Cosubstrate effects in reductive dehalogenation by Pseudomonas putida G786 expressing cytochrome P-450CAM

Abstract

Cytochrome P-450CAM was shown to be the primary catalyst mediating reductive dehalogenation of polychlorinated ethanes by Pseudomonas putida G786. Under anaerobic conditions, the enzyme catalyzed reductive elimination reactions in vivo with the substrates hexachloroethane, pentachloroethane, and 1,1,1,2-tetrachlorethane; the products were tetrachloroethylene, trichloroethylene, and 1,1-dichloroethylene, respectively. In vivo reaction rates were determined. No reaction was observed with 1,1,2,2-tetrachloroethane or 1,1,1-trichloroethane. Purified cytochrome P-450CAM was used to measure dissociation constants of polychlorinated ethanes for the enzyme active site. Observed rates and dissociation constants were used to predict the course of a reaction with the three substrates simultaneously. Data obtained from experiments with P. putida G786 generally followed the simulated reaction curves. Oxygen suppressed the reductive dechlorination reactions and, in the case of 1,1,1,2-tetrachlorethane, 2,2,2-trichloroacetaldehyde was formed. Significant rates of reductive dechlorination were observed at 5% oxygen suggesting that these reactions could occur under partially aerobic conditions. These studies highlight the potential to use an aerobic bacterium, P. putida G786, under a range of oxygen tensions to reductively dehalogenate mixed wastes which are only degraded at very low rates by obligately anaerobic bacteria.