Substrate-mediated oxygen activation by homoprotocatechuate 2,3-dioxygenase: Intermediates formed by a tyrosine 257 variant

Homoprotocatechuate (HPCA; 3,4-dihydroxyphenylacetate or 4-carboxymethyl catechol) and O₂ bind in adjacent ligand sites of the active site Fe^II of homoprotocatechuate 2,3-dioxygenase (FeHPCD). We have proposed that electron transfer from the chelated aromatic substrate through the Fe^II to O₂ gives both substrates radical character. This would promote reaction between the substrates to form an alkylperoxo intermediate as the first step in aromatic ring cleavage. Several active site amino acids are thought to promote these reactions through acid/base chemistry, hydrogen bonding, and electrostatic interactions. Here the role of Tyr257 is explored by using the Tyr257Phe (Y257F) variant, which decreases k_cat by about 75%. The crystal structure of the FeHPCD-HPCA complex has shown that Tyr257 hydrogen bonds to the deprotonated C2-hydroxyl of HPCA. Stopped-flow studies show that at least two reaction intermediates, termed Y257F _Int1^HPCA and Y257F_Int2^HPCA, accumulate during the Y257F-HPCA + O₂ reaction prior to formation of the ring-cleaved product. Y257F_Int1^HPCA is colorless and is formed as O₂ binds reversibly to the HPCA-enzyme complex. Y257F_Int2^HPCA forms spontaneously from Y257F _Int1^HPCA and displays a chromophore at 425 nm (ε₄₂₅ = 10 500 M^-1 cm^-1). Mössbauer spectra of the intermediates trapped by rapid freeze quench show that both intermediates contain Fe^II. The lack of a chromophore characteristic of a quinone or semiquinone form of HPCA, the presence of Fe^II, and the low O₂ affinity suggest that Y257F_Int1^HPCA is an HPCA-Fe^II-O₂ complex with little electron delocalization onto the O₂. In contrast, the intense spectrum of Y257F_Int2^HPCA suggests the intermediate is most likely an HPCA quinone-Fe^II-(hydro)peroxo species. Steady-state and transient kinetic analyses show that steps of the catalytic cycle are slowed by as much as 100-fold by the mutation. These effects can be rationalized by a failure of Y257F to facilitate the observed distortion of the bound HPCA that is proposed to promote transfer of one electron to O₂.