Involvement of cytochromes in the anaerobic biotransformation of tetrachloromethane by Shewanella putrefaciens 200

Shewanella putrefaciens 200 is an obligate respiratory bacterium that can utilize a variety of terminal electron acceptors, e.g., NO₃^-, NO₂^-, Fe(III), and trimethylamine N-oxide, in the absence of O₂. The bacterium catalyzed the reductive transformation of tetrachloromethane (CT) under anaerobic conditions. The only identified product was trichloromethane (CF), but CF production was not stoichiometric. No dichloromethane, chloromethane, or methane was produced. A chloride mass balance indicated that fully dechlorinated products were not formed. Studies with [¹⁴C]CT suggested that a portion of the transformed CT reacted with biomass to form unidentified soluble and insoluble products. Intermediate production of a trichloromethyl radical can explain observed product distribution without significant CO₂ formation. Evidence suggests that respiratory c-type cytochromes are responsible for the dehalogenation ability of S. putrefaciens 200. Previous growth under microaerobic conditions ([O₂], <2.5 μM) results in (i) a 2.6- fold increase in specific heme c content and (ii) a 2.3-fold increase in specific rates of anaerobic CT transformation. Manipulation of heme content by growth on iron-free medium or medium amended with δ-aminolevulinic acid showed that CT transformation rates increase with increases in specific heme c content. Transformation of CT is inhibited by CO. Dehalogenation studies with periplasmic, cytoplasmic, and membrane fractions indicated that only periplasmic and membrane fractions possessed dehalogenation ability. Cytochromes c were the predominant cytochromes present. Membranes were also found to contain smaller amounts of cytochrome b. Observed CT transformation patterns are consistent with a cometabolic description involving fortuitous CT reduction by reduced c-type cytochromes.