The hydroquinone 1,2-dioxygenases (HQDOs) are members of a large family of non-heme Fe(II)-containing enzymes that catalyze the oxidative cleavage of an aromatic ring, of which the catechol extradiol dioxygenases are by far the best known and best studied. PnpC1C2 is an HQDO from the soil bacterium Pseudomonas putida DLL-E1 that is involved in the catabolism of 4-nitrophenol. Its native substrate is unsubstituted hydroquinone, but it is active towards a variety of monosubstituted hydroquinones and (to a limited extent) 2,6-dimethylhydroquinone. This enzyme’s stability, high catalytic activity, and moderate promiscuity make it an ideal platform for enzyme engineering.1 We set out to reengineer it to be active towards a wider range of hydroquinone substrates, specifically chlorinated hydroquinones that could have relevance towards bioremediation of legacy chlorinated organic pollutants. As a first step, a series of nine mostly bulky hydrophobic residues that form the substrate binding pocket were varied by site saturation mutagenesis, and the resulting mutants were screened for improved activity towards 2,3-, 2,5- and 2,6-dimethylhydroquinone. Several mutants were found with significantly greater activity towards 2,5- and 2,6-dimethylhydrouinone. These variants were further characterized by steady-state kinetics and found to have excellent catalytic activity towards these substrates and even some activity towards chloromethyl- and dichlorohydroquinones. Although no single-site variants were found to be active towards 2,3-dimethylhydroquinone, some double mutants show promise as being active towards this substrate.