Homoprotocatechuate (HPCA; 3,4-dihydroxyphenylacetate or 4-carboxymethyl catechol) and O2 bind in adjacent ligand sites of the active site FeII of homoprotocatechuate 2,3-dioxygenase (FeHPCD). We have proposed that electron transfer from the chelated aromatic substrate through the FeII 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 kcat 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 Int1HPCA and Y257FInt2HPCA, accumulate during the Y257F-HPCA + O2 reaction prior to formation of the ring-cleaved product. Y257FInt1HPCA is colorless and is formed as O2 binds reversibly to the HPCA-enzyme complex. Y257FInt2HPCA forms spontaneously from Y257F Int1HPCA 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 FeII. The lack of a chromophore characteristic of a quinone or semiquinone form of HPCA, the presence of FeII, and the low O2 affinity suggest that Y257FInt1HPCA is an HPCA-FeII-O2 complex with little electron delocalization onto the O2. In contrast, the intense spectrum of Y257FInt2HPCA suggests the intermediate is most likely an HPCA quinone-FeII-(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.