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PnpC1C2 is an enzyme from the soil bacterium Pseudomonas putida DLL-E4 that is in the pathway for the oxidative catabolism of 4-nitrophenol. PnpC1C2 oxidatively cleaves hydroquinone into -hydroxymuconic semialdehyde. It belongs to the type II hydroquinone dioxygenase family, a relatively uncharacterized group of mononuclear nonheme Fe(II)-dependent enzymes that catalyze oxidative ring-cleavage reactions, which includes the well-studied catechol extradiol dioxygenases as well as the structurally unrelated 2,6-dichlorohydro-quinone dioxygenase (PcpA). Steady-state kinetics studies using UV/Vis spectroscopy were performed to characterize the enzyme specificity towards various substituted hydroquinones. In addition to its native substrate, PnpC1C2 was active towards a variety of monosubstituted hydroquinones. Methyl- and methoxyhydroquinone showed a moderately higher , and chloro- and bromohydroquinone showed a moderately lower , but all had a within an order of magnitude of unsubstituted hydroquinone. Likewise, only small differences in the rates of mechanism-based inactivation were observed among these substrates. Among disubstituted hydroquinones, only 2,6- and 2,5-dimethylhydroquinone showed any activity, with the latter only barely detectable. A variety of para-substituted phenols were found to be good inhibitors of PnpC1C2. NMR studies were performed to determine the regioselectivity of ring-cleavage with monosubstituted hydroquinones. All monosubstituted hydroquinones tested (methyl-, chloro-, bromo-, and methoxyhydroquinone) yielded exclusively the 1,6-cleavage product. Thus, PnpC1C2 shows notable differences in both its substrate specificity and the ring-cleavage regioselectivity compared to that of PcpA. These results provide an important basis for future comparison of structure-function correlations among the hydroquinone ring-cleaving dioxygenases.