MICROBIOLOGY - M.H. Serhiichuk - 2008

Chapter 6. PHYSIOLOGICAL CHARACTERISTICS OF ANAEROBIC MICROORGANISMS

Methanogenesis

Organisms capable of producing methane belong to a distinct group of living entities known as archaea. Let us examine their specific features in greater detail.

In the late 1970s, phylogenetic studies based on rRNA demonstrated that archaea (Archaea), previously referred to as archaebacteria, constitute a unique group of living organisms separate from Bacteria (Eubacteria) and Eukarya.

Archaea are divided into two main phylogenetic groups: Crenarchaea and Euryarchaea (Fig. 6.34). All Crenarchaea are thermophiles that utilize sulfur compounds in their METABOLISM, whereas Euryarchaea comprise several thermophiles (sulfur-utilizing), methanogens, and extreme halophiles.

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Fig. 6.34. Phylogeny of archaea (selected genera are shown; halophiles and methanogens are indicated by bold lines)

Archaea exhibit an extraordinary diversity of lifestyles and metabolic capabilities. Many species are adapted to extreme environmental conditions, such as high salinity, temperatures near the boiling point of Water, and extreme pH levels.

Phenotypically, archaea closely resemble bacteria. They are primarily small (0.5–5 µm) rods, cocci, spirilla, or filamentous forms, and they reproduce by fission. However, most archaea are thermophiles, and many are extreme thermophiles. They frequently possess an autotrophic metabolism or respire sulfur. Much like eukaryotes, archaea contain a high proportion of histone-like Proteins and possess DNA organized into nucleosome-like structures.

Methanogens (methanogenic archaea) are a group of archaea that produce methane (CH₄) as a byproduct of their metabolism. Methanogenesis is the exclusive pathway by which methanogenic archaea obtain energy for growth, and they are the only known organisms capable of generating methane as a catabolic end product.

Taxonomically, all methanogens belong to the ancient kingdom Euryarchaeota and are classified into five orders: Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, and Methanopyrales. Among these, only Methanosarcinales can ferment acetate to CH₄ and CO₂, and grow on methanol, methylthiols, and methylamines as sole Energy Sources. Hyperthermophilic species are found exclusively within the orders Methanobacteriales, Methanococcales, and Methanopyrales.

In addition to the cytoplasmic membrane and Cell wall characteristics common to all archaea, methanogens contain unique Coenzymes involved in The transfer of hydrogen and C₁ compounds during methanogenesis: coenzyme F₄₂₀, methanofuran, tetrahydromethanopterin, coenzyme M, factor B, coenzyme F₄₃₀, and methanophenazine.

Coenzyme F₄₂₀ is a flavin derivative whose Structure resembles the common coenzyme FMN, but it differs in The structure of the R-group, lacks a nitrogen atom in the middle ring, and lacks a methyl group on the benzene ring (Fig. 6.35).

Fig. 6.35. Oxidized and reduced forms of factor F₄₂₀

The Redox Potential of F₄₂₀ is quite low (E₀' = -380 mV). It interacts with several distinct methanogenic Enzymes, including Hydrogenase and NADP⁺ reductase. The oxidized form of F₄₂₀ absorbs light at 420 nm and fluoresces blue-green, allowing methanogens to be readily identified using Fluorescence Microscopy. Upon reduction, F₄₂₀ loses its color. Within The Cell, reduced F₄₂₀ performs the following Functions:

1. Transfers 2[H] to NADP⁺ and ferredoxin in biosynthetic reactions unrelated to methane formation.

2. Transfers 2[H] to carbon compounds during methanogenesis.

Methanofuran (MRF, MF) is a low-molecular-weight coenzyme that serves as

the primary acceptor of CO₂ in methanogenesis. Following the initial CO₂ reduction step, formylmethanofuran is produced. Methanofuran is composed of a phenol moiety, two glutamic acid residues, a long fatty acid chain, and a furan ring (Fig. 6.36).

Fig. 6.36. Structure of methanofuran

Tetrahydromethanopterin (THMP, MP, H4MPT) is a coenzyme containing a substituted pterin ring, structurally similar to the vitamin Folic acid (Fig. 6.37). It acts as a carrier of C₁ units throughout most stages of methanogenesis, ranging from the formyl to the methyl level.

Fig. 6.37. Structure of tetrahydromethanopterin

Coenzyme M (2-mercaptoethanesulfonic acid, (H-S-CoM)) functions as a methyl group carrier. It features a very simple structure (Fig. 6.38).

Fig. 6.38. Structure of cofactor M

Coenzyme M is a highly specific carrier in the methyl reduction reaction, which proceeds via the methylreductase-F₃₀ complex during The final stage of methanogenesis. Certain microorganisms, notably Methanobrevibacter ruminantium, cannot synthesize this compound independently and therefore require it as a growth factor. The coenzyme M analogue, bromoethanesulfonic acid, acts as a potent inhibitor of methanogenesis and can be used experimentally to selectively suppress methanogenic archaea when studying anaerobic processes. It is the smallest of all known coenzymes, found exclusively in methanogens.

Factor B (coenzyme B, H-S-CoB), or 7-mercaptoheptanoylthreonine phosphate (HTP-SH, HS-HTP), is a phosphorylated derivative of The amino acid Threonine, structurally similar to the vitamin pantothenic acid (Fig. 6.39).

Fig. 6.39. Structure of factor B

Much like coenzyme M, it participates in the terminal reactions of methanogenesis and also catalyzes the methylreductase system by serving as an electron donor for it. Factor B reacts with methyl-CoM to form the CoM-S-S-HTP disulfide.

Coenzyme F₄₃₀ is a yellow-colored, nickel-containing soluble tetrapyrrole (Fig. 6.40). It absorbs light at 430 nm, but unlike F₄₂₀, it is non-fluorescent. The high nickel content in methanogen Cells is associated with the presence of F430. This coenzyme is of great importance in the final stage of methanogenesis, as it forms part of the methylreductase system.

Fig. 6.40. Structure of coenzyme F₄₃₀

Methanophenazine (MP) is a low-molecular-weight, membrane-bound electron and hydrogen carrier (Fig. 6.41). It is reduced by F₄₂₀ dehydrogenase or hydrogenase and, in turn, reduces the heterodisulfide during the final stages of methanogenesis. It has been detected only in methanogens growing on methylated compounds.

Fig. 6.41. Structure of methanophenazine in its oxidized and reduced states

Metabolism of methanogens. Methanogens convert the following substrates into methane:

1. Compounds containing oxidized carbon: carbon dioxide CO₂, formate HCOOH, and carbon monoxide CO.

2. Compounds containing a methyl group: methanol CH₃OH, methylamine CH₃NH₃⁺, dimethylamine (CH₃)₂NH₂⁺, trimethylamine (CH₃)₃NH⁺, methyl mercaptan CH₃SH, and dimethyl sulfide (CH₃)₂S.

3. Acetic acid CH₃COOH.

These substrates are converted by methanogens with the release of energy According to the following equations:

СО₂ + 4Н₂ → СН₄ + 2Н₂О ΔG⁰'= -134 kJ/reaction

4НСООН → СН₄ + 3СО₂ + 2Н₂О ΔG⁰'= -281 kJ/reaction

4СО + 2Н₂О → СН₄ + 3СО₂ ΔG⁰'= -209 kJ/reaction

4СН₃ОН → 3СН₄ + СО₂ + 2Н₂О ΔG⁰'= -323 kJ/reaction

СН₃ОН + Н₂ → СН₄ + Н₂О ΔG⁰'= -133 kJ/reaction

4СНзКНзОІ + 2НзО → зСН4 + СО₂ + 4NH₄Cl ΔG⁰'= -230 kJ/reaction

СН₃СООН → СН₄ + СО₂ ΔG⁰'= -130 kJ/reaction

Methanogenesis is sometimes viewed as a process of Anaerobic Respiration and referred to as carbonate respiration, because when methanogens grow on a mixture of СО₂ and Н₂, molecular hydrogen serves as the electron donor, while carbon dioxide acts as the acceptor. At the same time, archaea lack a traditional

Respiratory Chain with the classical set of electron carriers. The term "methane Fermentation" is frequently found in literature. When methanogens grow on methanol, one substrate molecule is oxidized while another is reduced, resembling the amino acid fermentation process. However, it is known that during methanogenesis, ATP is synthesized through the generation of a proton and sodium ion gradient, which is not typical for classical fermentation. Methanogens lack substrate-level phosphorylation. Thus, methanogenesis can be regarded neither as anaerobic respiration nor as fermentation. It is a unique, specific energy-yielding process characteristic only of ancient living forms—archaea.

METHANE FORMATION DURING the growth of methanogens on a mixture of СО₂ and Н (Fig. 6.42). The formation of methane from a mixture of СО₂ and Н₂ involves eight reactions listed below:

As can be seen from the equations, СО₂ is reduced stepwise, and the resulting C₁ compounds are continuously bound to one of three carriers: methanofuran (MF), tetrahydromethanopterin (Н₄МРТ), and coenzyme M (H-S-KoM).

Reaction 1. The initial acceptor of СО₂ is methanofuran (MF). Carbon dioxide activated by methanofuran is reduced to the formyl level with the formation of formylmethanofuran. The reduction proceeds with hydrogen, although the hydrogen carrier in this reaction has not yet been precisely identified. The formation reaction of formyl-MF is endergonic.

Reaction 2. The formyl group is transferred from methanofuran to tetrahydromethanopterin.

Reaction 3. Dehydration of formyltetrahydromethanopterin (CHO-H₄МРТ) with the formation of methenyltetrahydromethanopterin (HC= Н₄МРT).

Reaction 4. Reduction of methenyltetrahydromethanopterin (H₂C=ЩМРT) to methylenetetrahydromethanopterin (H₂C=Н₄МРT). The hydrogen carrier in this reaction is coenzyme F₄₂₀.

Fig. 6.42. Scheme of methane formation during the growth of methanogens on a mixture of Н₂ and СО₂

Reaction 5. Reduction of methylenetetrahydromethanopterin (Н₂С=Н₄МРТ) to methyltetrahydromethanopterin (Н₃С-Н₄МРТ). The hydrogen carrier in this reaction is coenzyme F₄₂₀.

Reaction 6. The methyl group is transferred to coenzyme M. The process is accompanied by energy release (ΔG⁰' = -29.7 kJ/mol) and the export of Na+ ions into the periplasm. The pump for Na⁺ transport is the enzyme methyl-H4MPT:CoM methyltransferase, which is part of the central metabolic pathway of methanogens. Therefore, the export of Na⁺ into the periplasm is obligatory during methanogenesis and leads to the generation of a transmembrane Na⁺ gradient. The methyltransferase contains a cofactor (factor III or Co(III)) that participates in methyl conversion.

The Co(III) cofactor in its superreduced state is designated as Co(I). The Mechanism of sodium ion gradient generation (Δμ_Na+) is shown in Fig. 6.43.

Fig. 6.43. Scheme of the sodium ion transport mechanism involving methyl-Н₄МРТ: CoM methyltransferase

The transfer of the methyl group from Н₄МРТ to CoM occurs in two steps. In the first step, Co(I) attaches the methyl group, being thereby oxidized to Co(III) and forming the intermediate product СН₃-Co(III). In the second step, СН₃-Co(III) transfers the СН₃ group to coenzyme M, forming СН₃-S-CoM. It is precisely in the second step of the process that the translocation of sodium ions occurs, coupled with the demethylation reaction of the methyltransferase cofactor.

Reaction 7. Methyl-CoM is reduced to methane. The reduction and demethylation process is catalyzed by methyl-CoM reductase and proceeds with the participation of electrons supplied from reduced coenzyme B; cofactor F₄₃₀ is part of the methylreductase system. As a result, methane and the heterodisulfide of CoM and CoB (CoM-S-S-KoB) are formed.

Reaction 8. The CoM-S-S-KoB heterodisulfide is reduced by the heterodisulfide reductase enzyme complex. Electrons for its reduction are supplied from reduced methanophenazine (MP), a membrane-bound low-molecular-weight carrier.

This reaction is essential for completing the cycle and is critical for energy conservation. Heterodisulfide reductase is membrane-bound and functions as the terminal segment of several Electron Transport Chains.

Fig. 6.44 illustrates the relationship between reactions 7 and 8.

Fig. 6.44. Final reactions (7, 8) of methanogenesis:

7 — reaction of methane formation; 8 — reduction of the heterodisulfide CoM-S-S-CoB

The amount of energy released as a result of the reduction of methyl-CoM to CH₄ is sufficient for the translocation of protons into the periplasm, i.e., for the generation of a proton gradient (Δμ_Η+).

The Biosynthesis of Organic compounds in methanogens growing on a mixture of CO₂ and H₂ proceeds via a modified acetyl-CoA pathway (Fig. 6.45).

Fig. 6.45. Scheme of autotrophic metabolism in methanogens:

MF — methanofuran; H₄MPT — tetrahydromethanopterin; CoM — coenzyme M; COdh — CO dehydrogenase

During autotrophic growth on a gas mixture of CO₂ and H₂, methanogens assimilate CO₂ via the modified acetyl-CoA pathway, the end metabolite of which is acetyl-CoA, which is incorporated into the biosynthesis of all essential cellular components. Since the methyl group of acetyl-CoA is formed during methanogenesis, these microorganisms do not need to synthesize it de novo. Therefore, the modified acetyl-CoA pathway contains only a single branch in which CO₂ is reduced to a carbonyl group. The methyl group, in turn, is formed as an intermediate during Catabolism and, following activation by a Vitamin B12-containing coenzyme, enters the acetyl-CoA pathway.

STRUCTURE OF THE Electron Transport Chain in methanogens. The electron transport chain of methanogens may include the following enzymes and carriers:

- Hydrogenase — the entry point for electrons derived from molecular hydrogen into the respiratory chain when methanogens utilize H₂ as an electron donor.

- F₄₂₀ dehydrogenase — the entry point for electrons released during The oxidation of F₄₂₀H₂, a universal hydrogen carrier in methanogens.

- Heterodisulfide reductase — an enzyme involved in the reduction of the heterodisulfide CoM-S-S-CoB.

- Methanophenazine (MP) — a low-molecular-weight, membrane-bound electron and hydrogen carrier. It is reduced by F42o dehydrogenase or hydrogenase and, in turn, reduces the heterodisulfide CoM-S-S-CoB.

- Cytochromes of the b and c types.

Based on the composition of their membrane-bound electron carriers and the mechanism of proton gradient generation, methanogens can be divided into two groups:

1. Methylotrophic organisms, such as Methanosarcina barkeri, M. mazei (possessing various b- and c-type cytochromes and the methanophenazine carrier).

2. Hydrogen-utilizing methanogens, such as Methanobacterium thermoautotrophicum (lacking cytochromes, but containing polyferredoxins and flavoprotein analogs).

Methanogens utilize various mechanisms for proton gradient generation depending on the substrate and the composition of electron carriers. Possible variants of their respiratory chains are shown in Fig. 6.46.

Fig. 6.46. Hypothetical scheme of proton gradient generation in methanogens at the stage of heterodisulfide CoM-S-S-CoB reduction during growth on various substrates: MP — Methanophenazine

ATP Synthesis in methanogens. Methanogens are organisms that generate two ion gradients: a proton gradient (Δμ_Η+) and a sodium gradient (Δμ_Na+). Both of these gradients are involved in ATP synthesis. Protons flow back into the cell via ATP synthase (H⁺-ATPase), which is coupled to ATP synthesis. Regarding the Mechanism of ATP synthesis driven by the sodium ion gradient, two hypotheses exist. According to the first hypothesis, some methanogens, such as Methanobacterium thermoautotrophicum, possess a Na⁺-ATPase in addition to the H⁺-ATPase, which ensures ATP synthesis using the Na⁺ gradient. According to the second hypothesis, sodium ions re-enter the cell via a Na⁺/H⁺ antiporter, which drives the extrusion of protons. In this case, ATP synthesis is carried out solely by the H⁺-ATPase, while the Na⁺ gradient serves as an additional energy source.

Methane formation during growth of methanogens on methanol. Methanol (CH₃OH) occupies an intermediate oxidation state of carbon between CO₂ and CH₄, and therefore it can be both oxidized and reduced. The oxidation of methanol to carbon dioxide releases three pairs of electrons, 3[2H]. The anaerobic oxidation of the methyl group is a thermodynamically unfavorable process because electrons from the methyl group ($E^{0'}_{\text{methanol/formaldehyde}} = -180\text{ mV}$) must be transferred to the F₄₂₀ carrier ($E^{0'} = -340\text{ mV}$); thus, growth on methanol imposes an additional energy cost on methanogens. To balance the redox processes, the three pairs of electrons generated during the oxidation of the methyl group must reduce a suitable acceptor. Other methanol molecules serve as such acceptors. The reduction of one molecule of methanol to methane consumes one pair of electrons. Consequently, for the oxidation of one molecule of methanol, three molecules must be reduced.

The reaction of methane formation coupled with the reduction of the methyl group is linked to the generation of a proton gradient and ATP synthesis. The metabolic pathway of methanogens growing on methanol is illustrated in Fig. 6.47.

Fig. 6.47. Scheme of methanogenic metabolism during growth on methanol:

MF - methanofuran; H₄MPT - tetrahydromethanopterin; COdh - CO dehydrogenase; H-S-CoM - coenzyme M; B₁₂ - coenzyme with vitamin B₁₂

As shown in the figure, The conversion of methanol begins with the activation of the methyl group by a vitamin B₁₂-containing coenzyme. Then, one activated methyl group is oxidized to CO₂, generating three pairs of reducing equivalents and switching the carbon carrier to H4MPT and MF (left branch). The subsequent activated methyl groups are reduced to methane utilizing the reducing equivalents produced during the oxidation of the methyl group. The release of methane is coupled with ATP synthesis (right branch). Part of the methyl group from methanol and the CO₂ formed

during the oxidation reactions is channeled into biosynthesis via a modified acetyl-CoA pathway.

Methane production during growth of methanogens on acetate. Acetate is cleaved by methanogens using a large enzyme complex into CO and a methyl group, which at various Stages of the process is bound to different carriers. Subsequently, CO is oxidized to CO₂ by CO dehydrogenase. The released electrons reduce methyl-CoM to methane, establishing a proton gradient and ultimately ATP (Fig. 6.48). The overall reaction equation is as follows:

Since the Cleavage products of acetate act as electron Donors (CO) and acceptors (methyl-CoM), acetate-dependent methanogenesis resembles fermentation. However, ATP in this case is synthesized not via substrate-level phosphorylation, but rather driven by Δμ_H+.

Fig. 6.48. Scheme of methanogenic metabolism during growth on acetate: CoM - coenzyme M; COdh - CO dehydrogenase; B12 - coenzyme with vitamin B12