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Water Oxidation by a Cytochrome P450: Mechanism and Function of the Reaction

Water Oxidation by a Cytochrome P450: Mechanism and Function of the Reaction

  • Brinda Prasad, 
  • Derrick J. Mah, 
  • Andrew R. Lewis, 
  • Erika Plettner


P450cam (CYP101A1) is a bacterial monooxygenase that is known to catalyze the oxidation of camphor, the first committed step in camphor degradation, with simultaneous reduction of oxygen (O2). We report that P450cam catalysis is controlled by oxygen levels: at high O2 concentration, P450cam catalyzes the known oxidation reaction, whereas at low O2 concentration the enzyme catalyzes the reduction of camphor to borneol. We confirmed, using 17O and 2H NMR, that the hydrogen atom added to camphor comes from water, which is oxidized to hydrogen peroxide (H2O2). This is the first time a cytochrome P450 has been observed to catalyze oxidation of water to H2O2, a difficult reaction to catalyze due to its high barrier. The reduction of camphor and simultaneous oxidation of water are likely catalyzed by the iron-oxo intermediate of P450cam, and we present a plausible mechanism that accounts for the 1∶1 borneol:H2O2 stoichiometry we observed. This reaction has an adaptive value to bacteria that express this camphor catabolism pathway, which requires O2, for two reasons: 1) the borneol and H2O2 mixture generated is toxic to other bacteria and 2) borneol down-regulates the expression of P450cam and its electron transfer partners. Since the reaction described here only occurs under low O2 conditions, the down-regulation only occurs when O2 is scarce.


Cytochrome P450 enzymes (P450s or CYPs) belong to a family of heme-thiolate enzymes that couple the reduction of oxygen to the oxidation of non-activated hydrocarbons [1]. The catalytic cycle of cytochrome P450cam [2] (Fig. 1a) starts with binding of camphor to the resting enzyme 1 and expulsion of the axial water molecule to form 2. Enzyme-substrate complex 2 accepts two electrons from the nicotinamide cofactor (NADH) via two redox partner proteins: an iron-sulfur protein, putidaredoxin (PdX), and a flavoprotein, putidaredoxin reductase (PdR) [3]. P450 utilizes the two electrons to reduce oxygen, O2, in a stepwise manner, via intermediates 3 and 4 [4], [5]. This leads to the formation of peroxo complex 5, which is protonated to give hydroperoxo complex 6. Protonation of the distal oxygen of 6 and elimination of water gives rise to a high valent iron-oxo complex 7 known as compound I (Cpd I) [6] (Fig. 1a). The oxygen from 7 is then inserted into a C-H bond of the substrate, giving an alcohol product complexed to the iron, 8. The catalytic cycle is complete when water displaces the product.

Figure 1. The catalytic cycle of P450cam and the formation of the products, 10, 11 and 12.

a) R-H represents the substrate, camphor. i, ii, iii and iv represent the peroxide shunt reaction, four-electron uncoupling, two-electron uncoupling, and the loss of superoxide. b) Under highly oxygenated conditions, P450cam hydroxylates camphor 9 to 5-exo-hydroxy camphor 10 and further to 5-ketocamphor 11, whereas under low oxygen conditions, P450cam reduces camphor to borneol 12.

Instead of proceeding through the complete reduction and splitting of O2, P450 enzymes can be shunted to Cpd I by using oxidants such as cumene hydroperoxide or meta-chloroperbenzoic acid (m-CPBA) (Fig. 1a, path “i”) [7], [8]. Furthermore, there are three alternate pathways that lead to uncoupling of NADH from camphor oxidation. First, Cpd I can be reduced by two electrons, and protonated twice giving the substrate complex 2 and water. This reductive pathway is known as four-electron uncoupling because it requires two NADH equivalents (Fig. 1a, path “ii”) [9], [10], [11]. Second, two-electron uncoupling is the dissociation of H2O2 (Fig. 1a, path “iii”) from the ferric hydroperoxo species 6. Third, superoxide can dissociate from superoxo complex 4 (Fig. 1a, path “iv”) [1].

P450cam (CYP101A1) enables a strain of Pseudomonas putida (a soil bacterium) to use (1R)-(+)-camphor 9 (Fig. 1) as a carbon source, and it oxidises camphor at the 5th position to give 5-exo-hydroxycamphor 10 and 5-ketocamphor 11 (Fig. 1) [12]. Here we describe how P450cam can oxidize water to H2O2 and simultaneously reduce camphor to borneol 12 (Fig. 1b) under low O2 conditions, and how borneol regulates the expression of the P450cam system. Catalytic water oxidation is difficult to achieve, because the reaction is endothermic and has a large barrier. [13], [14], [15] To our knowledge, this is the first description of a cytochrome P450 oxidizing water.

We have observed that at low oxygen concentration, regardless of whether Cpd I forms via reduction of O2 or by shunting with oxidants, P450cam not only produces the oxidation products 10 or 11, but can also reduce camphor to borneol (Fig. 1b) [16]. We have interpreted this reaction to give P. putida an ecological advantage over other non-camphor metabolising bacteria because borneol is bactericidal to non-P450 containing bacteria, but not to P. putida [16]. In this paper, we present the mechanism of the camphor reduction reaction and the regulatory effect of borneol on the expression of P450cam.

Materials and Methods

I) Materials

All solvents were distilled prior to use. Nicotine adenine dinucleotide, reduced (NADH), dithiothreitol (DTT), lysozyme, DNase, RNase, vitamin B1, riboflavin, 5-aminolevulinic acid, hydrogen peroxide (used for assays), protease inhibitors leupeptin, aprotinin, and 4-(2-aminoethyl)-benzenesulfonyl fluoride, butylated hydroxytoluene (BHT), cytochrome P450 CYP3A4 (C-4982), superoxide dismutase (S5639), catalase (C-1345), glucose oxidase (G-2133) were purchased from Sigma. Ethylenediaminetetraacetic acid (EDTA) was purchased from Fisher Scientific. Ferrous sulphate (FeSO4) was purchased from Allied Chemical, Canada. Gas chromatography/mass spectrometry (GC-MS) was carried out on a Varian Saturn 2000 MS equipped with a 30-m SPB-5 column (Supelco, 0.25 mm ID; 0.25 µm film thickness) and the column was programmed as follows: 45°C (0.5 min), 7°C min−1 to 120°C (1 min), 50°C min−1 to 260°C (3 min). Electron impact (EI) spectra were obtained at an emission current of 30 µA, scanning from 50 to 365 amu, with ion storage (SIS mode) 49–375, trap temperature 170°C and transfer line 250°C. UV/Vis spectra were obtained on a Cary 300 Bio UV-visible double beam instrument. NADH utilization rates and hydrogen peroxide formation were measured on a thermostatted Hach DR/4000 U spectrophotometer. Activity assays were carried out at 22°C. Electrophoresis was performed on polyacrylamide gels (14%, 29∶1) with 0.5% SDS (SDS-PAGE). The samples were reduced by treating with 1 µL of DTT stock (31 mg/mL) before loading on gels. Gels were stained with Coomassie Brilliant Blue R (Sigma). Sonication was done using a Branson Ultrasonic sonicator. Centrifugations were carried out with a Beckmann Avanti J-26 XPI centrifuge, equipped with a JLA 8.1000 rotor.

The buffers used were: lysis (20 mM phosphate buffer (K+), pH 7.4 with 1 mM camphor; T-100 (50 mM Tris, 100 mM KCl, pH 7.4); T-400 (50 mM Tris, 400 mM KCl, pH 7.4). Buffers for nickel columns were: rinse buffer (20 mM Tris, pH 8.0); low imidazole buffer (5 mM imidazole, 20 mM Tris, 0.5 M NaCl, pH 8.0); strip buffer (0.1 M Ethylenediaminetetraacetic acid (EDTA)), 0.5 M NaCl, pH 8.0). For P450cam purifications, all buffers contained 1 mM camphor. Substrate-free P450 was prepared by passing the substrate bound enzyme over a Sephadex G-10 column equilibrated with 100 mM 3-(N-morpholino) propane sulfonic acid (MOPS, pH 7.0).

II) Methods

Deuterium (2H) NMR spectra were recorded on a Bruker AVANCE II 600 MHz spectrometer (operating at 92.124 MHz). A Bruker 5 mm TCI cryoprobe was used with samples maintained at a temperature of 298 K. 2H field-locking and field sweep were turned off. Samples were contained in 3 mm diameter MATCH nmr tubes filled to 40 mm (volume ca. 185 µL). Acquisition details: 10,240 transients summed, spectral width 15 ppm, transmitter offset 6.5 ppm, 11054 complex points acquired, 15 degree pulse with recycle delay of 1 s between transients, no decoupling of 1H during FID acquisition. Acquisition time was 14.2 h per spectrum.

The 17O NMR spectra were run on a Bruker AVANCE III 500 MHz NMR spectrometer (operating at 67.808 MHz) equipped with a Bruker 5 mm TBO probe and samples were maintained at a temperature of 298 K. Samples were contained in 5 mm diameter nmr tubes filled to 50 mm (volume ca. 600 µL). Acquisition details: 1,000 or 25,000 transients summed, spectral width 503 ppm, 17O transmitter offset 50 ppm, 1H transmitter offset 4.78 ppm, 32768 complex points acquired, 90 degree pulse with recycle delay of 1 s between transients, and inverse-gated WALTZ-16 composite pulse decoupling of 1H during FID acquisition. Acquisition time was 12 min per spectrum or 300 min (when catalase was present). The chemical shifts (δ) for all compounds are listed in parts per million using the NMR solvent as an internal reference (0 ppm for 17O or 4.78 ppm for 2H).

III) Protein Expression and Purification

E. coli strain BL-21 (DE 3) (Novagen) containing the appropriate plasmid [17] were grown in Luria Broth-ampicillin (LB-amp) medium at 37°C with shaking (250 rpm) to A600 = 0.9–1.0 [17]. At this point, cells were harvested by centrifugation, resuspended in fresh LB-ampicillin medium, and after 2 h of growth, IPTG (240 mg L−1) and trace additives were added. The cultures, except for PdR, were grown for 12 h at 27°C (PdR was grown for 6 h). The cells were harvested by centrifugation (30 min, 7000×g) and stored at −85°C until lysis. Additives were: FeCl2 (0.1 µM), 5-aminolevulinic acid (1 mM), Vitamin B1 (10 µM) for P450; FeCl2 (0.1 µM) and Na2S.9H2O (0.1 µM) for redoxin; riboflavin (1 mM) for reductase.

The lysis steps of P450 and PdR remained the same as described for P. putida [10] except that 1 mM camphor was added to the buffer in which P450 culture was lysed. The dialysed lysate of P450, or PdR was individually subjected to a 20% ammonium sulphate cut to remove the cell debris. The 20% supernatant was then carried forward to 45% ammonium sulphate saturation to isolate the protein. The 20–45% pellet was resuspended in T-100 buffer (50 mM Tris, 100 mM KCl, pH 7.4), camphor (1 mM) was added in the case of P450 and purified by DE-52 (anion exchange column) using a linear gradient with buffer T-100 to T-400, 1 mM camphor and 1 mM β-mercapto ethanol (P450 only) at 1 mL min−1. The fractions with high absorbances at λ392 (in the case of P450), λ454 (in the case of PdR) were checked with SDS-PAGE. The collected fractions were pooled and concentrated using an Amicon ultrafiltration cell equipped with a YM-10 membrane and the concentrated protein was individually loaded onto a S-100 column, eluted with T-100 buffer, 1 mM sucrose, 1 mM camphor (P450 only) at 1 mL min−1. SDS-polyacrylamide gel electrophoresis showed a single band for P450 and PdR.

In the case of PdX, cells from 2 L of culture were lysed in lysis buffer (0.25 M NaCl, 20 mM Tris/HCl, pH 8.0). Lysozyme (10 mg mL−1), DNase (2 mg, Sigma), and RNase (10 mg, Sigma) were added and the solution was stirred for 30 min at 4°C. The lysate was sonicated with 50% duty cycle for 10 minutes, stirred for 10 min at 4°C, and homogenized with a pestle. The homogenized cells were then harvested by centrifugation (10500×g, 30 min) and dialysed with frequent changes of lysis buffer followed by further purification by ammonium sulphate precipitation. The dialysed lysate was subjected to a 20% ammonium sulphate cut to remove the cell debris. The 20% supernatant was then carried forward to 90% ammonium sulphate saturation overnight to isolate the protein. The 20–90% pellet was resuspended in 5 mL of rinse buffer (20 mM Tris HCl, pH 8.5), dialysed against this buffer for 3 h and harvested at 5000 rpm for 5 min. The dialysed supernatant was loaded on a ∼5 cm Ni2+-His bind column and eluted with strip buffer (10 mL×3), low imidazole buffer (10 mL×2), high imidazole buffer (10 mL×4). The fractions with A280/A325<5.0 were pooled, dialysed with 100 mM Tris, 100 mM KCl, pH 7.4 and the concentrated PdX protein was frozen to −85°C. The concentrations of ferric P450 (with camphor), PdR and PdX were determined by their extinction coefficients (ε392 = 68.5 mM−1 cm−1, ε454 = 10 mM−1 cm−1, ε325 = 15.6 mM−1cm−1 respectively).

The procedures for the enzymatic assays with the recombinant proteins, as well as the superposition and docking procedures, using Molecular Operating Environment (MOE, Montréal, Canada) are included in Material S1.

Results and Discussion

I) Reaction Conditions Leading to Formation of Borneol

We have observed that borneol forms as a major product of P450cam when camphor is present and the O2 concentration is low (O2≤2 mg/L, ≤63 µM). In vivo, this occurs when cultures are poorly aerated [16] and, in vitro, this occurs when the buffer is sparged with argon in an open vial. In contrast, the known oxidation products 10 and 11 form at high O2 concentrations (∼9 mg/L = 284 µM). In vivo, this occurs when cultures are well aerated [16] and, in vitro, this occurs when pure O2 is bubbled into the buffer (Fig. 1b). To map the mechanism of the reduction, we have performed experiments with the recombinant proteins (P450cam, PdX, and PdR), isolated from expression in E. coli (Table 1). Assays were carried out in phosphate buffer (50 mM phosphate, 150 mM K+, pH 7.4), with NADH and camphor. Our extinction coefficient values were used for the calculation of the enzyme concentration (Table S1). Under high oxygenation (with pure O2 bubbled into the buffer), we observed 5-exo-hydroxy camphor as a major product (Table 1, entry 1). Similar experiments under mid-range oxygenated conditions (with air-treated buffer) yielded borneol as the only product (Table 1 entry 2). The formation of borneol under these conditions was 34-fold less compared to 5-exo-hydroxy camphor that formed under highly oxygenated conditions, and this could be because of the slower formation of iron-oxo species (Compound I).

Table 1. Assays with recombinant proteins: Formation of borneol, 5-ketocamphor and 5-exo- hydroxy camphor under various conditions.

Under poor buffer oxygenation, in the absence of NADH, P450cam shunted with m-CPBA (Fig. 1a, pathway “i”) reduced camphor to borneol (Table 1 entries 3 and 4). The observation that borneol formed in the absence of NADH indicates that NADH is not the source of electrons for the reduction reaction. Furthermore, shunted P450cam under high buffer oxygenation gave more 5-ketocamphor than borneol (Table 1 entry 5), indicating that O2 levels are important in the regulation of the reaction catalyzed by the enzyme.

II) Source of the 2-H in Borneol

Because NADH is not the source of electrons for the reduction of camphor, the source of the hydrogen attached to C-2 of borneol was further investigated in assays using deuterated phosphate buffer (50 mM phosphate in D2O, 150 mM K+, pD 7.4). Using recombinant proteins (P450cam, PdR, and PdX), under mid-range oxygenated conditions (with air), we detected the enzymatic conversion of camphor to 2-D-borneol 12D (Fig. 2a, Table S2) using 2H NMR. We also detected 5-ketocamphor, as well as the depletion of NADH (Table S2). Similar experiments using NADD (deuterated nicotinamide cofactor) in non-labeled phosphate buffer did not yield 2-D-borneol [18]. Enzymatic assays in deuterated buffer (with recombinant P450cam, shunted with m-CPBA, in the absence of NADH) also yielded borneol that was deuterated at C-2 (Hexo) (Fig. 2a, Table S2). All these experiments lead to the conclusion that water is the source of Hexo attached to C-2 in borneol formed by P450cam.

Figure 2. 2H NMR of the 2-D-borneol and 17O NMR in the detection of H217O2.

a) 2H NMR of the 2-D-borneol obtained from the recombinant proteins incubated in 50 mM deuterated phosphate buffer (pD = 7.4) with camphor and m-CPBA. The extracted product was backwashed with H2O. The peak at 7.26 ppm corresponds to CHCl3 in CDCl3. b) 17O NMR spectrum of the incubation mixture in 17O phosphate buffer (pH 6.3) containing: i) camphor, recombinant P450cam and m-CPBA, ii) camphor and recombinant P450cam (m-CPBA absent), iii) camphor and m-CPBA (enzyme absent), and iv) m-CPBA and recombinant P450cam (substrate absent). The peaks at 0 ppm and 178 ppm correspond to H217O and H217O2, respectively.

III) 17O NMR of H2O2

If camphor is reduced to borneol by electrons from water, then water should be oxidized to hydrogen peroxide. We observed H2O2 along with borneol, approximately in a 1∶1 stoichiometric ratio when P450cam was shunted with m-CPBA (Table 1, entries 3–5) or with other oxidants (Table S5). We prepared H217O [19] and incubated the reaction mixture containing 1 mM camphor, 1 mM m-CPBA and recombinant P450cam (0.1 µM) in 17O phosphate buffer (50 mM, 150 mM K+, pH 7.4 made with H217O) for 12 h to detect the formation of H217O2. To this assay mixture, P450cam (0.02 µM) and m-CPBA (0.2 mM) were added at 2 h intervals, to form detectable amounts of H217O2. A new resonance was observed at 178 ppm in the 17O NMR spectrum, (Fig. 2b(i)) which matched the chemical shift of H217O2 reported in the literature [20] and of our prepared standard [19]. The effect of pH on the chemical shift of hydrogen peroxide was also checked (Fig. S1). Controls (in the absence of m-CPBA, enzyme or substrate) were run simultaneously, and this resonance was not detected (Figs. 2b(ii), 2b(iii) and 2b(iv)), which led us to conclude that the new peak could not have come from the hydrolysis of m-CPBA. When catalase (an enzyme that disproportionates H2O2 to water and O2) was added to the reaction mixture, the resonance at 178 ppm disappeared (Fig. S2 b), confirming that the 178 ppm resonance is due to H217O2.

IV) Kinetic Isotope Effects (KIE)

The reaction catalyzed by P450cam, shunted with m-CPBA in D2O, gave 2-D-borneol at a much slower rate than the same reaction performed in normal water. The magnitude and temperature independence of the 1H/2H kinetic isotope effect (KIE) of ∼50 (Fig. 3a, Table S3) suggests that hydrogen transfer through tunnelling could occur at the rate-determining step in the reduction of camphor to borneol [21], [22], [23]. In contrast, the KIE (1H/2H) for hydrogen peroxide formation are much smaller, suggesting that this product does not form at the rate-limiting step (Fig. 3b, Table S4).

Figure 3. The Kinetic Isotope Effects for borneol and H2O2 and the Michaelis-Menten kinetics in their formation.

a) Ratios vH/vD at different temperatures for borneol and b) for H2O2 formation. c) Michaelis-Menten kinetics for borneol and d) 5-ketocamphor formation, under shunt conditions (with m-CPBA). To ensure a constant high O2 concentration for the 5-ketocamphor formation kinetics, reactions were run in vials fitted with septa and pressurized with pure O2.

V) Reduction Mechanism

Borneol formation under shunt conditions is saturable, with a KM = 699±88 µM and kcat = 426±20 min−1 for camphor (Fig. 3c). Similarly, ketocamphor formation under oxygenated shunt conditions is saturable with a KM = 83±10 µM and kcat = 461±14 min−1 for camphor (Fig. 3d). In D2O buffers, the formation of D-borneol was saturable with a KM = 802±107 µM and kcat = 9±0.4 min−1 for camphor (Fig. 3). Ketocamphor formation under oxygenated shunt conditions is saturable with KM = 118±6 µM and a similar kcat = 465±6 min−1 for camphor (Fig. 3). From control experiments we know that reducing P450cam and camphor with dithionite does not yield any borneol (Fig. S3). Therefore, borneol formation requires oxidation of P450cam, either through shunting or through intermediates 2 to 7 of the catalytic cycle (Fig. 1a). Therefore, Cpd I must be involved in both borneol and ketocamphor formation (Fig. 1a).

We propose that water reduces and protonates Cpd I as a first step in the borneol cycle, giving protonated Cpd II 13 and a hydroxyl radical (OH) (Fig. 4). The formation of OH in water has been estimated from electrochemical data [24], and the formation of species 13 from Cpd I has been estimated at ΔG° = –410 kJ/mol [25]. Therefore, the first step of the proposed reduction mechanism (Steps I and II, Fig. 4) involves the abstraction of a hydrogen atom from water by Cpd I to form the Fe(IV)-OH complex 13 (Fig. 4), which is favourable (ΔH∼−160 kJ/mol) (Fig. 4). Three water molecules are known to be poised above the Fe-porphyrin and are held in place by hydrogen bonds to Thr 252, Asp 251 and Glu 366 [26], so it is plausible that Cpd I could attack water instead of camphor. Next, we propose that the hydroxyl radical combines with the water molecule to yield hydrogen peroxide and a hydrogen atom (Step III). By our estimate, this step is highly unfavourable (ΔH° ≅ 570 kJ/mol, Material S1, section 2.9). Simultaneous transfer of the hydrogen atom from step III to the carbonyl group of camphor forms a borneol radical (Step IV, Fig. 4). A non-strained ketone such as acetone reacting with a hydrogen atom has a potential of approximately −2 V (ΔG° is +173 kJ/mol) [27]. However, because camphor is quite a strained ketone, and that strain is relieved by the reduction, we have estimated this reaction to be slightly favourable (ΔH°∼−79±8 kJ/mol, Material S1, section 2.9). Finally, the transfer of a hydrogen atom from protonated Cpd II to the borneol radical forms borneol and Cpd I (Steps V and VI, Fig. 4) (ΔH° ≅ 13 kJ/mol), completing the “borneol cycle”. The net reaction is endothermic, with ΔH° ≅ 305±8 kJ/mol (Material S1, section 2.9, Fig. S4a).

Figure 4. The proposed reduction mechanism and the Born-Haber estimates in the mechanism.

a) Proposed reduction mechanism of P450cam that accounts for the simultaneous formation of borneol 12 and H2O2, in a 1∶1 stoichiometry. b) Born-Haber cycle estimates of the reduction mechanism.

The involvement of OH-radicals in H2O2 formation has been proposed previously for electrolytic catalysts that oxidize water to O2 (via an intermediate peroxide) [13] and also for a recently discovered water oxidation catalyst that produces H2O2 during electrolysis [14], [15]. Interestingly, the latter MnOx catalyst stops the water oxidation process at H2O2, because the peroxide is solvated and stabilized by hydrogen bonding to ethylamine and/or water in the electrolyte. [14], [15] Analogously, here we propose that hydrogen bonding within the water cluster in the hydrophobic P450cam active site is essential for stabilization of the various reactive intermediates and of the H2O2 formed. The turnover numbers with regard to H2O2 formation we have observed are ∼7, whereas the electrocatalytic systems give turnover numbers of 20–1500 for complete water oxidation to O2. [13] This difference arises because P450cam only has access to thermal energy to perform this “uphill” reaction, whereas the electrocatalytic systems are run at overpotentials. [13], [14], [15].

The proposed mechanism accounts for our observation that borneol and hydrogen peroxide form in a 1∶1 stoichiometric ratio, provided 2-electron uncoupling is negligible (Table 1, Table S5). Given that Cpd I appears to be involved in the borneol cycle, our previous [16] and current data also suggest that Cpd I might be regulated by O2 levels: under high oxygenation, Cpd I sequentially hydroxylates camphor to 10, or 10 to 11 (Fig. 1b). Under poor oxygenation, Cpd I enters the borneol cycle that couples the oxidation of water to H2O2 to the simultaneous reduction of camphor to borneol (Fig. 1b). The borneol cycle is independent of how Cpd I is generated: through the reduction of O2 or the shunt pathway (Fig. 1a). Borneol formation was seen with all the shunt agents tested (m-CPBA, cumene hydroperoxide, periodate and bleach; Table S5).

In assays with CYP3A4 (a human cytochrome P450) under shunt conditions, 5-exo-hydroxy camphor formed as a major product. There were no detectable amounts of borneol, suggesting that the reduction cycle is specific to P450cam (Material S1, section 2.5). A BLAST search against the P450cam sequence revealed many other bacterial cytochromes P450 that show sequence identities for the three residues that hold a set of water molecules above the porphyrin (Asp 251, Thr 252 and Glu 366 in P450cam, Fig. S5), as well as for the hydrophobic residues that are involved in O2 binding (see below and Material S1, section 2.10). Superposition of P450cam (1DZ4, [28] on CYP3A4 (1TZN, [29] reveals that the active site of CYP3A4 is much larger and more polar than that of P450cam. In P450cam, camphor is surrounded by closely packed hydrophobic residues, which could form a cage around the reactive intermediates. (Fig. 5) The only water in the active site of the camphor-bound structure is in the water channel between Glu 366 and Thr 252, whereas the CYP3A4 active site can hold numerous water molecules in the absence of a ligand (Fig. 6). Docking of camphor into the active site of CYP 3A4 reveals the camphor bound near the porphyrin, capped by five phenylalanine residues and surrounded by Arg 212, Ser 119, Ile 120, Ile 301 and H-bonded to Arg 105 (Fig. 6). This more open arrangement may not provide the necessary stabilization for water oxidation to occur. Furthermore, the different positioning of the camphor within the active site may also preclude its utilization as an electron acceptor during the water oxidation and, therefore, the reaction was not observed in CYP3A4.

Figure 5. Superposition of P450cam and CYP3A4.

a) Top row: superimposed ribbon diagrams of P450cam (1DZ4) and CYP3A4 (1TQN). P450cam is shown with red helices and yellow sheets, whereas CYP3A4 is shown all in pink. The porphyrin for P450cam is shown in gray and the one for CYP3A4, in brown. The two views are orthogonal to each other. The substrate access channel (SAC) is marked, as is Helix I, the central pillar of the fold. b) Lower row: superimposed active sites of P450cam and CYP3A4. The porphyrin of P450cam is shown in gray, the one for CYP3A4 in brown. The camphor ligand of P450cam is shown in green. Residues from the two proteins are red (P450cam) and pink (CYP3A4). The two views are orthogonal to each other.

Figure 6. Sites in P450cam and in CYP3A4 with camphor docked.

a) Oxygen binding site in P450cam (residues shown in red), with superimposed residues in CYP3A4 shown in pink. The porphyrin of P450cam is gray, and the one for CYP3A4 is brown. b) Water channel in P450cam (residues shown in red), with superimposed residues in CYP 3A4 shown in pink. The view in a) and b) are from a similar angle, to emphasize the closeness of the O2 binding site and the water channel in P450cam. c) and d) Camphor docked into the active site of CYP3A4 (orthogonal views). The H-bond from Arg 105 to the camphor ketone can be seen in the lower right portion of d).

Amunom et. al. have stated that mammalian P450s can reduce 4-hydroxynonenal to 1,4-dihydroxynonenal under low oxygen conditions, [30] similar to our results presented in this paper. However, differences in the reaction mechanisms can be associated with the different reacting species of the P450. They proposed that the reduction they observed occurs via the ferrous (Fe (II)) species of P450, where the electron source is from NADPH through the NADPH-P450 reductase. We have shown that, in our case, the ferrous species is not involved (Fig. 4) We therefore propose that the reduction of camphor to borneol involves the iron-oxo species where the source of electrons is from water, and not from NADH. Kaspera et. al. have stated that P450BM-3 from Bacillus megaterium can reduce p-methoxy-benzaldehyde to methoxy-benzalcohol [31]. Electrons for this reaction are provided by a direct hydride transfer from NADPH to the aldehyde, or by NADPH reduction of the flavin mononucleotide in the reductase, which then reduces the substrate. In comparison, we found that the source of electrons in our case is clearly from water, and not from a direct hydride transfer.

VI) Control Experiments with Reactive O2 Species/quenchers

In vitro assays with P450 under shunt conditions were performed with a free radical quencher (BHT), a free metal chelator (EDTA), catalase and superoxide dismutase, to determine whether free reactive oxygen species are involved in borneol formation (Table 2). Under shunt conditions using m-CPBA, in Ar-sparged buffer, the enzyme formed more borneol than 5-ketocamphor (Table 1, entry 4; Table 2, entry 1). In the presence of catalase (Table 2, entry 2), the borneol formed was ∼50% lower due to the decomposition of H2O2 to O2. We confirmed that O2, but not H2O2, had an effect in lowering borneol formation by performing experiments with an O2 scavenging system (glucose/glucose oxidase) (Table 2, entry 3). With catalase alone, the O2 formed by the catalase-mediated decomposition of H2O2 regulated the enzyme such that it produced some ketocamphor. In contrast, in the presence of catalase and glucose oxidase/glucose, the O2 was destroyed and no ketocamphor formed. To check if superoxide plays a role in borneol formation, we performed experiments with superoxide dismutase and detected no significant effect in borneol formation (Table 2, entry 4). To check if the radicals proposed in the mechanism of the reduction (Fig. 4) can diffuse out of the P450’s active site, we experimented with BHT, and noticed no significant effect (Table 2, entry 5). This suggests that any radical species involved in the borneol cycle do not exist long enough to diffuse out of the active site of P450cam. To test if a metal impurity plays a role in our assays under shunt conditions, experiments were performed with EDTA, and we detected no effect on borneol formation (Table 2, entry 6). To check if free iron (outside of the active site) plays a role in reduction reaction, experiments were performed with ferrous sulphate and m-CPBA, in the absence of P450cam, and we did not detect borneol or 5-ketocamphor (Table 2, entry 7). These experiments suggest that the reduction of camphor to borneol is catalysed by P450cam alone, does not involve any adventitious metal species outside of the P450 active site and does not involve the diffusion of reactive oxygen species, other than the product H2O2, out of the active site.

Table 2. Tests for involvement of free reactive oxygen species: formation of borneol, 5-ketocamphor and H2O2.*

VII) Role of Oxygen in the Borneol Cycle

The reaction path taken by P450cam (camphor oxidation vs. borneol cycle, Fig. 1b) is controlled by oxygen concentration. Oxygen could exert its effect in two ways: 1) by affecting the interaction of P450 with its redox partners, or 2) by directly interacting with P450. Our results demonstrate that the former cannot be the case, because the effect was seen in the absence of PdX and PdR (Table 1). Therefore, P450cam must bind O2 not only for catalysis, but also for allosteric regulation.

Recently, cytochrome P450 2E1 has been shown to form endoperoxide rearrangement products when reacted with 1,1,2,2-tetramethylcyclopropane [32]. This suggests that there must be O2 bound in that enzyme near the active site, which reacts with the rearranged radical formed by H-atom abstraction from 1,1,2,2-tetramethylcyclopropane. Cytochromes P450 are known to be allosterically regulated by their substrates or co-substrates [33]. Studies with other O2-utilizing enzymes, such as diiron monooxygenases [34], laccase [35] and amine oxidase [36] have revealed that O2 can be bound in hydrophobic tunnels that are separated from the access channel for the other substrates of these enzymes. In P450cam, a hydrophobic O2 entry channel and two O2 binding cavities have been identified in Xe-treated crystals [37]. Two Xe atoms are bound near the porphyrin edge in a hydrophobic pocket lined by F163, A167, heme allyl, I220, A219, C242 and L245. The other two Xe atoms appear bound in a crevasse lined by L371, T370, L257, M261, water and S260 (first Xe) and I275, K372, T376, L375, L371, P278 and I281 (second Xe). This O2 binding site in P450cam is located near the edge of the porphyrin, near the water channel (Fig. 6 and 1 A. and B).

We have found a hydrophobic tunnel in P450cam that includes the Xe binding sites, using MOLEonline 2.0 [38] on the structure believed to represent the P450cam oxo complex (1DZ9). The binding sites are good candidates for O2 binding because they are hydrophobic and distinct from the substrate access route [11], [37]. Also, the sites are good candidates for allosteric regulation of P450 because they are near the plane of the porphyrin. It is plausible that an O2 molecule bound near the heme could affect the reactivity of Cpd I.

The O2 binding site in P450cam is closer to the porphyrin than the equivalent site in CYP3A4, and the O2 binding site is lined by different residues (Fig. 6). Furthermore, the O2 binding site in P450cam is close to the water channel, the only source of water in the camphor-filled active site of P450cam. It is reasonable to hypothesize that the O2 site, the porphyrin, the water channel and the tightly held camphor, all of which are near each other, could affect each other by allosteric effects in P450cam.

It is interesting to note that the KM for ketocamphor formation under high O2 concentration is 9-fold lower (see above) than that for borneol formation under low O2 concentration. This suggests that camphor binding and possibly positioning might be affected by O2 concentrations. Surprisingly, the kcat is the same for both reactions, even though there appears to be a larger barrier in the borneol cycle than in the normal oxidation reaction. This larger-than-expected kcat suggests that, consistent with the observed KIE, H-atom tunneling is occurring in the borneol cycle. Under high O2 concentrations using D2O as the solvent, 5-ketocamphor (Table S2) was detected as the only product suggesting that deuterium atoms from the solvent do not participate in that reaction. Steady-state kinetic assays for ketocamphor formation in D2O buffers resulted in similar kcat as in H2O buffers. In contrast, a 60-fold decrease in kcat (with a similar KM) was detected for borneol formation (Fig. 3). This illustrates that the solvent molecules participate only in the borneol formation, but not in ketocamphor formation.

There are two ways the cycle could end. Cpd I might oxidize a nearby enzymatic residue or, alternatively, the borneol radical might abstract a H-atom from water, giving borneol and OH, and the hydroxyl radical could rebind with the OH bound in Cpd II-H, to give a second H2O2 and the ferric enzyme (Fig. S4 b).

VIII) Adaptive Advantage of Borneol and H2O2 to P. putida

Previously we have determined the effect of borneol and camphor on the growth of P. putida and E. coli [16]. To determine the effects of hydrogen peroxide, we have tested the toxicity of H2O2 and a 1∶1 stoichiometric mixture of borneol and H2O2 on both P. putida and E. coli, a bacterium that lacks cytochrome P450 [39] (Figs. S6 and S7). The borneol/H2O2 mixture was lethal to E. coli and slightly toxic to P. putida (Fig. S7). The latter observation prompted us to explore whether borneol affects the expression of the P450cam system.

The camphor metabolism pathway, of which P450cam catalyzes the first step, is encoded on the Cam plasmid under the control of the Cam repressor. This repressor dissociates from the upstream control region of the Cam operon upon binding of camphor, ensuring that the entire operon is expressed when camphor is present [40]. To study this induction, we cultured P. putida in the absence of camphor for seven generations, then divided the culture and treated the sub-cultures as shown in Fig. 7a (with camphor, borneol or vehicle, dimethyl sulfoxide (DMSO)). We detected a steep increase in the characteristic absorption bands of P450cam, PdR, and PdX only in the culture induced with camphor, about 80 min after initial induction. Absorptions plummeted approximately 60 min after the addition of borneol to camphor-induced culture(s) (Fig. 7b, Figs. S8 and S9). This decrease in P450, PdR, and PdX expression must be due to the borneol addition, because the camphor-induced cultures that did not receive borneol expressed significantly higher levels of P450, PdR and PdX/CFU/mL than the borneol-treated cultures.

Figure 7. The effect of camphor, borneol and DMSO on the P450 expression.

a) Outline of the experiment used to determine the effect of camphor and borneol on P450cam, PdX and PdR expression. b) The effect of camphor, borneol and DMSO on the P450 expression by Pseudomonas putida (ATCC 17453). The concentration of P450cam was obtained from the Soret peak absorbances and was normalized against the number of colony forming units/mL. Points represent the average ± S. E. of three replicates.

The borneol down-regulation of P450cam, PdX, and PdR might be advantageous to P. putida during periods of low soil aeration. Because the camphor degradation pathway requires four O2/camphor (to reach 5-hydroxy-3,4,4-trimethyl-2-heptenedioic acid-δ-lactone), and the P450cam-catalyzed oxidation is the first committed step [41], it is advantageous to regulate camphor metabolism at the first step. When aeration increases, low levels of P450cam convert borneol back to camphor [16], and this frees the Cam operon from borneol down-regulation.


We describe the borneol cycle of P450cam, a cycle that occurs at low O2 concentration. The cycle connects to the known catalytic cycle via Cpd I which is regulated by O2 levels: at low O2 concentration, Cpd I oxidises water, whereas at high O2 concentration, Cpd I oxidises camphor. Under low O2 concentrations, the slower formation of compound I and high energy barrier with tunneling could account for lower formation of borneol.

The Cpd I-catalyzed reaction of P450cam proposed here (Fig. 4) is independent of the redox partner proteins (PdX and PdR) and of how Cpd I forms (O2 reduction or shunt). The reaction occurs both in vitro (this paper) and in vivo [16]. We show here that P450cam couples the oxidation of water to H2O2 and the reduction of camphor to borneol. We have presented evidence that: i) water is the source of the 2-H in the borneol; ii) water is oxidized to form H2O2 when camphor is reduced; and iii) the transfer of an H atom from water to C-2 of camphor occurs at a rate-limiting step in the borneol cycle. We propose that the reactivity of Cpd I is regulated by O2 concentration, and we have located a potential access channel where O2 might bind to P450cam to exert its allosteric control.

The borneol and H2O2 formed serve several ecological functions. First, borneol and H2O2 are not very toxic to P. putida, whereas the combination is lethal to bacteria such as E. coli which do not contain any P450 [16], and this may give P. putida an advantage in bacterial communities. Secondly camphor induces the expression of P450, PdX, and PdR, whereas borneol decreases the expression of these gene products in P. putida. These features of the P450cam may protect the bacteria from excessive exposure to borneol and reactive oxygen species during prolonged periods of low oxygen concentration.

Supporting Information

Figure S1.

17O NMR spectra of H217O2 (obtained by electrolysis of H217O) buffered at a) pH 10, b) pH 3, and c) pH 9.


Figure S2.

17O NMR spectra of the incubation mixture under shunt conditions using 1 mM m-CPBA in 17O phosphate buffer (final pH 6.3) containing 1 mM substrate camphor and recombinant P450cam.: a) before and b) after addition of catalase (1 unit). The peak at 178 ppm corresponds to H217O2 and that at 0 ppm is due to H217O.


Figure S3.

GC-MS traces of camphor incubated under shunt conditions using m-CPBA with: a) reduced P450cam, and b) with unreduced P450cam. 1-indanone was used as an internal standard.


Figure S4.

a) Summary of the borneol cycle steps and of the net reaction. b) Possible routes by which the borneol cycle could end.


Figure S5.

Alignment of microbial cytochromes P450 against P450cam (upper portion) and of vertebrate class II P450s, also against P450cam (lower portion). Microbial sequences used: gamma prot 1 = marine gamma proteobacterium HTCC2207 (ZP_01225512), Novo ar CYP = Novosphingbium aromaticivorans CYP 101D2 (PDB 3NV6), Sphingo echi = Sphingomonas echinoides ATCC14820 (ZP_10341012), Novo CYP 101D1 = a camphor hydroxylase from Novosphingobium aromaticivorans DSM 12444 (PDB 3LXI), Sphing chlor = Sphingomonas chlorophenolicum camphor hydroxylase (ZP_10341012), Azospir B510 = Azospirillium sp. B510 (YP_003451823), Azospir = (BAI74843), P450 Burk H160 = Burkholderia sp. H160 (ZP_03264429), P450 Burk MCO-3 Burkholderia cenocepacia MC0-3 = (YP_001774494), Sping Witt R = Sphingomonas wittichii RW1 (YP_001262244), Citromicrobi = Citromicrobium bathyomarinum JL354 (ZP_06860768), Novo CYP 101 = Novosphingobium aromaticivorans DSM12444 CYP 101C1 (PDB 3OFT_C), Sping E 14820 = Sphingomonas echinoides ATCC 14820 (ZP_10339023), gamma prot 2 = marine gamma proteobacterium NOR51-B (ZP_04956740), Sphingomonas = Sphingomonas sp. KC8 (ZP_09138048), Sphing chl L = Sphingobium chlorophenolicum L-1 (YP_004553185), P450 nor = Cytochrome P450nor from Fusarium oxysporum (BAA03390). Vertebrate P450s: Cyp lan deme = lanosterol 14-α demethylase isoform 1 precursor Homo sapiens (NP_000777), CYP 2C9 = human liver limonene hydroxylase (P11712), CYP 4A11 Homo sapiens (NP_000769), CYP 4F12 = fatty acyl Ω-hydroxylase Homo sapiens (NP_076433), CYP 4F2 = leukotriene-B(4) omega-hydroxylase 1 precursor Homo sapiens (NP_001073), CYP 3A5 form 1 = CYP 3A5 isoform 1 Homo sapiens (NP_000768), CYP 3A4 = CYP 3A4 isoform 1 Homo sapiens (NP_059488), CYP26B1 = retinoic acid hydroxylase Homo sapiens (NP_063938).


Figure S6.

IC50 determination of a) H2O2 and b) of a 1∶1 (molar) mixture of borneol and H2O2 against E. coli, a species of bacterium that lacks cytochrome P450.


Figure S7.

Effect of 16 h incubation of stationary E. coli (a) and P. putida (b) cultures with borneol: H2O2 (1∶1), borneol, or H2O2 (1 mM).


Figure S8.

Expression profile of PdX in P. putida, in the presence and absence of camphor or borneol (see experimental map and symbols in Fig. 7a). Points represent the average ± S. E. of three replicates.


Figure S9.

Expression profile of PdR in P. putida, in the presence and absence of camphor or borneol (see experimental map and symbols in Fig. 7a). Points represent the average ± S. E. of three replicates.


Table S1.

Calculated and literature values of P450cam extinction coefficients at selected wavelengths.


Table S2.

Formation of 2-D-borneol and 5-ketocamphor in D2O buffer, with the full P450cam system and with the shunted P450cam.


Table S3.

Assays with recombinant proteins at selected temperatures. Formation of borneol, D-borneol, under shunt conditions with the addition of m-CPBA.


Table S4.

Assays with recombinant P450cam, shunted with m-CPBA in H2O and D2O at selected temperatures. Formation of H2O2 or D2O2.


Table S5.

Formation of borneol and hydrogen peroxide from the P450 catalytic cycle using several shunt agents.


Material S1.

Supporting information for this paper. This file contains detailed descriptions of various assays, calculations, sequence alignments and P450cam system induction experiments. In addition, there are 9 supplemental Figures and 5 supplemental Tables.



We thank the second reviewer for discussion and good suggestions, Mr. Colin Zhang for running 2H NMR and the late Dr. Keith Slessor for discussion.

Author Contributions

Conceived and designed the experiments: BP DM ARL EP. Performed the experiments: BP DM ARL. Analyzed the data: BP DM ARL EP. Contributed reagents/materials/analysis tools: BP DM ARL EP. Wrote the paper: BP DM ARL EP.


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