Reaction cycle intermediates of Fe(II)-containing aromatic ring-cleaving extradiol dioxygenases are difficult to observe because the enzymes are colorless and EPR silent. Here, a new approach is used to reveal the these intermediates the first time. Homoprotocatechuate 2,3-dioxygenase (HPCD) catalyzes O₂-linked ring cleavage of both homoprotocatechuate (HPCA; 3,4-dihydroxyphenylacetate) and 4-nitrocatechol (4NC) in the same ring position to give semi-aldehyde products. Because 4NC is chromophoric, and the color depends on the ionization state and environment of the ring OH functions, its binding can be directly monitored using stopped flow techniques. Three steps are seen encompassing 4NC association and subsequent deprotonation of both OH functions to form an Fe(II) chelate complex. Three additional intermediates can be detected in 4 subsequent O₂ binding and reaction steps. Mutation of the active site histidine H200 slows the reaction with O₂ sufficiently to observe the oxy-Fe(II) complex for the first time in this enzyme class when using either 4NC or HPCA as the substrate. Crystal structures of substrate and product complexes show that H200 and its mutant forms are in position to interact with both the Fe-bound O₂ and the substrate OH function distal to the site of ring cleavage. Product characterization shows that reaction of the H200 mutant with HPCA gives the expected ring cleavage product, whereas reaction with 4NC gives a quinone. We propose that interaction of H200 with the Fe(II)- O₂ complex promotes formation of the Fe(III)-superoxide reactive form that attacks substrate. Then H200 can act as an acid to promote Criegee O-O bond cleavage and ring-insertion chemistry. This reaction is also promoted by electron donating ring substitutes as in HPCA. If neither electron donating substituents nor an acid catalyst is present, quinone formation rather than ring cleavage results. This highlights the joint roles the Fe, substrate and the active site residues in catalysis.