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 γ-hydroxymuconic semialdehyde. It belongs to the type II hydroquinone dioxygenase family, a relatively uncharacterized group of mononuclear non-heme 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-dichlorohydroquinone 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