Ng: i) camphor, recombinant P450cam and mCPBA, ii) camphor and recombinant P450cam (mCPBA absent), iii) camphor and mCPBA (enzyme absent), and iv) mCPBA and recombinant P450cam (substrate absent). The peaks at 0 ppm and 178 ppm correspond to H217O and H217O2, respectively. doi:10.1371/journal.pone.0061897.gPLOS A single | www.plosone.orgWater Oxidation by Cytochrome P5) or with other oxidants (Table S5). We ready H217O [19] and incubated the reaction mixture containing 1 mM camphor, 1 mM mCPBA and recombinant P450cam (0.1 mM) in 17O phosphate buffer (50 mM, 150 mM K, pH 7.four produced with H217O) for 12 h to detect the formation of H217O2. To this assay mixture, P450cam (0.02 mM) and mCPBA (0.2 mM) had been 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 inside the literature [20] and of our ready normal [19]. The effect of pH on the chemical shift of hydrogen peroxide was also checked (Fig. S1). Controls (in the absence of mCPBA, enzyme or substrate) had been 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 mCPBA. 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 because of H217O2.IV) Kinetic Isotope Effects (KIE)The reaction catalyzed by P450cam, shunted with mCPBA in D2O, gave 2Dborneol at a significantly slower price than the identical reaction performed in standard water. The magnitude and temperature independence on the 1H/2H kinetic isotope impact (KIE) of ,50 (Fig. 3a, Table S3) suggests that hydrogen transfer via tunnelling could happen at the ratedetermining step inside the reduction of camphor to borneol [21,22,23].1239319-91-5 uses In contrast, the KIE (1H/2H) for hydrogen peroxide formation are significantly smaller sized, suggesting that this product does not type at the ratelimiting step (Fig.Buy2-Hydroxy-4-(hydroxymethyl)benzaldehyde 3b, Table S4).PMID:24257686 V) Reduction MechanismBorneol formation under shunt circumstances is saturable, using a KM = 699688 mM and kcat = 426620 min21 for camphor (Fig. 3c). Similarly, ketocamphor formation below oxygenated shunt circumstances is saturable having a KM = 83610 mM and kcat = 461614 min21 for camphor (Fig. 3d). In D2O buffers, the formation of Dborneol was saturable having a KM = 8026107 mM and kcat = 960.four min21 for camphor (Fig. 3). Ketocamphor formation under oxygenated shunt circumstances is saturable with KM = 11866 mM as well as a comparable kcat = 46566 min21 for camphor (Fig. three). From handle experiments we know that lowering P450cam and camphor with dithionite doesn’t yield any borneol (Fig. S3). Consequently, borneol formation demands oxidation of P450cam, either via shunting or by way of intermediates 2 to 7 of your catalytic cycle (Fig. 1a). Thus, Cpd I must be involved in both borneol and ketocamphor formation (Fig. 1a). We propose that water reduces and protonates Cpd I as a initially step in the borneol cycle, providing protonated Cpd II 13 plus a hydroxyl radical (OHN) (Fig. 4). The formation of OHN in water has been estimated from electrochemical data [24], plus the formation of species 13 from Cpd I has been estimated at DGu = 410 kJ/mol [25]. Thus, the very first step of your proposed reduction mechanism (Steps I and II, Fig. four) requires the abstraction of a hydrogen atom from water by Cpd I to.
