Single-turnover kinetics of 2,3-dihydroxybiphenyl 1,2-dioxygenase reacting with 3-formylcatechol.

2,3-Dihydroxybiphenyl 1,2-dioxygenase (EC 1.13.11.39) from Pseudomonas sp. strain KKS102 (BphC) catalyzes the proximal extradiol cleavage of the catechol ring of 2,3-dihydroxybiphenyl (DHB), a key step in the biodegradation of polychlorinated biphenyl. Because the active site Fe(II) ion of the extradiol dioxygenase is colorless, it has been difficult to monitor the reaction cycle kinetics. Here, we have found that BphC binds strongly the chromophoric substrate 3-formylcatechol (3FC) as a monoanion (Kd=0.8 microM) and cleaves it two orders of magnitude slower compared to DHB under air-saturation conditions. By utilizing 3FC as a probe, the reaction cycle kinetics of BphC was monitored for the first time. The binding of 3FC occurred in a three-step process involving rapid deprotonation of 3FC. The bound monoanionic 3FC reacted slowly with O2 in three steps, occurring in sequence, the ring opening step being the slowest one.