Journal
BIOCHEMISTRY
Volume 40, Issue 37, Pages 11156-11167Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi011153h
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- NIGMS NIH HHS [GM 20877, GM 11106] Funding Source: Medline
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Cyclohexanone monooxygenase (CHMO), a bacterial flavoenzyme, carries out an oxygen insertion reaction on cyclohexanone to form a seven-membered cyclic product, epsilon -caprolactone. The reaction catalyzed involves the four-electron reduction Of O-2 at the expense of a two-electron oxidation of NADPH and a two-electron oxidation of cyclohexanone to form epsilon -caprolactone. Previous studies suggested the participation of either a flavin C4a-hydroperoxide or a flavin C4a-peroxide intermediate during the enzymatic catalysis [Ryerson, C. C., Ballou, D. P., and Walsh, C. (1982) Biochemistry 21, 2644-2655]. However, there was no kinetic or spectral evidence to distinguish between these two possibilities. In the present work we used double-mixing stopped-flow techniques to show that the C4a-flavin-oxygen adduct, which is formed rapidly from the reaction of oxygen with reduced enzyme in the presence of NADP, can exist in two states. When the reaction is carried out at pH 7.2, the first intermediate is a flavin C4a-peroxide with maximum absorbance at 366 nm; this intermediate becomes protonated at about 3 s(-1) to form what is believed to be the flavin C4a-hydroperoxide with maximum absorbance at 383 nm. These two intermediates can be interconverted by altering the pH, with a pK(a), of 8.4. Thus, at pH 9.0 the flavin C4a-peroxide persists mainly in the deprotonated form. Further kinetic studies also demonstrated that only the flavin C4a-peroxide intermediate could oxygenate the substrate, cyclohexanone. The requirement in catalysis of the deprotonated flavin C4a-peroxide, a nucleophile, is consistent with a Baeyer-Villiger rearrangement mechanism for the enzymatic oxygenation of cyclohexanone. In the course of these studies, the K-d for cyclohexanone to the C(4a)-peroxyflavin form of CHMO was determined to be similar to1 muM. The rate-determining step in catalysis was shown to be the release of NADP from the oxidized enzyme.
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