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The results indicate that oxidation of CO to CO 2 supplies electrons for reduction of CO 2 to a methyl group by steps and enzymes of the pathway for CO 2 reduction determined for other methane-producing species. However, proteomic and quantitative RT-PCR results suggest that reduction of the methyl group to methane involves novel methyltransferases and a coenzyme F 420H2:heterodisulfide oxidoreductase system that generates a proton gradient for ATP synthesis not previously described for pathways reducing CO 2 to methane. Biochemical assays support a role for the oxidoreductase, and transcriptional mapping identified an unusual operon structure encoding the oxidoreductase. The proteomic results further indicate that acetate is synthesized from the methyl group and CO by a reversal of initial steps in the pathway for conversion of acetate to methane that yields ATP by substrate level phosphorylation. The results indicate that M. acetivorans utilizes a pathway distinct from all known CO 2 reduction pathways for methane formation that reflects an adaptation to the marine environment. Finally, the pathway supports the basis for a recently proposed primitive CO-dependent energy-conservation cycle that drove and directed the early evolution of life on Earth.anaerobic ͉ Archaea ͉ carbon monoxide C arbon monoxide (CO), an atmospheric pollutant that binds tightly to hemoglobin, is held below toxic levels in part by both aerobic and anaerobic microbes (1). The microbial metabolism of CO is an important component of the global carbon cycle (1, 2), and CO is believed to have been present in the atmosphere of early Earth that fueled the evolution of primitive metabolisms (3-7). Investigations of aerobic species from the Bacteria domain have contributed important insights into microbial CO oxidation (8, 9), as have investigations of anaerobes from the Bacteria domain that conserve energy by coupling CO oxidation to H 2 evolution (10-12). Further understanding has been derived from studies of CO-using anaerobes from the Bacteria domain that conserve energy by oxidizing CO and reducing CO 2 to acetate (13,14) or reducing sulfate to sulfide (15). Far less is known for pathways of the few CO-using species in the Archaea domain that have been described. Methanothermobacter thermautotrophicus, Methanosarcina barkeri, and Methanosarcina acetivorans obtain energy for growth by converting CO to methane (16)(17)(18)(19)(20). Although methane formation from CO first was reported in 1947 (21), a comprehensive understanding of the overall pathway for any species has not been reported. It is postulated that M. barkeri oxidizes CO to H 2 , and the H 2 is reoxidized to provide electrons for reducing CO 2 to methane (16). It is postulated further that H 2 production is essential for ATP synthesis during growth on CO (16,22,23). M. acetivorans was isolated from marine sediments where giant kelp is decomposed to methane (24). The flotation bladders of kelp contain CO that is a presumed substrate for M. acetivorans in nature. M. acetivorans produ...

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The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio-and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. The naphthalene dioxygenase (NDO) enzyme system (EC 1.14.12.12) from Pseudomonas sp. strain NCIB 9816-4 catalyzes the first step in the aerobic degradation of naphthalene. In this reaction ( Fig. 1), NDO adds both atoms of oxygen to the aromatic nucleus of naphthalene, forming homochiral (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol) (30, 31). In addition, NDO catalyzes the oxidation of a wide variety of aromatic compounds to enantiomerically pure chiral products (8, 56). The NDO system consists of three components, each of which has been purified and characterized. An iron-sulfur flavoprotein reductase and an iron-sulfur ferredoxin transfer electrons from NAD(P)H to the catalytic oxygenase component (14,15,23,24). The oxygenase is composed of large and small subunits, ␣ and ␤, respectively, that are in an ␣ 3 ␤ 3 configuration (35). NDO is a member of a large family of oxygenases whose ␣ subunits contain a Rieske [2Fe-2S] center and mononuclear nonheme iron (10). In the NDO system, electrons are transferred from the Rieske center of the ferredoxin to the Rieske center of the oxygenase ␣ subunit. The reduced Rieske center in one ␣ subunit transfers an electron to mononuclear iron at the active site in an adjacent ␣ subunit (35, 50). His-208, His-213, and Asp-362 coordinate the active-site iron, forming a 2-His-1-carboxylate facial triad. This structural motif is found in other mononuclear nonheme iron enzymes, including tyrosine hydroxylase, isopenicillin synthetase, and 2,3-dihydroxybiphenyl 1,2-dioxygenase (26, 40). Asp-205 in the catalytic domain of the NDO ␣ subunit is hydrogen bonded to His-208 and to His-104 in the adjacent ␣ subunit (Fig. 2). His-104 is one of the Rieske center ligands. Asp-205 has been shown to be required for efficient electron transfer from the Rieske center to the active-site iron (50).Recent studies have shown that the oxygenase ␣ subunits are responsible for determining the substrate specificities of NDO and the related enzymes 2-nitrotoluene dioxygenase (2NTDO) from Pseudomonas sp. strain JS42 and 2,4-dinitrotoluene dioxygenase (DNTDO) from Burkholderia sp. strain DNT (48...

Comamonas sp. strain JS765 can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. We report here the sequence of the genes encoding nitrobenzene dioxygenase (NBDO), which catalyzes the first step in the degradation of nitrobenzene by strain JS765. The components of NBDO were designated Reductase NBZ , Ferredoxin NBZ , Oxygenase NBZ␣ , and Oxygenase NBZ␤ , with the gene designations nbzAa, nbzAb, nbzAc, and nbzAd, respectively. Sequence analysis showed that the components of NBDO have a high level of homology with the naphthalene family of Rieske nonheme iron oxygenases, in particular, 2-nitrotoluene dioxygenase from Pseudomonas sp. strain JS42. The enzyme oxidizes a wide range of substrates, and relative reaction rates with partially purified Oxygenase NBZ revealed a preference for 3-nitrotoluene, which was shown to be a growth substrate for JS765. NBDO is the first member of the naphthalene family of Rieske nonheme iron oxygenases reported to oxidize all of the isomers of mono-and dinitrotoluenes with the concomitant release of nitrite.Nitroaromatic compounds are used extensively as industrial feedstocks for many manufacturing processes, including the production of pesticides, dyes, and explosives (11). Due to improper storage, use, and disposal, nitroaromatic compounds have been released into the environment, where they are considered environmental pollutants. For example, nitrobenzene and 2,4-and 2,6-dinitrotoluene are included in the U.S. Environmental Protection Agency's list of priority pollutants (14).The biodegradation of aromatic hydrocarbons and related compounds by aerobic bacteria is often initiated by multicomponent dioxygenase systems that catalyze the addition of both atoms of molecular oxygen to the substrate. Nitroaromatic compounds, in general, are resistant to oxidative attack due to the electron-withdrawing nature of the nitro groups and the stability of the benzene ring (29,33). Only recently have aerobic bacteria been isolated that utilize nitroaromatic compounds as growth substrates (19,32). One example is Comamonas sp. strain JS765, which can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. Previous experiments showed that JS765 uses an oxidative pathway for the degradation of nitrobenzene, with the initial reaction catalyzed by nitrobenzene 1,2-dioxygenase (NBDO; Fig. 1) (18). Other nitroarene dioxygenase genes from aerobic bacteria have been cloned and sequenced; these include genes encoding 2-nitrotoluene dioxygenase from Pseudomonas sp. strain JS42 (20) and 2,4-dinitrotoluene dioxygenases (DNTDOs) from Burkholderia sp. strain DNT (35) and Burkholderia cepacia R34 (G. R. Johnson, B. E. Haigler, R. K. Jain, and J. C. Spain, Abstr. 98th Gen. Meet. Am. Soc. Microbiol., abstr. Q-83, p. 435, 1998). However, strains DNT and R34 are unable to grow with nitrobenzene and we have observed only slight growth of JS42 with nitrobenzene.The majority of nitroaromatic compounds are synthetic, and therefore they have been present in the environment for...