactors. Variant Q401A exhibits 67.1 ± 7.5 % activity of the parental enzyme, suggesting that the side chain of Q401 may play a role, but not strictly essential, in So P450-catalyzed reaction ( Fig. 5c ). This suggests that the electrons may bifurcate to another path such as one that comprises W591, A592, R99, H102 and then to the main chain of Q401 ( Fig. 5b ). Indeed, substituting R99 and H102 with Ala reduced the enzyme activity to 48 ± 1.9 % and 24.6 ± 0.4 %, respectively ( Fig. 5c ). This model also suggests that the CR I fragment of adequate length shall be required for the heme-domain to reach for the CPR-domain. Therefore, we constructed a series of variants with the CR I progressively truncated and measured their activity. Variant So P450/G84F-CR I -28 that retains 28 amino acids in the CR I segment, the same as that of P450BM3 ( Supplementary Fig. 10 ), exhibits activity comparable to the full-length protein ( Fig. 5d ). Further truncation led to progressive reduction in enzyme activity, suggesting that shorter CR I might constrain the optimal docking of heme-domain to the CPR-domain and that our model of a catalytic state of So P450 may be reliable. This is also the case for P450BM3, that the catalytic activity decreased as the CR I was shortened ( Fig. 5d ). Intriguingly, the length of CR I fragment of P450s tested in our study ranges from 26 ( Mt P450 from Marinactinospora thermotolerans ) to 41 amino acids ( So P450) ( Extended Data Table 1 ), and the shortest CR I that can be identified through a searching in the GenBank consists of 25 amino acids ( Supplementary Fig. 11 ). Altogether, 25 amino acids could be the minimal requirement for the CR I in P450BM3 homologous CPR-containing self-sufficient P450s to exert the optimal activity. Conclusion In this study, the cryo-EM and crystal structures of So P450 were resolved to reveal the unique dimeric organization of a P450BM3 homologous CPR-containing self-sufficient P450. The identification of the class 2 conformation that lacks one heme-domain suggests that the heme-domain is mobile and might relocate to reach the CPR-domain for electron acquisition. Accordingly, the catalytic state was proposed through modeling study, which reveals putative electron transfer paths from FMN to heme. Finally, the extensive CR I segment that connects the heme- and FMN-domain should govern the transition of the heme-domain between the resting and the catalytic state. While the association of the length of CR I segment and the catalytic activity is demonstrated, the correlation of other properties of this region (e.g., rigidity) remains to be explored. Given the high protein sequence identity between So P450 and P450BM3, the reported structures would be a reliable model for predicting the three-dimensional structure of the latter. Intriguingly, the indigo synthesis assay based on the Phe variant indicates that So P450 exhibits higher catalytic activity than P450BM3, illuminating further application potentials of So P450. These results are of fundamental importance to advance our understanding about the molecular basis of P450BM3 as well as other homologous P450s, but provide valuable information for engineering and applications of these enzymes. Data availability All data generated or analyzed during this study are included in the manuscript and the Supplementary Information files. The 3D cryo-EM density maps of So P450 (EMD-66257), So P450-class 2 (EMD-66258), So P450/NADPH (EMD-66267) and So P450-CR I -10 (EMD-66262) have been deposited in the Electron Microscopy Data Bank (EMDB) database. The atomic model by X-ray crystallography So P450 (PDB ID, 9X0N) has been deposited in the Protein Data Bank (PDB). Methods Plasmid construction and mutagenesis The genes that encode CPR-containing self-sufficient P450s containing His-tag, tobacco etch virus (TEV) protease cleavage site (ENLYFQG) and (AG) n linker on the N -terminus were chemically synthesized and cloned into expression vector pET-46 Ek/LIC. The amino acid sequences of all selected P450s are listed in Supplementary Table 1 . The polymerase chain reaction-based site-directed mutagenesis was conducted to construct variants by using plasmid that encodes So P450 or P450BM3 as a template with mutagenic oligonucleotides listed in Supplementary Table 2 . All plasmids were verified by direct sequencing. Protein expression and purification For protein expression, the plasmid was transformed into Escherichia coli BL21 (DE3). The transformants were initially cultured overnight in 100 mL Luria–Bertani (LB) medium at 37 °C containing 100 µg mL -1 ampicillin. The preculture was inoculated into 5 L LB medium containing 100 µg mL -1 ampicillin and grown at 37 °C with constant shaking at 220□rpm. Protein expression was induced when the optical density at 600 nm (OD600) reached 0.6 by adding 0.3 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) and 5 µM chlorhematin, followed by cultivation at 16 °C for 18 h. The cells were collected by centrifuging at 6,000 rpm for 10 min, and resuspended in 100□mL of lysis buffer containing 25□mM Tris-HCl (pH□7.5), 150□mM NaCl, and 20□mM imidazole. Cell disruption was achieved using a French press (JuNeng Biology & Technology), followed by centrifugation at 17,000 rpm for 60 min to pellet the debris. The supernatant was then loaded onto a Ni-NTA column using a protein chromatography system (Sepure Instruments, Suzhou, China) and eluted with a gradient of imidazole ranging from 20 to 500 mM. The fractions containing target protein were collected and dialyzed against a 25 mM HEPES buffer (pH 7.5) containing 150 mM NaCl. The proteins were supplemented with 10 mM DTT and then passed through a Superdex 200 10/300 GL column (GE Healthcare, Madison, WI). The peak fractions were collected, quantified and concentrated for further analyses. Cryo-EM sample preparation and data collection Prior to the sample preparation, the Electron Microscopy Sciences grids (CFlat, Au, R 1.2/1.3) were discharged with 15 mA at 0.39 mbar for 45 s. 4 μL of protein solution (1 mg mL -1 ) was applied to the grids, and 10 mM NADPH was supplemented for the preparation of the complex of So P450/NADPH. A Vitrobot Mark IV (Thermo Fisher Scientific) was used to blot the grid for 3 s at 4 °C and 100% humidity, followed by plunge-freezing into liquid ethane cooled by liquid nitrogen. The grids were then transferred into a box stored in liquid nitrogen before image acquisition. The datasets were collected with a Titan Krios electron microscope equipped with a Falcon 4 detector by using the EPU software (Thermo Fisher Scientific). The data collection parameters are outlined in Extended Data Table 2 . Cryo-EM data processing All datasets were processed using cryoSPARC v.4.6.0 64 , with the details outlined in Extended Data Fig. 1, 4 and 5 . In general, all movie frames underwent motion correction and contrast transfer function (CTF) prior to an automated particle picking. After multiple rounds of picking, ultimately yielded particles were subjected to ab-initio reconstruction with C1 symmetry imposed. After heterogeneous refinement to optimized data classification, select classes with high quantity for homogeneous refinement and non-uniform refinement. Further refined the correction of particle motion trajectories by reference-based motion correction, and repeated homogeneous refinement and non-uniform refinement were performed. For So P450, the final reconstructions were obtained at resolutions of 2.72 Å from a set of 740,228 particles (class 1 conformation, the dimer form) and 3.08 Å from a set of 154,436 particles (class 2 conformation, the one-heme-missing form) after local refinement. The resolution of the final maps was estimated using the gold-standard Fourier shell correlation (FSC) with a 0.143 criterion. For So P450-CR I -10, 4,826 movie stacks were collected and similarly processed. 4,684,184 particles were auto-picked and extracted from the preprocessed micrographs. 1,407,631 particles were selected for 3D reconstruction, and 1,180,657 particles were kept for producing the final density map determined at a resolution of 2.34 Å. For So P450/NADPH, 2,754 movies were acquired. The movie stacks were motion corrected using patch motion correction with a binning factor of 2 for further data processing (final pixel size of 0.926□Å on the sample level), and CTF estimation was performed with patch CTF estimation. After auto-picking, 437,136 particles were extracted for 2D classification, from which 139,104 particles were retained for reconstruction, yielding a density map at 2.68 Å resolution. Cryo-EM structure determination The initial models of each domain of So P450 were obtained by using AlphaFold3 algorithm 65 . These models were docked into the density map using UCSF Chimera 66 . The structure models were then refined against the sharpened maps with Phenix real-space refine 67 , ISOLDE 68 and Coot 69 . The models used for So P450-CR I -10, So P450-NADPH complex and the class-2 conformation of So P450 were constructed based on the dimeric So P450 model. Summary statistics for map reconstruction and model building are presented in Extended Data Table 2 . Crystallization, structure determination and refinement The initial crystallization screening for SoP450 was performed at 22 □ by the sitting-drop vapor-diffusion. Protein solution (1 µL) was mixed with 1 µL of reservoir solution and equilibrated against 100 µL of reservoir solution. Needle-shaped reddish-brown crystals were observed under the condition containing 0.1 M HEPES (pH 7.0), 15% PEG 20,000. After optimizing the crystallization conditions, reddish-brown strip-shaped crystals were obtained under 0.1 M HEPES (pH 7.5), 15% PEG 20,000. Prior to X-ray diffraction data collection, crystals were cryoprotected by soaking in a buffer containing 0.1 M HEPES (pH 7.5) and 20% PEG 20,000. The X-ray diffraction datasets were collected at beam line TPS 05A of the National Synchrotron Radiation Research Center (NSRRC, Hsinchu, Taiwan). Data was processed by using HKL2000 70 . The crystal structure of So P450 was solved by molecular replacement using the Phaser program 71 by using the cryo-EM structure of the dimeric So P450 as a search model. Prior to structure refinement, 5% of the reflections were randomly selected and set aside for calculating R free as a monitor of model quality. The model was manually adjusted using Coot 69 and refined with Refmac5 in CCP4 suit 72 and PHENIX 67 . The statistics of data collection and refinement are summarized in Extended Data Table 3 . Enzyme activity measurement For indigo formation assay, the reaction mixture (1 mL) containing purified enzyme (5 μM) and indicated concentration of indole were added in 0.1 M phosphate buffer (pH 8.0). The reaction was initiated by adding NADPH to a final concentration of 0.2 mM. The formation of indigo at 37 □ was monitored by measuring the absorbance at 670 nm at destined timepoints. Model preparation The reconstruction of CR I segment missing in the resolved structures of So P450 was performed by using the loop modeling module implemented in Rosetta 3.15 73 . In the absence of available homologous templates, the CR I segment was built de novo using the cyclic coordinate descent algorithm, which relies purely on physical principles 74 . To account for conformational diversity, at least 128 distinct conformations were generated for each loop region requiring completion. To model the relocated heme-domain approaching the FMN-domain (a “catalytic state”), protein–protein docking was performed using the local docking protocol in Rosetta 3.15. The most plausible pose—in which the heme-domain was positioned close to the FMN-domain to enable efficient electron transfer, and the loop-anchoring residues were spatially proximal to the heme-domain anchor points, thereby ensuring appropriate loop accessibility—was manually selected for subsequent loop modeling and molecular dynamics simulations. Molecular dynamics simulations The CR I -containing models and the relocated heme–FMN complex were subjected to molecular dynamics (MD) simulations using the AMBER22 software package 75 , with three independent replicas performed for each system to ensure reproducibility. During the MD process, the Amber FF14SB 76 force field was used for the protein and the TIP3P 77 model was employed for the solvent waters. The heme cofactor parameters were generated using the Metal Center Parameter Builder (MCPB) approach 78 . The restrained electrostatic potential (RESP) 79 charges of all ligands were calculated at the HF/6-31G* level using the Gaussian 16 package 80 , and the ligands were subsequently described using the AMBER GAFF□ force field, consistent with previous MD studies on cytochrome P450 systems 81 – 84 . All simulations were conducted under periodic boundary conditions, with cubic solvent boxes applied to each system. The initial coordinates and topology files were generated using the tleap module implemented in AMBER22. Sodium or chloride ions were added as needed to neutralize the total system charge. In total, each system comprised approximately 220,000 atoms. Energy minimization was performed in three stages: first on the solvent molecules, then on the protein side chains, and finally on all atoms. Each minimization stage consisted of 4000 cycles of steepest descent followed by 2000 cycles of conjugate gradient minimization. After minimization, each system was gradually heated from 0 K to 300 K under the NVT ensemble (with Langevin thermostat) 85 , followed by a 100 ps NPT ensemble density equilibration at 300 K and 1.0 atm (with Berendsen barostat) 86 . Subsequently, 100 ns production runs were carried out for each system—under the NVT ensemble with a 1 fs time step. A nonbonded cutoff of 10 Å was applied to both van der Waals (Lennard–Jones 12-6) and real-space electrostatic interactions, with long-range electrostatics treated by the particle mesh Ewald (PME) method 87 . The SHAKE 88 algorithm was applied to constrain all bonds involving hydrogen atoms, thereby removing the high-frequency stretching vibrations of X–H bonds during the MD simulations. To investigate the electron transfer chain, conformational clustering was performed for the relocated heme–CPR complex model based on protein backbone coordinates. The most populated cluster accounted for 83.7% of the total conformational ensemble ( Fig. 5a and 5b ). Trajectory analyses, including clustering, RMSD, and RMSF calculations, were carried out using the parallelized cpptraj module implemented in the AMBER 22 suite 89 , 90 . Competing interests The authors declare no competing interests. Download figure Open in new tab Extended Data Fig. 1 Workflow for image processing and structure reconstruction of cryo-EM analyses of So P450. a , A representative micrograph of So P450. Scar bar, 50 nm. b , Reference free 2D averages and cryo-EM maps at the various stages of processing. c , Local resolution estimation of the finalized cryo-EM maps and corrected curve of the global Fourier shell correlation (FSC) with 0.143 gold-standard criterion. Download figure Open in new tab Extended Data Fig. 2 The assignment of flavin-containing domains of So P450. a , The cartoon models, amino acids and electron density maps of CR II contoured at 5.0 σ of the cryo-EM structure of So P450 (PDB ID, 9WUC). b , The cartoon models of FMN- and FAD-domains of So P450 in two individual polypeptide chains. c , The CPR of So P450 in the dimeric configuration. d , One functional CPR-domain of So P450. Download figure Open in new tab Extended Data Fig. 3 Two possible models based on the direction of the CR I segment. a , The cartoon model of cryo-EM structure of So P450 (PDB ID, 9WUC) with two paths that connect heme-A to FMN-A or FMN-B indicated with green dashed lines. Green circle, the C-terminus of heme-A; black circles, the N-termini of FMN-A and FMN-B. b , The models based on two possible paths, with CR I fragments colored in green. The model construction processes are described in Supplementary Computation Information and Supplementary Fig. 12 . Download figure Open in new tab Extended Data Fig. 4 Workflow for image processing and structure reconstruction of cryo-EM analyses of So P450-CR I -10. a , A representative micrograph, reference free 2D averages, cryo-EM maps at the various stages of processing, local resolution estimation of the finalized cryo-EM maps and corrected curve of the global FSC with 0.143 gold-standard criterion. Scar bar, 50 nm. Top, the amino acid sequences of the full-length ( So P450) and CR I -truncated variant ( So P450-CR I -10) of So P450. b , The overall structure of So P450-CR I -10 (PDB ID, 9WUK) and electron density maps of prosthetic groups bound in the structure contoured at 5.0 σ are shown as described in Fig. 1b . Download figure Open in new tab Extended Data Fig. 5 Workflow for image processing and structure reconstruction of cryo-EM analyses of of So P450/NADPH. a , A representative micrograph, reference free 2D averages, cryo-EM maps at the various stages of processing, local resolution estimation of the finalized cryo-EM maps and corrected curve of the global FSC with 0.143 gold-standard criterion of the complex of So P450/NADPH (PDB ID, 9WUP). Scar bar, 50 nm. b , The structure superimposition of So P450 and So P450/NADPH. c , The electron density maps of prosthetic groups bound in So P450/NADPH contoured at 5.0 σ are shown as described in Fig. 1b . Download figure Open in new tab Extended Data Fig. 6 Interaction network of FMN, FAD and NADPH in So P450/NADPH (PDB ID, 9WUP) and P450BM3. Protein residues and cofactors are displayed in line and sticks, respectively. Proteins belong to two individual chains in the homodimer are colored in green and magenta. The drawings of P450BM3-FMN and P450BM3-FAD/NADP + are based on PDB entry 1BVY and 4DQL, respectively. Dashed lines, distance < 3.5 Å. View this table: View inline View popup Download powerpoint Extended Data Table 1. Basic information of CPR-containing self-sufficient P450s tested in this study. View this table: View inline View popup Download powerpoint Extended Data Table 2. Cryo-EM data collection, refinement and validation statistics View this table: View inline View popup Download powerpoint Extended Data Table 3. Data collection and refinement statistics of crystal structure of So P450 Acknowledgments This work was supported by the National Key Research and Development Program of China (2021YFC2104000), Hubei Hongshan Laboratory (2022hszd030), the National Natural Science Foundation of China (32371307, 32271318, 82341210, 22473118 and 82430108); and the Interdisciplinary Research Project of Hangzhou Normal University (2024JCXK02). We thank NSRRC (National Synchrotron Radiation Research Center, Taiwan) for access to beam lines TPS-05A and TPS-07A that contributed for the synchrotron data collection. Funder Information Declared National Natural Science Foundation of China , 32371307 , 32271318 , 82341210 , 22473118 , 82430108 Interdisciplinary Research Project of Hangzhou Normal University , 2024JCXK02 National Key Research and Development Program of China , 2021YFC2104000 References ↵ Bernhardt , R. & Urlacher , V. B . Cytochromes P450 as promising catalysts for biotechnological application: Chances and limitations . Appl Microbiol Biotechnol 98 , 6185 – 6203 , doi: 10.1007/s00253-014-5767-7 ( 2014 ). OpenUrl CrossRef PubMed ↵ Poulos , T. L . Heme enzyme structure and function . Chemical Reviews 114 , 3919 – 3962 , doi: 10.1021/cr400415k ( 2014 ). OpenUrl CrossRef PubMed Web of Science Smit , M. S. , Maseme , M. J. , van Marwijk , J. , Aschenbrenner , J. C. & Opperman , D. J . 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Share Full-length structure of CPR-containing self-sufficient cytochrome P450 Zhenzhen Xie , Ziwei Liu , Siyu Li , Kangwei Xu , Jian-Wen Huang , Jian Min , Qiru Li , Jingxue Zhai , Te Wang , Yutong Wang , Lu Yang , Junjie Duan , Junjie Chen , Ruibo Wu , Chun-Chi Chen , Rey-Ting Guo bioRxiv 2025.11.29.689641; doi: https://doi.org/10.1101/2025.11.29.689641 Share This Article: Copy Citation Tools Full-length structure of CPR-containing self-sufficient cytochrome P450 Zhenzhen Xie , Ziwei Liu , Siyu Li , Kangwei Xu , Jian-Wen Huang , Jian Min , Qiru Li , Jingxue Zhai , Te Wang , Yutong Wang , Lu Yang , Junjie Duan , Junjie Chen , Ruibo Wu , Chun-Chi Chen , Rey-Ting Guo bioRxiv 2025.11.29.689641; doi: https://doi.org/10.1101/2025.11.29.689641 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Synthetic Biology Subject Areas All Articles Animal Behavior and Cognition (7637) Biochemistry (17705) Bioengineering (13899) Bioinformatics (41970) Biophysics (21463) Cancer Biology (18605) Cell Biology (25526) Clinical Trials (138) Developmental Biology (13385) Ecology (19911) Epidemiology (2067) Evolutionary Biology (24329) Genetics (15615) Genomics (22514) Immunology (17743) Microbiology (40424) Molecular Biology (17194) Neuroscience (88650) Paleontology (667) Pathology (2835) Pharmacology and Toxicology (4827) Physiology (7648) Plant Biology (15160) Scientific Communication and Education (2046) Synthetic Biology (4302) Systems Biology (9825) Zoology (2271)