Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome of M. acetivorans.

Dismutation of superoxide has been shown previously to be catalyzed by stable nitroxide compounds. In the present study, the mechanism of superoxide (-O°) dismutation by various five-membered ring and six-membered ring nitroxides was studied by electron paramagnetic resonance spectrometry, UV-visible spectrophotometry, cyclic voltammetry, and bulk electrolysis. Electron paramagnetic resonance signals from the carbocyclic nitroxide derivatives (piperidinyl, pyrrolidinyl, and pyrrolinyl) were unchanged when exposed to enzymatically generated -02, whereas, in the presence of°2-and reducing agents such as NADH and NADPH, the nitroxides underwent reduction to their respective hydroxylamines. The reaction of 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (Tempol-H) with°2 was measured and, in agreement with earlier reports on related compounds, the rate was found to be too slow to be consistent with a mechanism of°2 dismutation involving the hydroxylamine as an intermediate.Voltammetric analyses of the carbocyclic nitroxide derivatives revealed a reversible one-electron redox couple at positive potentials. In contrast, oxazolidine derivatives were irreversibly oxidized. At negative potentials, all of the nitroxides studied exhibited a broad, irreversible reductive wave. The rate of°O dismutation correlated with the reversible midpoint redox potential. Bulk electrolysis at positive potentials was found to generate a metastable oxidized form of the nitroxide. The results indicate that the dismutation of°2 is catalyzed by the oxoammonium/nitroxide redox couple for carbocyclic nitroxide derivatives. In addition to the one-electron mitochondrial reduction pathway, the present results suggest the possibility that cellular bioreduction by a two-electron pathway may occur subsequent to oxidation of stable nitroxides. Furthermore, the cellular destruction of persistent spin adduct nitroxides might also be facilitated by a primary univalent oxidation.Stable nitroxide free radicals have found a wide range of applications in biology and medicine. These compounds have been used to monitor intracellular redox reactions (1), oxygen concentration (2), and pH (3), as well as electron paramagnetic resonance (EPR) microscopy of spheroids (4), as contrast agents in magnetic resonance imaging (5), and as probes in EPR imaging (6). Persistent nitroxide adducts resulting from reaction of a precursor nitrone (spin trap) with transient free radical species have been used to detect, characterize, and quantitate the production of free radicals in various in vitro and in vivo model systems (7). The cellular and in vivo pharmacology of stable nitroxides and persistent spin adduct nitroxides has been investigated in detail (8-10). Oneelectron reduction of stable nitroxides to the corresponding hydroxylamine is the primary metabolic pathway (11-15).Whereas the metabolic fate of spin adduct nitroxides is not clearly understood, oxidative degradation has been suggested (16,17).Previous studies have identified a stable five-membered nitroxi...

The methanogenic Archaea utilize a unique metabolic pathway for degradation of acetate under anaerobic conditions, and cleavage of acetate thereby accounts for a major proportion of the methane formed in the environment. The central reaction in this pathway is carried out by an unusual multienzyme complex, designated acetyl-CoA decarbonylase/synthase (ACDS), 1 which contains five different polypeptide subunits and accounts for as much as 25% of the soluble protein in species such as Methanosarcina thermophila and Methanosarcina barkeri growing on acetate. The ACDS complex catalyzes cleavage of the acetyl C-C bond using the substrates acetylCoA and tetrahydrosarcinapterin (H 4 SPt), a tetrahydrofolate analog which serves as methyl acceptor, and yields the products CoA, N 5 -methyltetrahydrosarcinapterin, CO 2 , and two reducing equivalents, as given in Reaction 1 (1).This overall reaction is made up of a series of partial reactions catalyzed by different protein subcomponents of the ACDS complex as shown in Scheme 1 (2). Acetyl-CoA binds to the ␤ subunit, and under low redox potential conditions, as required for activity, transfers the acetyl group to a nucleophilic center on the enzyme forming an acetyl-enzyme species and releasing CoA (Scheme 1, acetyl transfer) (2, 3). The acetyl intermediate then undergoes C-C bond cleavage by a reaction that is presumed to involve metal-based decarbonylation and/or methyl group migration (Scheme 1, cleavage). The nascent methyl group is then transferred to a corrinoid cofactor present on the ␥␦ subcomponent, which catalyzes subsequent methyl transfer to the substrate H 4 SPt (Scheme 1, methyl transfer) (2). The carbonyl group is oxidized to CO 2 by a process involving the ␣⑀ CO dehydrogenase subcomponent, with regeneration of the reduced form of the ␤ subunit. Previous studies on the ␤ subunit have focused on a C-terminally truncated form of the protein purified from the native ACDS complex following partial proteolytic digestion (2-4).The genes encoding the five ACDS subunits are arranged together in an operon along with one additional open reading frame in all species of Methanosarcina and in certain other methanogens as well (5-7, 9).2 The operon structure is shown in Scheme 2, with the designated genes and corresponding subunit molecular masses indicated for M. thermophila TM-1. The additional open reading frame encodes an accessory protein thought to be involved in nickel insertion, and nickel is present in both the large CO dehydrogenase subunit ␣ (CdhA) and in the ␤ subunit (CdhC) containing the active site for

Acetyl-CoA decarbonylase/synthase (ACDS) is a multienzyme complex that plays a central role in energy metabolism in Methanosarcina barkeri grown on acetate. The ACDS complex carries out an unusual reaction involving net cleavage of the acetyl C-C and thioester bonds of acetyl-CoA. The overall reaction is composed of several partial reactions, one of which involves catalysis of acetyl group transfer. To gain insight into the overall reaction, a study was carried out on the kinetics and mechanism of the acetyltransferase partial reaction. Analysis by HPLC was used to quantify rates of acetyl transfer from acetyl-CoA both to 3'-dephospho-CoA and, by isotope exchange, to 14C-labeled CoA. Acetyl transfer activity was observed only under strongly reducing conditions, and was half-maximal at -486 mV at pH 6.5. The midpoint activation potential became increasingly more negative as the pH was increased, indicating the involvement of a protonation step. Cooperative dependence on acetyl-CoA concentration was exhibited in reactions that contained incompletely reduced enzyme; however, under redox conditions supporting maximum activity, hyperbolic kinetics were found. A ping-pong steady state kinetic mechanism was established, consistent with formation of an acetyl-enzyme intermediate. Analysis of the inhibitory effects of CoA on acetyl transfer to 3'-dephospho-CoA provided values for KiCoA of 6.8 microM and for Kiacetyl-CoA of 45 microM; isotope exchange analyses yielded values of 32 and 120 microM, respectively. Two separate measures of stability yielded values for the free energy of hydrolysis of the acetyl-enzyme intermediate of -9.6 and -9.3 kcal/mol, an indication of a high-energy bonding interaction in the acetyl-enzyme species. Implications for the mechanism of C-C bond cleavage are discussed.