(10 mL62), high imidazole buffer (10 mL64). The fractions with A280/A325,five.0 have been pooled, dialysed with 100 mM Tris, one hundred mM KCl, pH 7.4 and also the concentrated PdX protein was frozen to 285uC. The concentrations of ferric P450 (with camphor), PdR and PdX had been determined by their extinction coefficients (e392 = 68.five mM21 cm21, 21 21 21 e454 = ten mM cm , e325 = 15.six mM cm21 respectively).PLOS One particular | www.plosone.orgII) Source of the 2H in BorneolBecause NADH is not the source of electrons for the reduction of camphor, the supply on the hydrogen attached to C2 of borneol was further investigated in assays applying deuterated phosphate buffer (50 mM phosphate in D2O, 150 mM K, pD 7.four). Employing recombinant proteins (P450cam, PdR, and PdX), under midrange oxygenated situations (with air), we detected the enzymatic conversion of camphor to 2Dborneol 12D (Fig. 2a, Table S2) working with 2H NMR. We also detected 5ketocamphor, too because the depletion of NADH (Table S2). Related experiments employing NADD (deuterated nicotinamide cofactor) in nonlabeled phosphate buffer did not yield 2Dborneol [18]. Enzymatic assays in deuterated buffer (with recombinant P450cam, shunted with mCPBA, inside the absence of NADH) also yielded borneol that was deuterated at C2 (Hexo) (Fig. 2a, Table S2). All these experiments result in the conclusion that water will be the supply of Hexo attached to C2 in borneol formed by P450cam.III)O NMR of H2OIf camphor is lowered to borneol by electrons from water, then water needs to be oxidized to hydrogen peroxide. We observed H2O2 together with borneol, roughly within a 1:1 stoichiometric ratio when P450cam was shunted with mCPBA (Table 1, entries 3Water Oxidation by Cytochrome PTable 1. Assays with recombinant proteins: Formation of borneol, 5ketocamphor and 5exo hydroxy camphor below different conditions.4e2 uncoupling (nmol min21 nmol21 P450)Enzymatic assayProducts (nmol min21nmol21 P450) Borneol 5keto camphor 11 2065 ND ND 1664 354612 5exohydroxy camphor 10 9506465 ND ND ND NDNADH consumed (nmol H2O2 formed (nmol min21 nmol 21 P450) min21 nmol 21 P450)O21 air2 rP450 mCPBA3 ArrP450 m CPBA3,4 O2 rP450 mCPBA3,7.564 2869 249628 40461913316270 335613 N/A N/A N/AND 2976103 291629 4446166606235 80610 N/A N/A N/AValues will be the typical of four replicates 6 S.BODIPY-FL Order E.Price of 751470-47-0 50 mM potassium phosphate buffer (pH 7.PMID:23927631 four) was utilised for each of the assays. Experimental facts are included in Material S1. ND = Not Detected; N/A = Not Applicable. 1 The reaction mixture contained recombinant P450cam, PdR and PdX and NADH. Oxygen (99 ) was bubbled into the buffer for 60 seconds prior to the assay. The 4e2 uncoupling was calculated by taking the difference among the total NADH expected and observed. two The reaction mixture contained recombinant P450cam, PdR, PdX, and NADH. Air (charcoal filtered) was bubbled in to the buffer just before the assay. three The assay was performed working with recombinant P450cam and mCPBA as a shunt agent. 4 The buffer was sparged with argon (99 ). five The buffer was treated with oxygen (99 pure, Sigma Aldrich) and assays have been performed working with camphor. doi:ten.1371/journal.pone.0061897.tFigure 2. 2H NMR with the 2Dborneol and 17O NMR within the detection of H217O2. a) 2H NMR of your 2Dborneol obtained from the recombinant proteins incubated in 50 mM deuterated phosphate buffer (pD = 7.4) with camphor and mCPBA. The extracted product was backwashed with H2O. The peak at 7.26 ppm corresponds to CHCl3 in CDCl3. b) 17O NMR spectrum of your incubation mixture in 17O phosphate buffer (pH 6.three) containi.