INTRODUCTION

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Ever since archaea have been studied, their ability to thrive in unusual habitats under extremely harsh conditions has stimulated interest in the molecular mechanisms that confer heat stability on proteins at temperatures above 100°C, tolerance of extreme pH values and salt concentrations, and unique metabolic functions not found in bacteria, such as methanogenesis or rhodopsin-linked energy and signal transduction. Actually, ever since Archaea was identified as a third evolutionary kingdom (606-608) presumably located relatively near the hypothetical root of the evolutionary tree, it has been speculated that the structural organization and metabolic pathways of archaea might reflect more ancestral organisms whose essential properties differ from those of bacteria and eucarya. In this regard, one has to realize with respect to biological energy conservation that all existing forms of life rely on the universal principle of chemiosmotic energy transduction (366, 367), which in phylogenetic terms should have evolved very early. In fact, the origin of cellular life must have been connected with the permanent manifestation of mechanisms allowing the transduction of energy between exergonic and endergonic processes and with the development of transitory and long-term energy stores.

The definition of Archaea as a separate domain of organisms was based on the comparative analysis of 16S rRNA sequences (405), which led to a result different from classical taxonomy. The term Archaea reflects an earlier idea that these organisms descended from life forms that existed prior to the division into the bacterial and eukaryal domains. However, based on the sequences of universally present proteins (176), Archaea has been placed on the branch also leading to Eucarya. A feature that distinguishes Archaea from Bacteria is the structure of archaeal ribosomes (435), which in halophiles were first recognized to contain acidic rather than basic proteins (42). In addition, the transcriptional machinery is unique as to the structure of DNA-dependent RNA polymerases (133, 626). With respect to subunit structure, a closer relationship to eukaryotes than to bacteria was found (300). Another feature distinguishing Archaea from Bacteria is the specific composition of archaeal surface layers (266), which do not contain peptidoglycans. Their glycoprotein surface layers can form quasicrystalline structures (97, 360) that are firmly attached to the plasma membrane, thus leaving practically no periplasmic space (39).

Nevertheless, although archaea are located on a distinct evolutionary branch as depicted in Fig. 1, they represent, with regard to their primary energy-transducing mechanisms, a very heterogeneous domain comprising chemolithoautotrophic as well as organotrophic species. In addition to obligate anaerobes such as the methanogens, a second group that performs various types of aerobic or anaerobic respiration can be distinguished. Further, for some halobacteria we have to consider archaeal phototrophic energy transformation in addition to respiratory mechanisms.

View larger version (38K): [in this window] [in a new window]   FIG. 1.   Phylogenetic tree. The scheme demonstrates the division into Crenarchaeota and Euryarchaeota and shows the position of the major archaeal genera. The tree was redrawn according to references 88, 175, and 608. Stars denote archaeal species for which specific bioenergetic information has been found.

In contrast to this diversity, archaeal membrane structures reveal a comparatively homogeneous phenotype, significantly different from that of other prokaryotes. Diether and tetraether lipids are uniformly used as building blocks for archaeal plasma membranes (266). Obviously, the low ion permeability of membranes formed from these bipolar monolayer-forming lipids (136, 571) contributes significantly to the stability of chemiosmotic charge separation in archaea, particularly at high temperatures and/or at extremely low pH values.

Interestingly, neither oxygenic nor anoxygenic "green" photosynthesis has been found in the archaeal kingdom. This latter observation served as an argument in favor of the respiration-first hypothesis, suggesting that the formation of the basic structure of terminal oxidase complexes preceded the occurrence of chlorophyll-based water-splitting and charge-separating systems (93, 94).

It is the aim of this review to introduce the reader to archaeal bioenergetics by a critical state-of-the-art report and to demonstrate similarities and distinguishing features by contrast to bacterial and eucaryal systems. It will not be possible in all cases to give an unambiguous answer to the question of what is typical or genuine for the archaeal domain, because, especially within respiratory electron transport, we will find a number of chimeric functional complexes. In fact, one has to assume that during early evolution, i.e., prior to the division into three urkingdoms, the barriers against lateral gene transfer were much lower than they are now (605).

Of the various bioenergetic mechanisms in archaeal organisms, the present review focuses specifically on the primary energy conservation that involves membrane-residing chemiosmotic processes. Therefore, purely fermentative energy transduction by substrate-level phosphorylation as well as secondary active-transport systems for solutes will not be discussed. As an exception among secondary energy transducers, the ATP synthase complexes will be dealt with because they apparently possess a unique and ubiquitously conserved mechanism, irrespective of the primary energy converter which provides the electrochemical ion gradient to be used as the driving force for high-energy bond formation during ADP phosphorylation.

Another limitation is the great diversity of the archaeal domain, much greater than that suggested by Fig. 1. What we presently know about diversity within the archaeal domain is probably only the tip of the iceberg. By means of molecular studies based on rRNA-directed probes, new archaea are constantly being discovered not only in deep-sea vents or solfataric fields (37, 89, 379) but also in mesophilic and even low-temperature environments. Unfortunately, only a few of these isolates or new species identified by DNA hybridization will prove amenable to cultivation. Thus, the diversity of bioenergetic systems may well exceed the number of classes reviewed in this comprehensive study.

Primary energy conservation by membrane-residing systems is characterized by the formation of an electrochemical potential of hydrogen ions or sodium ions. According to the work of Mitchell (367), the free energy stored in this gradient is described by equations 1 and 2 for the proton motive force:

(1)

(2)

The primary pumps may be driven by redox systems, by methyl transfer reactions as in methanogenesis, or by light as in photophosphorylating halobacteria.

As illustrated by some representative examples below, whole-cell experiments with various genera of Archaea have proven that ATP synthesis is driven, according to the chemiosmotic theory, at the expense of such ion gradients. These experiments were of significance because minimal systems such as inverted plasma membrane vesicles or spheroplasts which are easily prepared from several bacterial organisms are essentially inaccessible in the case of Archaea. The rigid structure and extremely tight adhesion or interdigitation of the glycoprotein cell walls covering archaeal plasma membranes (39, 98, 181, 270, 514) represent an invincible obstacle. For the same reason, the preparation of intact complexes of energy-transducing membrane proteins is quite difficult. In addition, other factors are frequently responsible for the failure to purify catalytically active complexes, such as ATP synthase or terminal oxidases. Such factors can be the absence of the high pH gradient to which membrane proteins are exposed in vivo, extreme salt concentrations, hypersensitivity toward oxygen, and cold dissociation even at room temperature. Also, the determination of energetic parameters such as pH or by direct monitoring or by distribution of diffusible molecular probes is very limited at ambient pH values below 3, the physiological environmental pH for many extreme acidophiles.

Extremely acidophilic organisms, including the archaeon Thermoplasma, were shown to create an inverted membrane potential (30, 364) in order to prevent acidification of the cytosol by influx of H⁺ at the prevailing pH. This does not generally apply to all acidophiles, however. The membrane potential of thermoacidophilic archaea such as Sulfolobus may be rather low (approximately 30 mV), and most of the proton motive force is maintained by a large pH gradient of >3 (335, 370, 467). Chemiosmotic H⁺ cycling (370) with H⁺/O ratios of 3 and a strict correlation of proton motive force (p) with cellular ATP levels could be established for Sulfolobus. Dissipation of p by protonophores caused an immediate collapse of ATP synthesis; at p  0, a persisting residual pH was counterbalanced by an inverted membrane potential, in which the inside was positive (335). In the same experiments, external proton pulses that lowered the pH from 6.1 to 3.4 produced an increase of p from 94 to 170 mV with a concomitant rise of intracellular ATP. For experimental reasons, the reported data was determined at 45°C at an ambient pH of 3.5 and thus may assume slightly different values at the optimal growth temperature of the cells. A review of strategies to cope with extremely low pH values is given in reference 465.

With Halobacterium halobium, the coupling of either a light- or a respiration-induced electrochemical proton gradient with intracellular ATP has been established (361-363). Interestingly, by cation counter transport considerable energy can be stored in the form of a potassium gradient also. Photophosphorylation is a backup system under oxygen limitation in extremely halophilic archaea. This is corroborated by recent studies of the haloalkaliphile Natronobacterium pharaonis (604) demonstrating full recovery of p under oxygen-limiting conditions during illumination. Actually, in these latter archaea the main contribution to the proton motive force is made by the membrane potential of  = 225 to 280 mV, and pH is influenced only marginally by oxygen limitation. Under these conditions, the high membrane potential is generated by an outwardly directed chloride gradient produced by the light-activated chloride pump halorhodopsin (HR); it is insensitive to protonophores and uncouplers and can even be increased by the Cl/OH exchanger triphenyltin (604).

The membrane potential can contribute approximately 90% to the proton motive force (56) in methanogenic archaea also, as shown with Methanosarcina barkeri. Evidence for H⁺- and Na⁺-mediated chemiosmotic energy transduction in methanogens has been compiled previously (124); thereby, the sodium and proton gradients may be linked by Na⁺-H⁺ antiporters (382). A methanogenic strain, Gö1 (now classified as Methanosarcina mazei), is the only known case in which the successful preparation of archaeal vesicular membrane systems has provided a useful experimental model for the study of energy transduction (58, 59).

The coexistence of proton- and sodium ion-coupled energy converters in anaerobes as well as the branching of electron transport pathways in aerobic archaea is difficult to resolve because these organisms either lack or are insensitive to the site-specific inhibitors known to function in bacteria or eucarya. In addition, genetic systems for directed mutagenesis or gene disruption in archaea have scarcely been developed or are unavailable.

The scheme of Fig. 2 illustrates the generation of ion gradients by primary pumps and their utilization by secondary processes. In the following sections, molecular properties of the known functional complexes are discussed separately for methanogenic, respiring, or photophosphorylating archaea; it should be noted, however, that current complete genome projects have predicted the existence of additional functional complexes which have not yet been verified at the protein or mRNA level.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   Primary energy-transducing processes and coupling principles in membrane bioenergetics. The top scheme illustrates the processes found in archaea that contribute to the formation of either proton or sodium ion potentials across the plasma membrane. Details are discussed throughout this review. The bottom schemes illustrate three mechanisms by which an ion gradient can be produced: (a) chemical charge separation (only electrons are transferred through the membrane); (b) a mobile membrane-integral cofactor like the quinones or methanophenazine functioning as proton transporter (examples are bc1 complexes); and (c) redox-driven pumps like cyt c oxidase. All schemes are drawn for an H+/e ratio of 1. Scheme d illustrates the proton-driven ATP synthase of the FoF1 or A1Ao type as an example for a secondary energy transducer. D, electron donor; Ac, electron acceptor.

Methanogens are a phylogenetically diverse but nutritionally rather uniform group of strictly anaerobic archaea. They are able to grow by the conversion of a small number of compounds to methane. This rather simple pathway is not coupled to substrate-level phosphorylation but, instead, to the generation of ion gradients across the membrane that are used to drive the synthesis of ATP. Interestingly, the pathway of methane formation is coupled to the simultaneous generation of primary gradients of both protons and sodium ions. Although methanogens are nutritionally rather similar and employ identical pathways, they differ significantly with respect to the components involved in the proton motive electron transport chain and, therefore, most likely employ different mechanisms to generate the proton gradient. For example, methylotrophic methanogens, such as M. mazei Gö1, contain cytochromes, whereas hydrogenotrophic methanogens, such as Methanobacterium thermoautotrophicum, do not. Since most of our current knowledge derives from studies using methylotrophic methanogens, in particular M. barkeri and M. mazei, this section focuses on these organisms. For a more thorough discussion of the pathways and the biochemistry of methanogenesis, the reader is referred to recent reviews (58, 124, 381, 561).

Central to all pathways of methane formation is the intermediate methyl coenzyme M (2-methylthioethanesulfonate; CoM), the ultimate precursor of methane (Fig. 3). It is reductively demethylated by the methyl-CoM reductase with electrons derived from reduced CoB (7-mercaptoheptanoylthreonine phosphate), to give rise to methane and a heterodisulfide of CoM and CoB (CoM-S-S-CoB; henceforth referred to as the heterodisulfide), in a reaction involving the cofactor F₄₃₀ (reaction 6 in Fig. 3). To complete the cycle, the heterodisulfide is reduced by the heterodisulfide reductase complex (reaction 7 in Fig. 3); this reaction is most important in terms of energy conservation (561). The heterodisulfide reductase is membrane bound and operates as the final limb of several membrane-bound electron transport chains (124). Depending on the substrate, the electron donor used is different. Hydrogenase is employed during growth on H₂ plus CO₂, CO dehydrogenase (or reduced ferredoxin:heterodisulfide oxidoreductase) is used during growth on acetate, and F₄₂₀ dehydrogenase (F₄₂₀, a 5'-deazaflavin, is the universal electron carrier in methanogens) and formylmethanofuran (formyl-MF) dehydrogenase are used during growth on methyl group-containing C₁ substrates.

View larger version (28K): [in this window] [in a new window]   FIG. 3.   Pathways of methanogenesis. Reactions involved in energy conservation are boxed. The reduction of methyl-CoM (reactions 6 and 7) is common to all methanogenic substrates. During methane formation from H2 plus CO2, reactions 1 to 5 proceed in the direction of CO2 reduction. The methyl groups of methanol and acetate enter the central pathway at the level of H4MPT. During methanogenesis from methanol, one-fourth of the methanol is oxidized to CO2 by the reversal of reactions 1 to 5; the six reducing equivalents gained are used to reduce 3 mol of methanol to methane. During methanogenesis from acetate, the carboxyl group is oxidized to CO2 and the electrons gained are used to reduce the methyl group to acetate. F420, oxidized form of coenzyme F420; F420H2, reduced form of F420; HS-CoM, CoM (2-mercaptoethanesulfonate); HS-CoB, CoB (7-mercaptoheptanoylthreonine phosphate); CoM-S-S-CoB, heterodisulfide of HS-CoM and HS-CoB. Enzymes: 1, formyl-MF dehydrogenase; 2, formyl-MF:H4MPT formyltransferase and methenyl-H4MPT cyclohydrolase; 3, F420-dependent methylene-H4MPT dehydrogenase; 4, F420-dependent methylene-H4MPT reductase; 5, methyl-H4MPT:CoM-methyltransferase; 6, methyl-CoM reductase; 7, heterodisulfide reductase system (different electron donor systems are indicated).

H₂-dependent reduction of the heterodisulfide, as catalyzed by inverted vesicles of M. mazei Gö1, was accompanied by H⁺ translocation into the lumen of the vesicles (Fig. 4). Protonophores inhibited ATP formation but stimulated electron transport, i.e., heterodisulfide reduction. Electron transport and ATP synthesis were inhibited by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD), but inhibition was relieved by the addition of protonophores. These effects are clearly reminiscent of respiratory control as observed in mitochondria and can be taken as evidence that the µ_H⁺ generated drives the synthesis of ATP from ADP and P_i. Washed everted vesicles exhibited stringent coupling between heterodisulfide reduction and ATP synthesis, with maximal stoichiometries of 1H⁺ translocated/e and 1ATP synthesized/4e (120).

View larger version (21K): [in this window] [in a new window]   FIG. 4.   Tentative scheme of electron flow and proton translocation during heterodisulfide reduction with H2 as electron donor. This reaction sequence is part of methanogenesis from H2-CO2. This scheme is valid for methylotrophic methanogens only, for hydrogenotrophic methanogens do not contain cytochromes and the presence of methanophenazine (MP) has not been verified. The heterodisulfide reductase is not indicated to be a proton pump, but this cannot be ruled out a priori. This scheme is based on the experimentally derived stoichiometry of 3 to 4 H+ translocated/methyl group reduced. P, periplasm; CM, cytoplasmic membrane; C, cytoplasm. For explanations, see the text.

The F₄₂₀H₂-dependent heterodisulfide reduction was also shown to drive proton translocation into the lumen of everted vesicles of M. mazei Gö1, resulting in the generation of a µ_H⁺ (Fig. 5) Protonophores stimulated the heterodisulfide reduction but prevented µ_H⁺ formation and ATP synthesis. The ATP synthase inhibitor DCCD decreased the rate of F₄₂₀H₂-dependent heterodisulfide reduction. The reversal of this DCCD-mediated inhibition by protonophores and the stimulation of the F₄₂₀H₂-dependent heterodisulfide reduction by ADP indicate stringent coupling between electron transport and ATP synthesis. The F₄₂₀H₂-dependent heterodisulfide reductase system displayed stoichiometries of 1 H⁺ translocated/e and 0.8 ATP synthesized/4e (122).

View larger version (21K): [in this window] [in a new window]   FIG. 5.   Tentative scheme of electron flow and proton translocation during heterodisulfide reduction with F420H2 as electron donor. This reaction sequence is part of methanogenesis from methanol, methylamines, and formate. This scheme is valid for methylotrophic methanogens only (see the legend to Fig. 4). F420, coenzyme F420; MP, methanophenazine; P, periplasm; CM, cytoplasmic membrane; C, cytoplasm. For explanations, see the text.

Evidence that the conversion of CO to CO₂ and H₂ (G°' = 20 kJ/mol) by resting cells of M. barkeri is coupled to the synthesis of ATP has been presented (70, 71). The cleavage of acetyl-CoA as catalyzed by carbon monoxide dehydrogenase yields enzyme-bound CO and an enzyme-bound methyl group (150, 308). The latter is transferred via a corrinoid protein to tetrahydromethanopterin (H₄MPT). Enzyme-bound CO undergoes ferredoxin-dependent oxidation to carbon dioxide, catalyzed by carbon monoxide dehydrogenase (151, 560). In a reconstituted system consisting of purified CO dehydrogenase, heterodisulfide reductase, and ferredoxin, CO oxidation was coupled to heterodisulfide reduction. However, the rate of heterodisulfide reduction was increased 10-fold by addition of membranes, indicating a membrane-bound electron transport chain from ferredoxin to the heterodisulfide (Fig. 6) (420, 512).

View larger version (19K): [in this window] [in a new window]   FIG. 6.   Tentative scheme of electron flow and proton translocation coupled to heterodisulfide reduction with CO as electron donor. This reaction sequence is part of methanogenesis from acetate. The presence of methanophenazine (MP) in acetate-grown cells has not been verified. Fd, ferredoxin; P, periplasm; CM, cytoplasmic membrane; C, cytoplasm. For explanations, see the text.

Methyl group oxidation proceeds via the reversal of CO₂ reduction (reactions 1 to 5 of Fig. 3 in the oxidative direction). There are indications that formyl-MF oxidation is accompanied by the generation of an electrochemical ion potential across the membrane, either protons or sodium ions (262, 603). The formyl-MF-dependent heterodisulfide reduction is associated with a large G°' of 58 kJ/mol. In contrast, G°' of the electron transfer from F₄₂₀H₂ to the heterodisulfide is considerably smaller (29 kJ/mol). Since the physiological electron acceptor employed in the oxidation of formyl-MF to CO₂ is unknown, the G°' of the formyl-MF oxidation cannot be calculated. However, the midpoint potential at pH 7 (E_m,7) of the CO₂-formyl-MF couple of 500 mV indicates that a low-potential electron carrier can be reduced (47).

Heterodisulfide reductase. The reaction catalyzed by the heterodisulfide reductase resembles a polysulfide reduction catalyzed by some bacteria and archaea. The S-S bonds of polysulfide can be reduced by H₂ as external electron donor, and this reaction is coupled with energy conservation (480).

Hydrogenases. The hydrogenases are the entry point for electrons derived from molecular hydrogen. Of the four types of hydrogenases isolated from methanogens to date, one was clearly shown to be involved in energy conservation. The F₄₂₀-reactive hydrogenase reacts with F₄₂₀ and viologen dyes, whereas the F₄₂₀-nonreactive hydrogenase reacts with viologen dyes only. The latter enzyme is therefore often referred to as methyl viologen-reactive hydrogenase. The function of the F₄₂₀-reactive hydrogenase in energy metabolism is still a matter for debate (14, 78, 338, 385). On the other hand, there is clear evidence that the F₄₂₀-nonreactive hydrogenase is the electron donor for a membrane-bound electron transport chain. In methylotrophic methanogens, the F₄₂₀-nonreactive hydrogenase is found in the particulate fraction. The enzyme as purified from M. mazei Gö1 was composed of only two subunits containing redox-active Ni and iron-sulfur clusters (123). A molecular analysis revealed that M. mazei Gö1 contains two operons encoding isoenzymes designated vho for viologen-reactive hydrogenase 1 and vht for viologen-reactive hydrogenase 2. Both operons encode the structural subunits of the hydrogenase (VhoG, VhtG, VhoA, and VhtA) and a gene coding for cyt b (VhoC and VhtC); this indicates that these b cytochromes are the natural electron acceptors of the two F₄₂₀-nonreactive isoenzymes. The small subunit contains a leader peptide, which suggests that the catalytic part of the enzyme faces the periplasm (121). Interestingly, the C termini of the two b cytochromes are not homologous, indicating that they interact with different proteins. Indeed, Northern blot analysis revealed that the expression of the isoenzymes is substrate dependent. vho was apparently constitutively expressed, whereas vht was expressed only during growth on H₂-CO₂ or methanol (119). Therefore, it was speculated that the vho gene products are part of the heterodisulfide reductase system whereas the vht gene products are involved in electron flow to and from CO₂ in the course of the formyl-MF dehydrogenase reaction (124).

F₄₂₀ dehydrogenase. The F₄₂₀H₂ dehydrogenase is the entry point for the electrons derived from F₄₂₀H₂ oxidation. The enzyme was first isolated from Methanolobus tindarius after solubilization from membranes with detergents (182). The apparent molecular mass of the native enzyme was 120 kDa; it consisted of five different subunits of 45, 41, 22, 18, and 17 kDa. The enzyme contained 16 mol of nonheme iron and 16 mol of acid-labile sulfur per mol, but flavin was not detected. The gene encoding the 40-kDa subunit (ffdB) was cloned (600). Sequence analysis, primer extension, and reverse transcription-PCR indicated that ffdB is part of an operon harboring three additional genes (ffdA, ffdC, and ffdD). FfdA is similar to the F₄₂₀-dependent methylene-H₄MPT reductase. The first 90 amino acids of FfdB are similar to numerous ferredoxins, suggesting the likely presence of at least two iron-sulfur centers. FfdC and FfdD are similar to proteins of unknown function of Methanococcus jannaschii and Archaeoglobus fulgidus. FfdD appears to be very hydrophobic and is likely to be the membrane anchor. Recently, F₄₂₀ dehydrogenases were purified from M. mazei Gö1 and the sulfate-reducing archaeon A. fulgidus (3, 297). Flavin was detected in both enzymes. Therefore, it is likely that flavin is also present in the enzyme from M. tindarius but lost during purification.

With respect to their membrane-integral electron carriers, and thus probably with respect to the mechanism of proton translocation, methanogens can be divided into two groups: the methylotrophic organisms, in which a variety of b- and c-type cytochromes were found, and the hydrogenotrophic methanogens, which are devoid of cytochromes (260, 295). In hydrogenotrophic methanogens, the situation is far from settled; polyferredoxins described above and a recently described flavoprotein encoded by the gene fpaA (394) are the only electron carriers identified so far.

In methylotrophic methanogens, there are several lines of evidence for the involvement of cytochromes in electron transport from the F₄₂₀-nonreactive hydrogenase to the heterodisulfide in methylotrophic methanogens. First, membranes of acetate-grown cells catalyze an H₂-dependent reduction of cytochromes (275, 560), and second, hdrE (designated cyt b₂) is part of the heterodisulfide reductase operon and was expressed during growth on H₂-CO₂ (296). The vho operon encoding cyt b₁ along with the structural subunits of the F₄₂₀-nonreactive hydrogenase was also expressed during growth on H₂-CO₂ (119). Therefore, an electron flow from the F₄₂₀-nonreactive hydrogenase via cyt b₁ and b₂ to the heterodisulfide can be envisaged (Fig. 4).

Experiments performed with M. mazei Gö1 strongly suggest that one or several cytochromes also participate in electron transport from F₄₂₀H₂ to the heterodisulfide (264). Membranes of M. mazei Gö1 contain two b- and two c-type cytochromes with midpoint potentials (E_m,7) of 135 and 240 mV (b-type cytochromes) and 140 and 230 mV (c-type cytochromes). The cytochromes were reduced by F₄₂₀H₂ and oxidized by the heterodisulfide at high rates. Addition of the heterodisulfide to reduced cytochromes and subsequent low-temperature spectroscopy showed the oxidation of cyt b₅₆₄. This indicates the involvement of cytochromes in electron transport from F₄₂₀ via cyt b to the heterodisulfide (Fig. 5).

A different class of membrane-bound electron carriers was discovered recently (1). Membranes of methanogens do not contain typical quinones found in bacteria or aerobic archaea. However, extraction of membranes from methanol-grown M. mazei Gö1 with isooctane yielded a fraction containing a redox-active, low-molecular-weight compound identified as a phenazine derivative, methanophenazine. The structure and reactivity of methanophenazine are given in Fig. 7. Methanophenazine is reduced by F₄₂₀ dehydrogenase or hydrogenase, and reduced methanophenazine then reduces the heterodisulfide (2, 40). In a reconstituted system consisting of purified F₄₂₀ dehydrogenase and heterodisulfide reductase, methanophenazine mediated the electron transfer from F₄₂₀ to the heterodisulfide (40). Methanophenazine was isolated from methanol-grown M. mazei Gö1, but it is probably also involved in electron transport to the heterodisulfide from other donors, i.e., formyl-MF and CO. The most interesting question, whether methanophenazine is also present in hydrogenotrophic methanogens, remains to be solved.

View larger version (16K): [in this window] [in a new window]   FIG. 7.   Structure and reactivity of methanophenazine, a membrane-integral electron and hydrogen carrier of methanogens.

In methylotrophic methanogens, the F₄₂₀-nonreactive hydrogenase is localized in the periplasm, as inferred from its leader sequence and its homology to membrane-bound, cytochrome-containing bacterial hydrogenases. Formation of a proton potential can be easily envisaged, because the uptake of H₂ and transfer of electrons to an electron acceptor would lead to the liberation of scalar protons on the outside of the cytoplasmic membrane. However, the H⁺/CH₄ stoichiometry of 3 to 4 (measured in M. barkeri during methanogenesis from methanol-H₂ [57]) cannot be accounted for by scalar protons only. That leaves us with the question of the nature of the vectorial proton pump. Electron flow from F₄₂₀H₂ to methanophenazine, as well as from the reduced methanophenazine to the heterodisulfide, is coupled to proton translocation, indicating the presence of two coupling sites (2). The F₄₂₀H₂ dehydrogenase and the H⁺-translocating bacterial NADH dehydrogenase have in common a complex structure and the presence of flavins and iron-sulfur centers. Therefore, it is tempting to speculate that the F₄₂₀H₂ dehydrogenase, like the NADH dehydrogenase, is a proton pump. It is not known whether the heterodisulfide reductase itself is a proton pump. The genes encoding the hydrogenase, the heterodisulfide reductase, and part of the F₄₂₀ dehydrogenase are known, but the similarities of the deduced proteins to subunits of NADH dehydrogenases or cytochrome oxidases are too low to allow identification of polypeptides involved in proton transport.

With the discovery of methanophenazine, another possibility has emerged. By analogy with the ubiquinone cycle in the bc₁ complex, it is likely that electron transfer from cyt b₁ to methanophenazine is coupled to proton uptake from the cytoplasm. The reduced methanophenazine then donates its electrons to cyt b₂, and the protons are liberated into the periplasm. The H⁺/e stoichiometry of such a mechanism would be fixed at 1H⁺/e.

Apart from the proton motive electron transport chain, methanogens have a primary sodium ion pump, the methyl-H₄MPT:CoM methyltransferase (44, 149, 384). This enzyme is part of the central pathway, and therefore Na⁺ transport is obligatory for methane formation. This enzyme represents the first example of a methyltransferase catalyzing ion transport across a membrane. Because the central pathway is reversible, this enzyme functions as generator of a sodium ion potential during methanogenesis from CO₂ or acetate but as an endergonic reaction driven by the sodium ion potential in the course of methyl group oxidation, which has to be carried out during methanogenesis from methyl group-containing C₁ compounds (380). Unlike the cytochromes and the resulting differences in the electron transport chains, the methyltransferase is found in every methanogen, and there is no reason to assume different reaction mechanisms.

The energetics of the methyltransferase was first investigated by using cell suspensions of M. barkeri and the substrate combination H₂-HCHO. Upon addition of the substrate, sodium ions were actively extruded from the cytoplasm, resulting in the generation of a transmembrane Na⁺ gradient of 60 mV. Na⁺ translocation was not inhibited by protonophores or inhibitors of the Na⁺-H⁺ antiporter, indicating a primary mechanism. This process resulted in the generation of a protonophore-resistant membrane potential of 60 mV; correspondingly, protonophores elicited formation of a reversed pH (inside acidic) of the same magnitude as the (384). A Na⁺-formaldehyde stoichiometry of 3 to 4 was determined with cell suspensions (261). By the use of everted vesicles of M. mazei Gö1, the methyltransferase was identified as a Na⁺ pump (44); this was later corroborated with the purified enzyme reconstituted into liposomes. These proteoliposomes catalyzed an electrogenic Na⁺ transport with a stoichiometry of 1.7 mol of Na⁺ per mol of methyl-H₄MPT demethylated (317).

The methyltransferase contains the cofactor Co-[-(5-hydroxybenzimidazolyl)]-cobamide (factor III), which is involved in methyl transfer (163, 164, 427). The cofactor in its superreduced Co(I) form accepts the methyl group from methyl-H₄MPT, giving rise to a methyl-Co(III) intermediate. In the second partial reaction, this methyl-Co(III) is subjected to a nucleophilic attack, probably by the thiolate anion of CoM, to give rise to methyl-CoM and regenerated Co(I) (149, 164):

(3)

(4)

Reaction 3 has a free-energy change of 15 kJ/mol and was not stimulated by sodium ions. On the other hand, demethylation of the enzyme-bound corrinoid (reaction 4) is also accompanied by a free-energy change of 15 kJ/mol, and this reaction was sodium ion dependent, with half-maximal activity obtained at approximately 50 µM Na⁺. This finding indicates that the demethylation of the enzyme-bound corrinoid is coupled to sodium ion translocation (599) (Fig. 8).

View larger version (13K): [in this window] [in a new window]   FIG. 8.   Tentative scheme of the reaction mechanism of the Na+-translocating methyl-H4MPT:CoM-methyltransferase. The enzyme is a multisubunit enzyme consisting of eight nonidentical subunits in unknown stoichiometry. The reaction can be divided into two partial reactions, methylation and demethylation of an enzyme-bound corrinoid cofactor. The demethylation reaction is apparently coupled to Na+ transport. Co(I) and Co(III) denote different valence states of the enzyme-bound corrinoid cofactor. HS-CoM, CoM (2-mercaptoethanesulfonate); P, periplasm; CM, cytoplasmic membrane; C, cytoplasm. For explanations, see the text.

The methyltransferase was purified from M. thermoautotrophicum and M. mazei Gö1 (163, 317). In the latter, six subunits were found, with apparent molecular masses of 34, 28, 20, 13, 12, and 9 kDa; it contains a [4Fe-4S] cluster with an E₀' of 215 mV and a base-on cobamide with a standard reduction potential of 426 mV for the Co²⁺/¹⁺ couple (324). In M. thermoautotrophicum, eight subunits were found, with apparent molecular masses of 34 (MtrH), 28 (MtrE), 24 (MtrC), 23 (MtrA), 21 (MtrD), 13 (MtrG), 12.5 (MtrB), and 12 (MtrF) kDa. The purified enzyme contains 2 mol of corrinoid, 8 mol of nonheme iron, and 8 mol of acid-labile sulfur (163, 192). The encoding genes have been sequenced from a number of methanogens; they are organized in an operon in the order mtrEDCBAFGH. Hydrophobicity plots indicate that all of the subunits except MtrA and MtrH are hydrophobic and potentially membrane bound. Very recently, the membrane localization of MtrD was confirmed experimentally for M. mazei Gö1, M. thermoautotrophicum, and M. jannaschii (456). This subunit may be directly involved in Na⁺ transport (318).

MtrA was overexpressed, purified from Escherichia coli, and successfully reconstituted with cobalamin. Electron paramagnetic resonance (EPR) spectroscopic studies indicate that the cobalamin is in the base-off form and that the axial ligand is a histidine residue of MtrA (191). From this observation, a hypothetical mechanism was formulated for coupling the methyl transfer reaction to ion transport via a long-range conformational change in the protein (191). It is known that cob(II)alamin and cob(III)alamin, but not cob(I)alamin, carry an axial ligand. Methylation of cob(I)alamin gives rise to a methylcob(III)alamin, which is then able to ligate the histidine residue; demethylation leads to a reversal of this reaction. It is easily conceivable that binding and dissociation of the histidine residue with the corrinoid lead to a conformational change in the hydrophilic part of the enzyme. This change is then transmitted to the membrane-bound subunits, giving rise to Na⁺ transport. Work on the structure and function of this interesting enzyme is just emerging but is apparently well on its way.

Methanogens are the only microorganisms known to produce two primary ion gradients, µ_Na⁺ and µ_H⁺, at the same time. They are, therefore, confronted with the problem of coupling both ion gradients to the synthesis of ATP (124). How this is achieved is still a matter for debate. There have been conflicting reports regarding µ_Na⁺-driven ATP synthesis in the hydrogenotrophic archaeon M. thermoautotrophicum. Smigan and coworkers (515, 516) had indications of a Na⁺-ATPase along with a H⁺-ATPase, whereas Kaesler and Schönheit favored a mechanism in which the µ_Na⁺ established by the methyltransferase reaction is converted to a secondary proton gradient that then drives synthesis of ATP via a H⁺-translocating A₁A_o ATPase (262). The latter hypothesis is supported by the finding that the genomes of the hydrogenotrophic methanogens M. jannaschii and M. thermoautotrophicum contain genes that encode the A₁A_o ATPase but lack those of F₁F_o ATPase (87, 517). On the other hand, differential inhibitor studies indicated the simultaneous presence of both A₁A_o and F₁F_o ATP synthases in M. mazei Gö1 (43). The A₁A_o enzyme may be coupled to H⁺ transport, whereas the F₁F_o ATPase may be Na⁺ coupled. However, no F₁F_o ATPase could be purified from M. mazei Gö1, nor have the encoding genes been detected. In another organism, M. barkeri MS, a gene cluster encoding an F₁F_o ATPase has been identified in addition to the archaeal A₁A_o ATPase genes (549); however, the deduced  subunit is very unusual and presumably nonfunctional, and no gene encoding subunit  was found. Since an mRNA transcript could not be detected in cells grown on methanol, it is doubtful that the F₁F_o-like genes are expressed in M. barkeri (312). The presence of both F₁F_o and A₁A_o was also proposed for halobacteria (227, 230). Most likely, the F₁F_o ATPase genes definitely present at least in M. barkeri MS have arisen from horizontal gene transfer. In line with this argument is the discovery of V₁V_o ATPases in bacteria (433, 620). However, the presence of the F₁F_o ATPases in Methanosarcina species still has to be proven biochemically, and, if present, their contribution to energy metabolism has to be clarified. Apparently, the mechanism for µ_Na⁺-driven ATP synthesis differs among the methanogens. The structure and function of the A₁A_o ATPases are discussed below ("Secondary Energy Converters").

One of the major differences between the anaerobic bacteria and the archaea that employ the acetyl-CoA pathway is the way that CO₂ is activated. In bacteria, this requires the action of formate dehydrogenase and formyltetrahydrofolate synthase, at the expense of ATP hydrolysis. In the reverse reaction, oxidation of formyltetrahydrofolate is coupled to ATP synthesis by substrate-level phosphorylation (320). In methanogens, the low redox potential of the [CO₂ + MF]/[formyl-MF] couple of approximately 500 mV (47) is overcome, not by ATP hydrolysis, but by a reversed electron flow driven by the transmembrane ion (H⁺ or Na⁺) potential (262).

Whereas all methanogens tested so far require Na⁺ for growth and methane formation (422, 423), homoacetogens can be divided into two groups with respect to their energy metabolism, the proton organisms and the sodium ion organisms. In the latter, an as yet unidentified primary sodium ion pump is operative. Since these organisms have membrane-bound corrinoids, it is speculated that the methyltransferase is the sodium ion pump (382). If this is the case, this would allow a study of the evolution of Na⁺-translocating methyltransferases.

The Na⁺ gradient established in methanogens is coupled to ATP synthesis, but the mechanisms involved are still controversial and may differ among the various methanogens. In homoacetogens, H⁺-translocating ATPases are found in proton organisms (112, 113), but a Na⁺-translocating F₁F_o ATP synthase was found in the Na⁺-dependent homoacetogen Acetobacterium woodii (214, 439). The finding of Na⁺-ATPases in homoacetogens strengthens the assumption that Na⁺-ATPases are also present in methanogens.

ENERGETICS OF RESPIRATION

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Aerobiosis and Other Respiration Forms in Archaea

Obligate aerobes are relatively uncommon among the archaea. Given the phylogenetic position of Archaea, this may reflect the prevalence of anaerobic energy-transducing reactions at early stages of evolution; likewise, all organisms branching off at the bottom of the phylogenetic tree are hyperthermophiles, in conformity with the assumption that life originated in hot environments (6, 540).

Table 1 gives an overview of the archaea that grow obligately or facultatively with oxygen or other high-potential terminal electron acceptors. Only those for which sufficient data is available are included. The genus Acidianus displays obligate chemolithoautotrophic growth with CO₂ as the sole carbon source. Most other species are facultative or obligate heterotrophs. The autotrophic growth of S. acidocaldarius with sulfur as electron donor, as reported for the original isolates (82), has to be questioned since the deposited type strains (DSM 639 and ATCC 33909) are incapable of such growth.

Members of the genus Sulfolobus are obligate aerobes. Interestingly, some Sulfolobus isolates were found to reduce ferric ions or molybdate as terminal acceptors under low oxygen tension (80, 83). In addition, Sulfolobus and Acidianus strains have been shown to grow aerobically by the oxidation of molecular hydrogen (Knallgas reaction) (236) at low oxygen concentrations (0.2 to 0.5%).

Alternatively, Acidianus species can derive energy from the reduction of sulfur by molecular hydrogen under anoxic conditions. This sulfur respiration appears to be the preferential energy source: Acidianus is thus classified as a facultative aerobe.

With the exception of Pyrobaculum, the extreme thermophiles in Table 1 are also extreme acidophiles (optimal growth at pH 1 to 3.5), which imposes a bioenergetic challenge regarding the maintenance of a nearly neutral cytosol. In contrast, the members of the aerobic order Halobacteriales are either neutrophilic or alkaliphilic. However, only halobacteria can synthesize purple membranes and use light as an additional energy source. It has been proposed that respiration was the primary energy-transducing mechanism of halobacteria and that the light-driven ion pumps might reflect later adaptations to low oxygen tension (513), an inevitable consequence of the extremely high salinity of their natural habitat.

Not only oxygen reduction but also various forms of anaerobic respiration have been reported as energy sources for archaea. Table 2 summarizes the free-energy changes of the respiratory redox systems used as primary energy sources in archaea. Haloferax mediterranei, Haloferax denitrificans, and Haloferax volcanii are capable of reducing nitrate (as terminal acceptor) to nitrogen (407, 566, 567); several halobacterial nitrate reductases have been described (16, 50, 229). The hyperthermophile Pyrobaculum aerophilum also reduces nitrate under anoxic conditions (586). Moreover, some members of the halobacterial family perform fumarate respiration (406) or grow fermentatively on arginine (193). Purely anaerobic respiration was reported for the genera Thermoproteus, Pyrodictium, Desulfurococcus, Archaeoglobus, and Thermodiscus (7, 483, 540).

In contrast to oxygen respiration, none of the alternate electron transport systems has yet been elucidated in detail at the level of genes or proteins.

(i) NADH dehydrogenases. Older reports on the characterization and partial purification of NADH dehydrogenase activities from halobacteria are reviewed in reference 226. These activities were usually measured with redox dyes as electron acceptors; however, inhibition by the quinone analog 2-heptyl-4-hydroxyquinoline-N-oxide has been interpreted to indicate the in vivo transfer of electrons to the quinone pool (302). Sulfolobus membranes oxidize NADH with low activity in a cyanide-sensitive reaction (21, 592). None of these activities was sensitive to rotenone, amytal, piericidine, or antimycin. From the inhibition of cell respiration by acridone carbonic acid derivatives (402), it was concluded that S. acidocaldarius has an NDH-II type enzyme (463). This activity is only loosely associated with the membrane.

(ii) SDHs and a novel complex II. Succinate-stimulated respiration and SDH activities with redox dyes as electron acceptors have been reported for several aerobic archaeal species (10, 20, 477, 592).

View larger version (20K): [in this window] [in a new window]   FIG. 9.   Gene organization within archaeal SDH operons. Numbers indicate the calculated molecular masses (in kilodaltons) of the gene products. In all cases, subunit A is the flavin-containing dehydrogenase polypeptide; subunit B refers to the iron-sulfur protein in complex II or fumarate reductase, respectively. The columns on the right indicate the type of quinone used as terminal electron acceptor in vivo and the number of heme B molecules present in the complex. MQ, menaquinone; TQ, Thermoplasma quinone, CQ, caldariella quinone. tmh, putative transmembrane helices. E.coli, E. coli (256); B.sub., B. subtilis (353); T.ac., T. acidophilum (29); S.ac., S. acidocaldarius (254) (accession no. Y09041); N.ph., N. pharaonis (accession no. Y07709). The assignment of the open reading frames was done by analyzing sequence similarities. From Thermoplasma, only a partially sequenced operon has been reported; ORF-1 has homology to sdhB and frdB; ORF-2 is a putative diheme cyt b.

(i) The SoxABCD complex. Figure 10 illustrates schematically the composition of a novel terminal oxidase complex, SoxABCD, discovered in S. acidocaldarius (92, 329). It was found to combine features of what are usually separate respiratory complexes III and IV, although its cyt aa₃ subunit (SoxB) could be isolated as a functional quinol oxidase by harsh detergent treatment (23). The integrated complex is transcribed from an operon encoding four polypeptides, designated SoxA to -D. Of these, SoxB represents a subunit I equivalent of typical heme-Cu oxidases; its binuclear center contains two hemes A and one Cu_B, which have been well characterized by optical, EPR, and resonance-Raman spectroscopy (168, 173, 224). SoxA, like subunit II in the quinol oxidase of E. coli (339, 388), lacks the typical mixed-valence binuclear Cu_A ligands of cyt c oxidases. In total, the complex contains four hemes A_S. The two additional hemes are the reaction centers of SoxC; judged by its primary and secondary structural elements, it represents a cyt b homolog and can be identified spectroscopically as cyt a⁵⁸⁷-I. When examined in intact membranes, the complex reveals CO binding kinetics similar to those of mitochondrial cyt c oxidase; the cyt a⁵⁸⁷ and cyt aa₃ centers exhibit coordinated redox behavior on the millisecond time scale (172). Interestingly, with tetramethyl-p-phenylenediamine (TMPD) as electron donor, the turnover of the integrate