Lipid composition and macromolecular crowding effects on CYP2J2‐mediated drug metabolism in nanodiscs

Lipid composition and macromolecular crowding are key external effectors of protein activity and stability whose role varies between different proteins. Therefore, it is imperative to study their effects on individual protein function. CYP2J2 is a membrane‐bound cytochrome P450 in the heart involved in the metabolism of fatty acids and xenobiotics. In order to facilitate this metabolism, cytochrome P450 reductase (CPR), transfers electrons to CYP2J2 from NADPH. Herein, we use nanodiscs to show that lipid composition of the membrane bilayer affects substrate metabolism of the CYP2J2‐CPR nanodisc (ND) system. Differential effects on both NADPH oxidation and substrate metabolism by CYP2J2‐CPR are dependent on the lipid composition. For instance, sphingomyelin containing nanodiscs produced more secondary substrate metabolites than discs of other lipid compositions, implying a possible conformational change leading to processive metabolism. Furthermore, we demonstrate that macromolecular crowding plays a role in the lipid‐solubilized CYP2J2‐CPR system by increasing the K_m and decreasing the V_max, and effect that is size‐dependent. Crowding also affects the CYP2J2‐CPR‐ND system by decreasing both the K_m and V_max for Dextran‐based macromolecular crowding agents, implying an increase in substrate affinity but a lack of metabolism. Finally, protein denaturation studies show that crowding agents destabilize CYP2J2, while the multidomain protein CPR is stabilized. Overall, these studies are the first report on the role of the surrounding lipid environment and macromolecular crowding in modulating enzymatic function of CYP2J2‐CPR membrane protein system.     

Nanodomains of Cytochrome b6f and Photosystem II Complexes in Spinach Grana Thylakoid Membranes

The cytochrome b₆f (cytb₆f) complex plays a central role in photosynthesis, coupling electron transport between photosystem II (PSII) and photosystem I to the generation of a transmembrane proton gradient used for the biosynthesis of ATP. Photosynthesis relies on rapid shuttling of electrons by plastoquinone (PQ) molecules between PSII and cytb₆f complexes in the lipid phase of the thylakoid membrane. Thus, the relative membrane location of these complexes is crucial, yet remains unknown. Here, we exploit the selective binding of the electron transfer protein plastocyanin (Pc) to the lumenal membrane surface of the cytb₆f complex using a Pc-functionalized atomic force microscope (AFM) probe to identify the position of cytb₆f complexes in grana thylakoid membranes from spinach (Spinacia oleracea). This affinity-mapping AFM method directly correlates membrane surface topography with Pc-cytb₆f interactions, allowing us to construct a map of the grana thylakoid membrane that reveals nanodomains of colocalized PSII and cytb₆f complexes. We suggest that the close proximity between PSII and cytb₆f complexes integrates solar energy conversion and electron transfer by fostering short-range diffusion of PQ in the protein-crowded thylakoid membrane, thereby optimizing photosynthetic efficiency.     

Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

The membrane heme protein cytochrome b₅ (b₅) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b₅ and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b₅, and an androgen-forming lyase reaction that is facilitated 10-fold by b₅. NMR chemical shift mapping of b₅ titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b₅ residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b₅ binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b₅ on the two CYP17A1-mediated reactions and, thus, communication between the superficial b₅ binding site and the buried CYP17A1 active site, CYP17A1/b₅ complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b₅ interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b₅ binding site.     

The α-helical membrane spanning domain of cytochrome b5 interacts with cytochrome P450 via nonspecific interactions

Cytochrome b₅ (cyt b₅) is an amphipathic membrane-bound heme protein found in the endoplasmic reticulum of eukaryotes. It consists of three domains, an N-terminal cytosolic, hydrophilic domain containing the heme, a short flexible linker and an α-helical membrane-spanning domain. This study investigated whether there are specific side chain helix–helix packing interactions between the COOH-terminal membrane anchor of cyt b₅ and cytochrome P450 (cyt P450) 2B4 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b₅. Insertion of alanine into an α-helix causes all amino acids at its carboxyl terminus to be rotated by 100°. The ability of the alanine insertion mutants of cyt b₅ to bind to cyt P450 2B4 was similar to that of the wild-type protein as was the ability of the mutant cyts b₅ to stimulate the metabolism of the anesthetic, methoxyflurane. These results demonstrate that the C-terminal hydrophobic α-helix of cyt b₅ does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions.     

Heterologous expression of fungal cytochromes P450 (CYP5136A1 and CYP5136A3) from the white-rot basidiomycete Phanerochaete chrysosporium: Functionalization with cytochrome b5 in Escherichia coli

Cytochromes P450 from the white-rot basidiomycete Phanerochaete chrysosporium, CYP5136A1 and CYP5136A3, are capable of catalyzing oxygenation reactions of a wide variety of exogenous compounds, implying their significant roles in the metabolism of xenobiotics by the fungus. It is therefore interesting to explore their biochemistry to better understand fungal biology and to enable the use of fungal enzymes in the biotechnology sector. In the present study, we developed heterologous expression systems for CYP5136A1 and CYP5136A3 using the T7 RNA polymerase/promoter system in Escherichia coli. Expression levels of recombinant P450s were dramatically improved by modifications and optimization of their N-terminal amino acid sequences. A CYP5136A1 reaction system was reconstructed in E. coli whole cells by coexpression of CYP5136A1 and a redox partner, NADPH-dependent P450 reductase (CPR). The catalytic activity of CYP5136A1 was significantly increased when cytochrome b₅ (Cyt-b₅) was further coexpressed with CPR, indicating that Cyt-b₅ supports electron transfer reactions from NAD(P)H to CYP5136A1. Notably, P450 reaction occurred in E. coli cells that harbored CYP5136A1 and Cyt-b₅ but not CPR, implying that the reducing equivalents required for the P450 catalytic cycle were transferred via a CPR-independent pathway. Such an “alternative” electron transfer system in CYP5136A1 reaction was also demonstrated using purified enzymes in vitro. The fungal P450 reaction system may be associated with sophisticated electron transfer pathways.     

Protein-protein interactions in the systems of cytochromes P450 3A4 and 3A5

Molecular interactions between protein partners of the monooxygenase system involved in drug biotransformation (cytochromes P450 3A4, 3A5 and cytochrome b ₅) have been investigated. Human cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) form complexes with various forms of cytochrome b ₅, including microsomal (b ₅ mc) and mitochondrial (b ₅ om) forms of this protein, as well as two chimeric constructs (b ₅(om-mc),(b ₅(mc-om). Interestingly, significant differences were observed only during interactions with b ₅ om. Electroanalytical characteristics of electrodes with immobilized hemoproteins have been determined for CYP3A4, CYP3A5, b ₅ mc, b ₅ om, b ₅ mc(om-mc), and b ₅ mc(mc-om). The electrochemical analysis revealed that these proteins are characterized by close reduction potentials ranged from ?0.435 V to ?0.350 V (vs. Ag/AgCl). Cytochrome b ₅ mc stimulated the electrocatalytic activity of CYP3A4.     

Site-Directed Mutagenesis of Cytochrome b5 for Studies of Its Interaction with Cytochrome P450

We have shown earlier that microsomal cytochrome b ₅ can form a specific complex with mitochondrial cytochrome P450 (cytochrome P450scc). The formation of the complex between these two heme proteins was proved spectrophotometrically, by affinity chromatography on immobilized cytochrome b ₅, and by measuring the cholesterol side-chain cleavage activity of cytochrome P450scc in a reconstituted system in the presence of cytochrome b ₅. To further study the mechanism of interaction of these heme proteins and evaluate the role of negatively charged amino acid residues Glu42, Glu48, and Asp65 of cytochrome b ₅, which are located at the site responsible for interaction with electron transfer partners, we used sitedirected mutagenesis to replace residues Glu42 and Glu48 with lysine and residue Asp65 with alanine. The resulting mutant forms of cytochrome b ₅ were expressed in E. coli, and full-length and truncated forms (shortened from the C-terminal sequence due to cleavage of 40 amino acid residues) of these cytochrome b ₅ mutants were purified. Addition of the truncated forms of cytochrome b ₅ (which do not contain the hydrophobic C-terminal sequence responsible for interaction with the membrane) to the reconstituted system containing cytochrome P450scc caused practically no stimulation of catalytic activity, indicating an important role of the hydrophobic fragment of cytochrome b ₅ in its interaction with cytochrome P450scc. However, full-length cytochrome b ₅ and the full-length Glu48Lys and Asp65Ala mutant forms of cytochrome b ₅ stimulated the cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc by 100%, suggesting that residues Glu48 and Asp65 of cytochrome b ₅ are not directly involved in its interaction with cytochrome P450scc. The replacement of Glu42 for lysine, however, made the Glu42Lys mutant form of cytochrome b ₅ about 40% less effective in stimulation of the cholesterol side-chain cleavage activity of cytochrome P450scc, indicating that residue Glu42 of cytochrome b ₅ is involved in electrostatic interactions with cytochrome P450scc. Residues Glu42 and Glu48 of cytochrome b ₅ appear to participate in electrostatic interaction with microsomal type cytochrome P450.     

Human Cytochrome P450 17A1 Conformational Selection: MODULATION BY LIGAND AND CYTOCHROME b5*

Crystallographic studies of different membrane cytochrome P450 enzymes have provided examples of distinct structural conformations, suggesting protein flexibility. It has been speculated that conformational selection is an integral component of substrate recognition and access, but direct evidence of such substate interconversion has thus far remained elusive. In the current study, solution NMR revealed multiple and exchanging backbone conformations for certain structural features of the human steroidogenic cytochrome P450 17A1 (CYP17A1). This bifunctional enzyme is responsible for pregnenolone C17 hydroxylation, followed by a 17,20-lyase reaction to produce dehydroepiandrosterone, the key intermediate in human synthesis of androgen and estrogen sex steroids. The distribution of CYP17A1 conformational states was influenced by temperature, binding of these two substrates, and binding of the soluble domain of cytochrome b₅ (b₅). Notably, titration of b₅ to CYP17A1·pregnenolone induced a set of conformational states closely resembling those of CYP17A1·17α-hydroxypregnenolone without b_5, providing structural evidence consistent with the reported ability of b₅ to selectively enhance 17,20-lyase activity. Solution NMR thus revealed a set of conformations likely to modulate human steroidogenesis by CYP17A1, demonstrating that this approach has the potential to make similar contributions to understanding the functions of other membrane P450 enzymes involved in drug metabolism and disease states.     

The role of cytochrome b 5 structural domains in interaction with cytochromes P450

To understand the role of the structural elements of cytochrome b ₅ in its interaction with cytochrome P450 and the catalysis performed by this heme protein, we carried out comparative structural and functional analysis of the two major mammalian forms of membrane-bound cytochrome b ₅ — microsomal and mitochondrial, designed chimeric forms of the heme proteins in which the hydrophilic domain of one heme protein is replaced by the hydrophilic domain of another one, and investigated the effect of the highly purified native and chimeric heme proteins on the enzymatic activity of recombinant cytochromes P4503A4 and P45017A1 (CYP3A4 and CYP17A1). We show that the presence of a hydrophobic domain in the structure of cytochrome b ₅ is necessary for its effective interaction with its redox partners, while the nature of the hydrophobic domain has no significant effect on the ability of cytochrome b ₅ to stimulate the activity of cytochrome P450-catalyzed reactions. Thus, the functional properties of cytochrome b ₅ are mainly determined by the structure of the hemebinding domain.     

The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5)

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b₅. In both instances, product formation occurs. When the second electron is donated by cytochrome b₅, catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b₅ has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b₅ on cytochrome P450 2B4 reactivity can explain how cytochrome b₅ is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b₅ to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b₅ to cytochrome P450 reductase, cytochrome b₅ inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b₅ are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b₅ stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.     

Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.     

Effect of homomeric P450-P450 complexes on P450 function

Previous studies have shown that the presence of one P450 enzyme can affect the function of another. The goal of the present study was to determine if P450 enzymes are capable of forming homomeric complexes that affect P450 function. To address this problem, the catalytic activities of several P450s were examined in reconstituted systems containing NADPH-POR (cytochrome P450 reductase) and a single P450. CYP2B4 (cytochrome P450 2B4)-, CYP2E1 (cytochrome P450 2E1)- and CYP1A2 (cytochrome P450 1A2)-mediated activities were measured as a function of POR concentration using reconstituted systems containing different concentrations of P450. Although CYP2B4-dependent activities could be explained by a simple Michaelis-Menten interaction between POR and CYP2B4, both CYP2E1 and CYP1A2 activities generally produced a sigmoidal response as a function of [POR]. Interestingly, the non-Michaelis behaviour of CYP1A2 could be converted into a simple mass-action response by increasing the ionic strength of the buffer. Next, physical interactions between CYP1A2 enzymes were demonstrated in reconstituted systems by chemical cross-linking and in cellular systems by BRET (bioluminescence resonance energy transfer). Cross-linking data were consistent with the kinetic responses in that both were similarly modulated by increasing the ionic strength of the surrounding solution. Taken together, these results show that CYP1A2 forms CYP1A2-CYP1A2 complexes that exhibit altered catalytic activity.     

The cytochromes P450 and b5 and their reductases—Promising targets for structural studies by advanced solid-state NMR spectroscopy

Members of the cytochrome P450 (cyt P450) superfamily of enzymes oxidize a wide array of endogenous and xenobiotic substances to prepare them for excretion. Most of the drugs in use today are metabolized in part by a small set of human cyt P450 isozymes. Consequently, cyt P450s have for a long time received a lot of attention in biochemical and pharmacological research. Cytochrome P450 receives electrons from cytochrome P450 reductase and in selected cases from cytochrome b₅ (cyt b₅). Numerous structural studies of cyt P450s, cyt b₅, and their reductases have given considerable insight into fundamental structure-function relationships. However, structural studies so far have had to rely on truncated variants of the enzymes to make conventional X-ray crystallographic and solution-state NMR techniques applicable. In spite of significant efforts it has not yet been possible to crystallize any of these proteins in their full-length membrane bound forms. The truncated parts of the enzymes are assumed to be α-helical membrane anchors that are essential for some key properties of cyt P450s. In the present contribution we set out with a basic overview on the current status of functional and structural studies. Our main aim is to demonstrate how advanced modern solid-state NMR spectroscopic techniques will be able to make substantial progress in cyt P450 research. Solid-state NMR spectroscopy has sufficiently matured over the last decade to be fully applicable to any membrane protein system. Recent years have seen a remarkable increase in studies on membrane protein structure using a host of solid-state NMR techniques. Solid-state NMR is the only technique available today for structural studies on full-length cyt P450 and full-length cyt b₅. We aim to give a detailed account of modern techniques as applicable to cyt P450 and cyt b₅, to show what has already been possible and what seems to be viable in the very near future.     

Productive and non-productive complexes in cytochrome P450-containing systems

The equilibrium dissociation constants K_D, the complex association / dissociation rate constants (k _on /k _off) and lifetimes of the complexes of redox partners were measured for three cytochrome P450-containing monooxygenase systems (P450cam, P450scc, and P450 2B4) under hydroxylation conditions. The Q parameter representing the ratio of protein-protein complex lifetime (τ _lT ) to time required for a single hydroxylation cycle (τ_turnover) was introduced for estimation of productivity of complexes formed within the systems studied. The Q parameter was insignificantly changed upon transition from the oxidation to hydroxylation conditions. Lifetimes (τ _lT ) for the binary complexes formed within the P450cam and the P450scc systems obligatory requiring an intermediate electron transfer protein between the reductase and cytochrome P450 could not realize hydroxylation reactions for substrates with known τ_turnover and so they were non-productive while the binary complexes formed within the P450 2B4 system, not requiring such intermediate electron-transfer protein, appeared to be productive. Formation of ternary complexes was demonstrated under hydroxylation conditions in all three systems. Analysis of Q values led to the conclusion that the ternary complexes formed within the P450cam and the P450scc systems were productive. In the case of the P450 2B4 system, more than half (about 60%) ternary complexes were also found to be productive.     

Immobilization and activity assay of cytochrome P450 on patterned lipid membranes

We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s.     

Cytochrome b5 involvement in cytochrome P450 monooxygenase activities in house fly microsomes

The involvement of cytochrome b₅ in different cytochrome P450 monooxygenase and palmitoyl CoA desaturase activities in microsomes from insecticide-resistant (LPR) house flies was determined using a specific polyclonal antiserum developed against house fly cytochrome b₅. Anti-b₅ antiserum inhibited the reduction of cytochrome b₅ by NADH-cytochrome b₅ reductase. The antiserum also inhibited palmitoyl CoA desaturase, methoxycoumarin-O-demethylase (MCOD), ethoxycoumarin-O-deethylase (ECOD), and benzo[a]pyrene hydroxylase (aromatic hydrocarbon hydroxylase, AHH) activities. However, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethy-lase (EROD) activities were not affected by this antiserum. These results demonstrate that cytochrome b₅ is involved in fatty acyl CoA desaturase activities and in certain cytochrome P450 monooxygenase activities (i.e., MCOD, ECOD, and AHH) in LPR house fly microsomes. Other cytochrome P450 monooxygenase activities (i.e., MROD and EROD) may not require cytochrome b₅. The results suggest that cytochrome b₅ involvement with cytochrome P450 monooxygenase activities is dependent upon the cytochrome P450 isoform involved. © 1994 Wiley-Liss, Inc.     

Beta sheet 2–alpha helix C loop of cytochrome P450 reductase serves as a docking site for redox partners

As a promiscuous redox partner, the biological role of cytochrome P450 reductase (CPR) depends significantly on protein–protein interactions. We tested a hypothesized CPR docking site by mutating D113, E115, and E116 to alanine and assaying activity toward various electron acceptors as a function of ionic strength. Steady-state cytochrome c studies demonstrated the mutations improved catalytic efficiency and decreased the impact of ionic strength on catalytic parameters when compared to wild type. Based on activity toward 7-ethoxy-4-trifluoro-methylcoumarin, CYP2B1 and CPR favored formation of an active CYP2B1•CPR complex and inactive (CYP2B1)₂•CPR complex until higher ionic strength whereby only the binary complex was observed. The mutations increased dissociation constants only for the binary complex and suppressed the ionic strength effect. Studies with a non-binding substrate, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) suggest changes in activity toward cytochrome c and CYP2B1 reflect alterations in the route of electron transfer caused by the mutations. Electrostatic modeling of catalytic and binding parameters confirmed the importance of D113 and especially the double mutant E115 and E116 as mediators in forming charge–charge interactions between CPR and complex partners.