Elucidation of the catalytic mechanisms of the non-haem iron-dependent catechol dioxygenases: synthesis of carba-analogues for hydroperoxide reactionintermediates

The catalytic mechanisms of the non-haem iron-dependent intradiol and extra diol catechol dioxygenases are thought to involve transient hydroperoxide r eaction intermediates, formed by reaction of a catechol substrate with diox ygen. The synthesis of carba-analogues of these intermediates is described in which the hydroperoxide functional group (-OOH) is replaced by a hydroxy methyl group (-CH2OH), and the cyclohexadienone skeleton simplified to a cy clohexanone. Analogues of the "proximal" hydroperoxide in which the hydroxy methyl group was positioned axially with respect to the ring were found to act as reversible competitive inhibitors (K-i 0.7-7.6 mM) for the extradiol enzyme 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) from Escherich ia coli, whereas analogues in which the hydroxymethyl group was positioned equatorially showed no inhibition. In contrast, assays versus the intradiol -cleaving protocatechuate 3,4-dioxygenase from Pseudomonas sp. showed inhib ition only by an analogue containing an equatorial hydroxymethyl group (IC5 0 9.5 mM). These data support the existence of a proximal hydroperoxide int ermediate in the extradiol catechol dioxygenase mechanism, and suggest that the conformation adopted by the hydroperoxide reaction intermediate may be an important determinant in the reaction specificity of the extradiol and intradiol dioxygenases.