Organohalide-respiring microorganisms can use a variety of persistent pollutants, including trichloroethene (TCE), as terminal electron acceptors. The final two-electron transfer step in organohalide respiration is catalyzed by reductive dehalogenases. Here we report the x-ray crystal structure of PceA, an archetypal dehalogenase from Sulfurospirillum multivorans, as well as structures of PceA in complex with TCE and product analogs. The active site harbors a deeply buried norpseudo-B12 cofactor within a nitroreductase fold, also found in a mammalian B12 chaperone. The structures of PceA reveal how a cobalamin supports a reductive haloelimination exploiting a conserved B12-binding scaffold capped by a highly variable substrate-capturing region.

Tetrachloroethene reductive dehalogenase from the tetrachloroethene-utilizing anaerobe, Dehalospirillum multivorans, was purified approximately 100-fold to apparent homogeneity. The purified dehalogenase catalyzed the reductive dechlorination of tetrachloroethene (PCE) to trichloroethene and of trichloroethene to cis-1,2-dichloroethene with reduced methyl viologen as the electron donor at a specific activity of 2.6 microkatal/mg. The apparent Km values for tetrachloroethene and trichloroethene were 0.20 and 0.24 mM, respectively. The apparent molecular mass of the native enzyme was determined by gel filtration to be 58 kDa. Sodium dodecyl sulfate-gel electrophoresis revealed a single protein band with a molecular mass of 57 kDa. One mol of dehalogenase contained 1.0 mol of corrinoid, 9.8 mol of iron, and 8.0 mol of acid-labile sulfur. The pH optimum was about 8.0. The enzyme had a temperature optimum of 42 degrees C. It was slightly oxygen-sensitive and was thermolabile above 50 degrees C. The dechlorination of PCE was stimulated by ammonium ions. Chlorinated methanes severely inhibited PCE dehalogenase activity.

Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B 12 ) was studied. Fractionation was largest during the reaction with cyanocobalamin with ␣C ‫؍‬ 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (␣C ‫؍‬ 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (␣C ‫؍‬ 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, ␣C values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.The chlorinated ethenes tetrachloroethene (PCE) and trichloroethene (TCE) have been used as solvents in the drycleaning industry and as metal degreasing agents and are among the most common groundwater contaminants due to spillage and leakage (12). The assessment of in situ biodegradation of groundwater contaminants is difficult since a decrease in the concentration can be a result of many factors, including dilution, sorption, and biological conversion. Monitoring and assessment of in situ microbial degradation activities of contaminants at polluted sites is therefore a major challenge. In the last few years, the application of stable isotope techniques has been suggested for assessment of the in situ biodegradation of contaminants (for a review, see references 17 and 33).Stable isotope fractionation of PCE and TCE has been observed under field conditions in contaminated aquifers as well as in laboratory studies by mixed microbial cultures and microcosms (2, 9, 39, 40); however, the factors affecting the isotope fractionation have not yet been studied systematically.Several factors may influence the degree of stable isotope fractionation, including biodegrading microorganisms, properties of the dehalogenating enzymes, and the reaction mechanism. Cobalamins are important cofactors of reductive dehalogenases in organisms capable of dehalorespiration (8,10,15,20,25,26). The microbial dehalogenation by cobalamin-containing dehalogenases has been proposed to proceed by alkylating a superreduced corrinoid containing a Co(I) species at the reactive site with the chloroethene (26). The chemical mechanism of the reductive dehalogenation of chlorinated ethenes catalyzed by cobalamin has been the subject of previous studies and has been suggested to occur via a single elect...

No abstract

A structure is proposed for F430M, a non-cristalline methanolysis product of isolates of the nickel-containing, porphinoid factor F430 from Methanobacterium thermoautotrophicum.Crucial to the structure determination are five incorporation experiments with M . thermoautotrophicum (strain Marburg) in which the specifically m~n o -~~C -l a beled biosynthetic precursors (2-13C)-, (3-I3C)-, (4-I3C)-, (5-I3C) ALA (ALA = 6-amino-levulinic acid) and ~-(methyl-'~C)methionine were incorporated into F430 with high efficiency. The I3C-NMR.-spectra of the specifically labeled F430M samples derived therefrom, together with the UV./VIS. spectral data of F430M, contain all the information necessary for the deduction of the constitution of the F430M chromophore, assuming the established pattern of porphinoid biosynthesis to be operative in F430 biosynthesis. IH-NMR. spectroscopy and, in particular, 'H-NMR.-NOE-difference spectroscopy corroborates and completes the constitutional assignments and, furthermore, makes possible an almost complete derivation of the molecule's relative configuration. Schemes 3 and 4 summarize the results of 'H-NMR. spectroscopy, presenting them within the context of the proposed structure for F430M. The assignment of absolute configuration implied in the formula is given preference because of F430M's very close structural and (assumed) biosynthetic relationship to sirohydrochlorin and vitamin BI2 (with respect to ring C, the assignment is based on degradative evidence).According to the proposed structure, the nickel complex F430M possesses an uroporphinoid (Type 111) ligand skeleton with an additional carbocyclic ring and a chromophore system not previously encountered among natural porphinoids. It can be considered to be a (tetrahydro) derivative of the corphin system, combining structural elements of both porphyrins and corrins. I ) Arbeitsgruppe f u r physikalische organische Chemie (Leitung Prof. J . F. M. Oh), Organisch-chemisches Laboratorium der ETHZ. 0018-019X/82/3/0828-38$01.00/0 0 1982 Schweizerische Chemische Gesellschaft HELVETICA CHIMICA ACTA -Vol. 65, Fasc. 3 (1982) -Nr. 81 829 14) Die Strukturen I, J und K zeigen die hier als UV./VIS.-Modellsysteme massgebenden Chromophortypen. u b e r deren UV./VIS.-Spektren vgl. (151. I J K