Abstract
13(S)-Hydroperoxyoctadeca-9(Z),11(E),15(Z)-trienoic acid (13-HPOT) was used to probe the mechanism of the hydroperoxide O-O bond cleavage catalyzed by solubilized and partially purified soybean peroxygenase. When reacted with this ferrihemoprotein, it was converted to 13(S)-hydroxyoctadeca-9(Z),11(E), 15(Z)-trienoic acid (13-HOT) and a single epoxide regio-isomer, i.e. 15,16-cis-epoxy-13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid (15,16-EHOD). In the absence of co-oxidizable substrates, such as oleic acid or thiobenzamide, this latter compound accounted for about two-thirds of the reaction products. 13-HOT and 15,16-EHOD are products of heterolytic scission of the O-O bond of 13-HPOT; no products arising by homolytic scission could be detected. Therefore, soybean peroxygenase catalyzes hydroperoxide reduction exclusively by a heterolytic mechanism leading to a ferryl-oxo complex analogous to peroxidase compound I. In similar experiments, 13(S)-hydroperoxyoctadeca-9(Z),11(E)-dienoic acid gave 13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid and 9,10 epoxy-13(S)-hydroxyoctadec-11(E)-enoic acid. Experiments with 18O-labeled 13-HPOT indicated that about 83% of the oxygen atom incorporated into the epoxide group of 15,16-EHOD, originated from the hydroperoxide group. Moreover, using mixtures of unlabeled and 18O-labeled 13-HPOT, it was established that this transfer takes place predominantly (about 3:1) by an intramolecular process. In the intermolecular reaction 13-HOT, formed after reduction of the hydroperoxide, diffuses from the active site and, after reassociation, is epoxidized at the 15,16-double bond. A unifying mechanistic scheme, which takes into account all of the reactions catalyzed by the peroxygenase, is proposed.
Highlights
0-0 bond of 13-HPOT; no products arisingby homo- this enzyme
Ithas been demonstrated that both homolytic (White et al, 1980; from the active site and, after reassociationi,s epoxi- Blake and Coon, 1981) and heterolytic cleavages take place dized at the 15,lS-doubbloend
We have first established that 13-HPOT was able to act as a n oxygen donor
Summary
Acid (56 Ci/mol) were purchased from Du Pont-New England Nu- Analytical Procedures-Products obtained after reaction of [ 1-14C]. ' 13-HPOTwith peroxygenase were separated by analytical HPLC on The abbreviations used are: 13-HPOD, 13(S)-hydroperoxyocta- a Altech Partisil Silica 10 pm (4.5 X 250 mm) column using a Varian deca-9(2),11(E)-dienoicacid13-HPOT, 13(S)-hydroperoxyocta- 9010 pump. Enzymatic Assays-In a typical procedure to study the transformation of 13-HPOTcatalyzed by peroxygenase,-6 gg of enzyme was incubated a t 26 "C, in 10 mM sodium acetate buffer, pH 5.5, containing 20% glycerol and 0.1% emulphogene (final volume, 200 PI), in the presence of (1-'4C]13-HPOT (300 g ~wi)th or without oleic acid (300 p M ) or thiobenzamide (300 p ~ as) substrates. After the incubation time,the reaction products were extractedwithdichloromethane (more than 95%of the initial radioactive compounds were routinely recovered) and analyzed: (i) by TLC onsilica gel plates using solvent A or (ii) by HPLC as describedabove. Thiobenzamide sulfoxide formation was monitored spectrophotometrically a t 370 nm
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