Nitrogenase is the metalloenzyme responsible for the biological reduction of N2 to NH3. Nitrogenase has been shown to reduce a variety of substrates in addition to N2 and protons. General properties of alternative substrates for nitrogenase are the presence of N-N, N-O, N-C, and C-C triple or double bonds. In the present work, we demonstrate that Azotobacter vinelandii nitrogenase can reduce both C-S and C-O bonds. Nitrogenase was found to reduce carbonyl sulfide (COS), to CO and H2S at a maximum rate of 37.2 +/- 2.0 nmol min-1 (mg of protein)-1 with a Km of 3.1 +/- 0.6 mM. The formation of CO from nitrogenase reduction of COS was monitored spectrophotometrically in real time by following the formation of carboxyhemoglobin. In this assay, the change in the visible absorption spectrum of reduced hemoglobin upon binding CO provided a sensitive way to quantify CO formation and to remove CO, which is a potent inhibitor of nitrogenase, from solution. COS reduction by nitrogenase required the molybdenum-iron protein (MoFeP), the iron protein (FeP), and MgATP. The reduction reaction was inhibited by MgADP, acetylene, and N2, while H2 was not an inhibitor of COS reduction. The pH optimum for COS reduction was 6.5. Nitrogenase was also found to reduce carbon dioxide (CO2) to CO and H2O. CO2 was reduced at a maximum rate of 0.8 +/- 0.07 nmole min-1 (mg of protein)-1 with a calculated Km for CO2 of 23.3 +/- 3.7 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

effect on the cells; and (iii) compounds which were cooxidized and produced a turnover-dependent inactivation of ammonia oxidation by N. eluropaea. MATERIALS AND METHODS Growth and preparation of cells. Cells of N. europaea ATCC 19718 were grown in batch cultures (1 to 2 liters) and harvested by centrifugation as described previously (15).

Methanopterin is a folate analog involved in the C 1 metabolism of methanogenic archaea, sulfate-reducing archaea, and methylotrophic bacteria. Although a pathway for methanopterin biosynthesis has been described in methanogens, little is known about the enzymes and genes involved in the biosynthetic pathway. The enzyme ␤-ribofuranosylaminobenzene 5-phosphate synthase (␤-RFAP synthase) catalyzes the first unique step to be identified in the pathway of methanopterin biosynthesis, namely, the condensation of p-aminobenzoic acid with phosphoribosylpyrophosphate to form ␤-RFAP, CO 2 , and inorganic pyrophosphate. The enzyme catalyzing this reaction has not been purified to homogeneity, and the gene encoding ␤-RFAP synthase has not yet been identified. In the present work, we report on the purification to homogeneity of ␤-RFAP synthase. The enzyme was purified from the methane-producing archaeon Methanosarcina thermophila, and the N-terminal sequence of the protein was used to identify corresponding genes from several archaea, including the methanogen Methanococcus jannaschii and the sulfate-reducing archaeon Archaeoglobus fulgidus. The putative ␤-RFAP synthase gene from A. fulgidus was expressed in Escherichia coli, and the enzymatic activity of the recombinant gene product was verified. A BLAST search using the deduced amino acid sequence of the ␤-RFAP synthase gene identified homologs in additional archaea and in a gene cluster required for C 1 metabolism by the bacterium Methylobacterium extorquens. The identification of a gene encoding a potential ␤-RFAP synthase in M. extorquens is the first report of a putative methanopterin biosynthetic gene found in the Bacteria and provides evidence that the pathways of methanopterin biosynthesis in Bacteria and Archaea are similar.Methanopterin is a folate analog involved in the C 1 metabolism of methanogenic archaea, sulfate-reducing archaea, and methylotrophic bacteria (5,7,19,31,39). This coenzyme is used during the production of methane by methanogenic archaea and during the oxidation of growth substrates by sulfurmetabolizing archaea and certain methylotrophic bacteria. The recent discovery of methanopterin in the bacterium Methylobacterium extorquens (5) was surprising because methanopterin had been thought to be exclusive to Archaea. This discovery has raised interesting questions about the evolutionary relationships between archaea and bacteria that use methanopterin. While M. extorquens is the only bacterium in which methanopterin itself has been detected, methanopterin-dependent enzyme activity has been observed in a number of other methylotrophic bacteria (34).One approach to investigating the evolutionary relationships among organisms that use methanopterin is to compare the enzymes and genes involved in methanopterin biosynthesis. At present, the pathway of methanopterin biosynthesis in the Bacteria is unknown. However, a methanopterin biosynthetic pathway has been described for the methane-producing archaeon Methanosarcina thermophila (38, 39). To date, the g...

We have investigated the substrate specificity of ammonia monooxygenase in whole cells of the nitrifying bacterium Nitrosomonas europaea for a number of aliphatic halogenated hydrocarbons. To determine the effect of the halogen substituent and carbon chain length on substrate reactivity, we measured the rates of oxidation of the monohalogenated ethanes (fluoroethane, chloroethane, bromoethane, and iodoethane) and n-chlorinated Cl to C4 alkanes by whole cells of N. europaea. For monohalogenated ethanes, acetaldehyde was the major organic product and little or none of any of the alternate predicted products (2-halogenated alcohols) were detected. The maximum rate of haloethane oxidation increased with decreasing halogen molecular weight from iodoethane to chloroethane (19 to 221 nmol/min per mg of protein). In addition, the amount of substrate required for the highest rate of haloethane oxidation increased with decreasing halogen molecular weight. For the n-chlorinated alkanes, the rate of dechlorination, as measured by the appearance of the corresponding aldehyde product, was greatest for chloroethane and decreased dramatically for chloropropane and chlorobutane (118, 4, and 8 nmol of aldehyde formed per min per mg of protein, respectively). The concentration profiles for halocarbon oxidation by ammonia monooxygenase showed apparent substrate inhibition when ammonia was used as the reductant source. When hydrazine was used as the electron donor, no substrate inhibition was observed, suggesting that the inhibition resulted from reductant limitation.Nitrosomonas europaea is an obligate, chemolithotrophic, nitrifying bacterium which derives all of its energy for growth from the oxidation of ammonia to nitrite (23). The oxidation of ammonia in N. europaea is initiated by the enzyme ammonia monooxygenase (AMO) through a reductant-dependent process, as shown in the following equation (23): NH3 + 02+ XH2 --NH20H + H20 + X In vivo, the reductant for AMO-catalyzed reactions is provided by the oxidation of hydroxylamine to nitrite by hydroxylamine oxidoreductase, as shown by the following equation (23): NH2OH + H20 -NO2-+ 5H+ + 4e-In addition to oxidizing ammonia, whole cells of N. europaea are capable of cooxidizing a broad range of hydrocarbon substrates, including alkanes and alkenes (4, 8, 10, 12, 21), methanol (20), benzene (9), phenol (9), CO (14), and halogenated hydrocarbons (halocarbons) (1,11,19). These oxidations are mediated by AMO. One group of alternative AMO substrates which are of considerable interest are the aliphatic halocarbons. Many of these compounds are industrial chemicals recognized by the U.S. Environmental Protection Agency as priority pollutants (22). At present, there is concern about the presence of these chemicals in domestic water supplies since some of these compounds are potential human carcinogens (2, 16). The ability of N. europaea to degrade halocarbons (1,11,19) clearly points to their possible use in bioremediation schemes, in which the activity of these widely distributed bacteria c...

A central step in the biosynthesis of the modified folate methanopterin is the condensation of p-aminobenzoic acid (pAB) and 5-phospho-alpha-D-ribosyl-1-pyrophosphate (PRPP) which produce 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate (beta-RFA-P) [White, R. H. (1996) Biochemistry 35, 3447-3456]. This reaction, catalyzed by the enzyme beta-RFA-P synthase, is unique among known phosphoribosyltransferases in that a decarboxylation of one of the substrates (pAB) occurs during the reaction and a C-riboside rather than an N-riboside is the product. In this work, the reaction catalyzed by the enzyme from Methanosarcina thermophila is shown to be analogous to other phosphoribosyltransferase reactions in that pyrophosphate is released as a product of the reaction, which is dependent upon magnesium ions. The molecular weight of the enzyme was estimated to be 65 000 using gel filtration chromatography, and the pH optimum was 4.8. Kinetic analysis indicated that the reaction involved a sequential pattern of substrate binding. Benzoic acid and several para-substituted benzoic acids inhibited beta-RFA-P synthase activity, while aniline, 4-aminobenzamide, and the methyl ester of pAB did not, indicating that an ionized carboxylic group plays a role in the binding of pAB. The observation that the enzyme was not inhibited by carbonyl reagents and that 4-hydroxybenzoic acid served as an alternate substrate, producing 4-(beta-D-ribofuranosyl)hydroxybenzene 5'-phosphate as the product, indicated that pyridoxal phosphate was not directly involved in the reaction mechanism. Incubation of the enzyme with PRPP and either pAB or 4-aminothiobenzoic acid in the presence of sodium cyanoborohydride led to the decreased production of beta-RFA-P and the accumulation of a reduced form of the proposed cyclohexadienimine reaction intermediates. These compounds were characterized by their acid-catalyzed decomposition which produces beta-D-ribofuranosylbenzene 5'-phosphate. On the basis of these results, a concerted mechanism is proposed for beta-RFA-P synthase in which an SN1-like reaction produces oxonium ion character at C-1 of PRPP which undergoes an ipso electrophilic aromatic substitution reaction at the carboxylic acid-bound carbon of pAB. Decarboxylation of the resulting cyclohexadienimine intermediate leads to the formation of beta-RFA-P.