Isolation and Characterization of Methanophenazine and Function of Phenazines in Membrane-Bound Electron Transport of Methanosarcina mazei Gö1

Abstract

A hydrophobic, redox-active component with a molecular mass of 538 Da was isolated from lyophilized membranes of Methanosarcina mazei Gö1 by extraction with isooctane. After purification on a high-performance liquid chromatography column, the chemical structure was analyzed by mass spectroscopy and nuclear magnetic resonance studies. The component was called methanophenazine and represents a 2-hydroxyphenazine derivative which is connected via an ether bridge to a polyisoprenoid side chain. Since methanophenazine was almost insoluble in aqueous buffers, water-soluble phenazine derivatives were tested for their ability to interact with membrane-bound enzymes involved in electron transport and energy conservation. The purified F₄₂₀H₂ dehydrogenase from M. mazei Gö1 showed highest activity with 2-hydroxyphenazine and 2-bromophenazine as electron acceptors when F₄₂₀H₂ was added. Phenazine-1-carboxylic acid and phenazine proved to be less effective. The K_m values for 2-hydroxyphenazine and phenazine were 35 and 250 μM, respectively. 2-Hydroxyphenazine was also reduced by molecular hydrogen catalyzed by an F₄₂₀-nonreactive hydrogenase which is present in washed membrane preparations. Furthermore, the membrane-bound heterodisulfide reductase was able to use reduced 2-hydroxyphenazine as an electron donor for the reduction of CoB-S-S-CoM. Considering all these results, it is reasonable to assume that methanophenazine plays an important role in vivo in membrane-bound electron transport of M. mazei Gö1.

The formation of methane from H₂ + CO₂, formate, methanol, methylamines, or acetate is the characteristic feature of methanogenic archaea. The metabolic pathways leading to the generation of CH₄ are unique and involve novel enzymes and coenzymes such as tetrahydromethanopterin, methanofuran, coenzyme M (CoM-SH), and CoB-SH. Methyl-S-CoM is the central intermediate in all methanogenic pathways and is reductively demethylated to methane catalyzed by the methyl-CoM reductase. The two electrons required for the reduction derive from CoB-SH, resulting in the formation of a heterodisulfide (CoB-S-S-CoM) of CoM-SH and CoB-SH (10, 25). An energy-conserving step in the metabolism of methylotrophic methanogens is the reduction of CoB-S-S-CoM with either hydrogen or reduced F₄₂₀ (8). In recent years, the membrane-bound electron transfer of Methanosarcina mazei Gö1 has been analyzed in detail, resulting in the discovery of two proton-translocating systems referred to as H₂:heterodisulfide oxidoreductase and F₄₂₀H₂:heterodisulfide oxidoreductase (4, 5). It has been shown that a membrane-bound, F₄₂₀-nonreducing hydrogenase, cytochromes, and the heterodisulfide reductase are involved in the first of these electron transport systems. Electron transport from F₄₂₀H₂ to CoB-S-S-CoM is mediated by an F₄₂₀H₂ dehydrogenase which channels electrons via unknown electron carriers to the heterodisulfide reductase. Recently, the F₄₂₀H₂ dehydrogenase from M. mazei Gö1 was purified (1). The native enzyme (115 kDa) contains iron-sulfur clusters and flavin adenine dinucleotide and is composed of five different subunits with molecular masses of 40, 37, 22, 20, and 17 kDa. Different compounds such as methylviologen, flavins, and quinones could act as electron acceptors. The most interesting question concerns the nature of the electron carriers which are responsible for electron transfer from F₄₂₀H₂ dehydrogenase to the heterodisulfide reductase. In this publication, we report on the identification of a small hydrophobic component referred to as methanophenazine which was extracted from the cytoplasmic membrane of M. mazei Gö1. Furthermore, it is shown that phenazine derivatives are able to interact with enzymes which are involved in membrane-bound electron transport.

MATERIALS AND METHODS

Abbreviations.

CoM-SH, 2-mercaptoethanesulfonate; F₄₂₀, (N-l-lactyl-γ-l-glutamyl)-l-glutamic acid phosphodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5′-phosphate; F₄₂₀H₂, reduced F₄₂₀; CoB-SH, 7-mercaptoheptanoylthreonine phosphate; HPLC, high-performance liquid chromatography; MOPS, morpholinepropanesulfonic acid; EI-HRMS, electron impact high-resolution mass spectra; NMR, nuclear magnetic resonance.

Growth of cells.

M. mazei Gö1 (DSM 3647) was grown on 100 mM methanol in a 100-liter fermentor in the medium described previously (3). Cells from the late exponential growth phase were harvested by continuous centrifugation at 20°C, frozen in liquid nitrogen, and stored at −70°C.

Preparation of washed membranes and of F₄₂₀H₂ dehydrogenase.

Membranes were prepared under anaerobic conditions. Cells (about 30 g [wet weight]) were lysed by suspension in 25 mM MOPS buffer (pH 7) containing 2 mM dithioerythritol and 1 mg of resazurin per liter (referred to as buffer A) and centrifuged at 8,000 × g for 10 min. The crude extract was centrifuged at 120,000 × g for 1 h. The resulting membrane pellet was resuspended in buffer A and washed twice by centrifugation at 120,000 × g for 30 min. Finally, the membrane pellet was diluted with 30 ml of buffer A. The F₄₂₀H₂ dehydrogenase was purified as described by Abken and Deppenmeier (1).

Extraction and purification of methanophenazine.

For the isolation of methanophenazine, washed membranes were lyophilized overnight and extracted five times with 25 ml of isooctane. The organic solvent was evacuated and flushed with nitrogen to remove oxygen before use. The extracts were combined and purified by HPLC with an HPLC system composed of Kontron (Neufahrn, Germany) 422 pumps, a Kontron 425 gradient former, a Kontron 322 UV detector, and Kontron Data System 450-MT2 HPLC software. For analytical separations, a LiChroCART column (4 by 125 mm with LiChrospher Si-60, 5 μm; Merck, Darmstadt, Germany) was used at a flow rate of 1 ml/min (detection, 260 nm; mobile phase A, cyclohexane; mobile phase B, ethyl acetate; gradient profile, ethyl acetate concentration of 5% at 0 min increasing from 5 to 100% at 10 to 20 min). Preparative separations were performed by using a Kontrosorb 10 SIL column (10 by 250 mm, 10 μm; Kontron) (detection, 260 nm; mobile phase A, cyclohexane; mobile phase B, ethyl acetate; flow rate, 4 ml/min; gradient profile, ethyl acetate concentration of 5% at 0 min increasing to 30% at 15 min and 100% at 22 min). From 10 g (dry weight) of cells, 1 mg of methanophenazine corresponding to a cofactor concentration of 186 nmol/g (dry weight) was isolated. During purification and chemical analysis, the cofactor was not exposed to daylight.

Mass spectroscopy.

The electron impact mass spectra and the EI-HRMS were obtained with a Finnigan MAT 95 mass spectrometer (70 eV, direct insert, high resolution with perfluorokerosine as the standard).

NMR.

The ¹H, ¹³C, and ¹H,¹H shift-correlated NMR spectra were recorded on a Varian VRX 500 instrument. The samples were prepared by dissolving purified methanophenazine either in C₆D₆ or in CD₃OD. Chemical shifts are expressed in δ values (ppm) and are given relative to values for benzene (δ_H = 7.15, δ_C = 128.0) or methanol (δ_H = 3.30, δ_C = 49.0).

UV spectra.

The optical absorption spectra of the oxidized and reduced forms of methanophenazine were monitored with a Uvikon photometer (model 810; Kontron) from 200 to 600 nm. Methanophenazine (final concentration, 7 μM) was diluted in 0.5 ml of acetic acid-ethanol (1:1, vol/vol) containing a few crystals of insoluble platinum(IV) oxide under an atmosphere of hydrogen. Immediately, the spectrum of the oxidized form of the cofactor was monitored. After 1.5 h, the reduction of methanophenazine was completed and the reduced form was analyzed spectroscopically. From the spectra, an extinction coefficient of 2.3 mM⁻¹ cm⁻¹ was determined at 425 nm.

Synthesis of 2-hydroxyphenazine.

Hydroxyquinone was synthesized as described by Willstädler and Müller (27) by using 1,2,4-trihydroxybenzene (Aldrich, Steinheim, Germany) and silver oxide (Aldrich). 2-Hydroxyphenazine was obtained by reaction of hydroxyquinone with o-phenylenediamine (18) and was purified by flash chromatography (24) using BAKERBOND silica gel (eluents, diethyl ether-petroleum ether [10:1]). Mass spectra (70 eV): m/z = 196 (100%) [M⁺], 168 (7%) [M⁺ − CO], 140 (3%) [168 − N₂], 98 (4%) [C₆H₁₂N⁺], and 76 (2%) [C₆H₄⁺]. Other phenazine derivatives were purchased from Sigma (Deisenhofen, Germany).

Assays.

F₄₂₀ and F₄₂₀H₂ were prepared as described previously (4). F₄₂₀H₂ oxidation was assayed under N₂ at room temperature in 1 ml of buffer A in 1.6-ml glass cuvettes closed with rubber stoppers. After the addition of 10 μl of F₄₂₀H₂ (final concentration, 20 μM), 10 to 15 μg of the membrane fraction or 0.2 μg of the purified F₄₂₀H₂ dehydrogenase was added to each cuvette and the cuvettes were incubated for an additional 5 min until a stable baseline was reached. The reaction was started by the addition of electron acceptors as indicated and was monitored photometrically at 420 nm (ɛ = 40 mM⁻¹ cm⁻¹). 2-Hydroxyphenazine was diluted in ethanol (10 mM stock solution). The other phenazine derivatives were dissolved in dimethylformamide (20 mM stock solution).

The membrane-bound hydrogenase was assayed as described above with the exception that the cuvettes were gassed with hydrogen. The reaction was started by the addition of electron acceptors as indicated. The H₂-dependent 2-hydroxyphenazine reduction was monitored photometrically at 425 nm (ɛ = 4.5 mM⁻¹ cm⁻¹ for buffer A). 2-Hydroxyphenazine (final concentration, 0.25 mM) was reduced in buffer A containing platinum(VI) oxide (2 mg/40 ml) under a hydrogen atmosphere. After reduction was completed, the catalyst was removed by centrifugation in an anaerobic chamber and the resulting solution was kept under nitrogen. For the determination of membrane-bound heterodisulfide reductase activity, buffer A and the reduced 2-hydroxyphenazine stock solution were mixed to the indicated concentration (final volume, 1 ml). After washed membranes were added, the reaction was started by the addition of CoB-S-S-CoM to a final concentration of 180 μM and monitored at 425 nm.

RESULTS

Isolation, characterization, and structure of methanophenazine.

A number of proteins which are involved in the energy-conserving electron transport system of Methanosarcina strains, such as membrane-bound hydrogenases, the heterodisulfide reductase, F₄₂₀H₂ dehydrogenases, and cytochromes, have been purified and characterized (1, 6, 7, 14). An interesting question concerns the nature of the electron carriers that mediate electron transfer between the proteins mentioned above. Membranes of methanogenic archaea do not contain typical quinone components such as ubiquinone or menaquinone (2, 17). Only minor amounts of α-tocopherolquinone have been detected (15). To search for any other redox-active, lipid-soluble components, washed membranes of M. mazei Gö1 were lyophilized and extracted with isooctane. Examination of the isooctane extract by analytical HPLC revealed the presence of several UV-absorbing components (Fig. 1). Some compounds present in minor amounts eluted within the first 2 min and at about 14.1 min. Furthermore, a large symmetric peak was observed at 4.5 min. On examination by UV spectroscopy, the major component displayed absorption maxima at 250 and 365 nm with shoulders at 300, 330, and 400 nm (Fig. 2). After reduction with Pt(IV) oxide under an atmosphere of hydrogen, the absorption at 250 nm increased and new peaks at 295 and 500 nm appeared, whereas the peak at 365 nm became a shoulder. These results showed that a redox-active component was isolated.

FIG. 1.

HPLC of isooctane extract prepared from lyophilized membranes of M. mazei Gö1. For separation conditions, see Materials and Methods.

FIG. 2.

UV-Vis spectrum of methanophenazine in the oxidized (curve 1) and reduced (curve 2) forms. For assay conditions, see Materials and Methods.

Chemical structure.

To analyze the chemical structure of the component, membranes were prepared and extracted with isooctane as described in Materials and Methods. The pure compound was characterized spectroscopically, the molecular formula was determined by EI-HRMS, and the basic structure was elucidated by detailed analysis of the ¹H and ¹³C NMR spectra, as well as the ¹H,¹H shift-correlated NMR spectrum.

The isolated compound was obtained as a yellow oil. In the mass spectrum, the base peak occurred at m/z = 538 and was attributed to the molecular ion. The EI-HRMS data (accurate experimental mass, 538.3930; theoretical mass, 538.3923) pointed to a molecular formula of C₃₇H₅₀N₂O. Several peaks of lower intensity at m/z = 470, 402, 334, and 265 were due to the sequential loss of isoprene units.

In the ¹H NMR spectrum (Fig. 3), a complex resonance in the δ = 7.30 to 8.20 region indicated the presence of 7 aromatic protons. Furthermore, 4 olefinic protons at δ = 4.95 to 5.20, 14 allylic protons at δ = 1.80 to 2.20, and 15 methyl protons at δ = 1.50 to 1.65, as well as 3 methyl protons at δ = 1.05, were detected. The characteristic resonance at δ = 4.20 to 4.35 corresponded to a methylene group. The downfield shift of this signal indicated the proximity of an oxygen atom. The structure elucidation in the aliphatic region between δ = 1.10 and δ = 2.20 was complicated by superimposed signals due to the presence of 5 other protons. Inspection of the ¹³C NMR and the ¹³C attached protein test (APT) NMR spectra (Fig. 4) showed five quaternary carbon atoms at δ = 160.75, 142.63, 144.12, 141.36, and 145.90 which represent C-2, C-4a, C-5a, C-9a, and C-10a of the structure shown in Fig. 4 (inset). The highfield shift of C-1 (δ = 105.84) indicates an alkoxy substitution at C-2.

FIG. 3.

¹H NMR spectrum of methanophenazine from M. mazei Gö1 (CD₃OD; 499.9 MHz). (Inset) Assignment of the ¹H NMR signals.

FIG. 4.

¹³C APT NMR spectrum of methanophenazine from M. mazei Gö1 (C₆D₆; 125.71 MHz). (Inset) Assignment of the ¹³C NMR signals.

Evaluation of the ¹H,¹H shift-correlated NMR spectrum (data not shown) and detailed analysis of the splitting pattern and comparison with published spectra (22, 23) revealed the aromatic structure as a phenazine derivative connected at C-2 to an unsaturated side chain via an ether bridge (Fig. 3 and 4 insets). Together with the information from the interpretation of the mass spectra, an isoprenoid side chain is assumed. The structure of this moiety was not elucidated in detail.

Analysis of the interaction of methanophenazine and phenazine with membrane-bound enzymes.

Recently, the F₄₂₀H₂ dehydrogenase was purified from M. mazei Gö1. To identify the electron acceptor of the enzyme, the membranes were solubilized and the proteins were fractionated by anion-exchange chromatography. It was found that F₄₂₀H₂ oxidation did not occur when the F₄₂₀H₂ dehydrogenase was supplemented with fractions obtained from the anion-exchange column. It was concluded that the direct electron acceptor was probably not a protein. Therefore, the question of whether methanophenazine or other phenazine derivatives are able to function as electron acceptors of the purified enzyme arose.

With phenazine, 2-hydroxyphenazine, 2-bromophenazine, or phenazine-1-carboxylic acid as the electron acceptor, F₄₂₀H₂ was oxidized by the F₄₂₀H₂ dehydrogenase, as shown in Table 1. Specific activities of 8.8 and 8.4 U/mg of protein were obtained with 2-hydroxyphenazine and 2-bromophenazine, respectively. These specific activities were comparable to the rate obtained with methylviologen plus metronidazole (Table 1), which, however, had to be added in a much higher concentration. The enzyme was less active with phenazine and phenazine-1-carboxylic acid. When the formation of F₄₂₀ (λ = 420 nm) and the reduction of phenazine (λ = 365 nm) were monitored simultaneously, it was found that the cofactors reacted concomitantly and stoichiometrically according to the following equation: F₄₂₀H₂ + phenazine → F₄₂₀ + phenazine H₂. A methanophenazine-dependent F₄₂₀H₂ oxidation was not observed under the assay conditions employed. The electron carrier is a very hydrophobic component that is almost insoluble in water. Concentrations lower than 1 μM led to a variable turbidity of the reaction mixture, making photometric analysis of the reaction impossible. Therefore, it is reasonable to assume that the interaction of the purified enzyme and the cofactor is probably very low or even impossible in aqueous buffers. Instead of methanophenazine, the phenazine derivatives mentioned above, which are soluble in the enzyme assay without disturbing the optical measurement, provide efficient electron acceptors.

TABLE 1.

F₄₂₀H₂-dependent reduction of phenazine derivatives as catalyzed by the purified F₄₂₀H₂ dehydrogenase

Electron acceptor	Concn (μM)	Sp act (U/mg of protein)
Methylviologen plus metronidazole	300a	10.0
2-Hydroxyphenazine	53	8.8
2-Bromophenazine	53	8.4
Phenazine-1-carboxylic acid	53	4.6
Phenazine	53	1.8
Methanophenazine	NDb	<0.1

^aConcentration of each element of the combination. 

^bND, not determined. 

F₄₂₀H₂ oxidation as catalyzed by the purified F₄₂₀H₂ dehydrogenase followed simple Michaelis-Menten kinetics with phenazine (V_max = 11.2 U/mg of protein) or 2-hydroxyphenazine (V_max = 11.3 U/mg of protein) as the electron acceptor. These values correlate with the V_max determined for the artificial electron acceptor methylviologen plus metronidazole (1). The apparent K_m values of 2-hydroxyphenazine and phenazine amounted to 35 μM (k_cat/K_m = 6.2 × 10⁵ M⁻¹ s⁻¹) and 250 μM (k_cat/K_m = 8.6 × 10⁴ M⁻¹ s⁻¹), respectively. Since 2-hydroxyphenazine reacted directly with F₄₂₀H₂ dehydrogenase and represents a water-soluble analog of methanophenazine, all further experiments were performed with this electron carrier.

2-Hydroxyphenazine also interacts with enzymes involved in membrane-bound electron transport (Table 2). The component was reduced by the membrane-bound form of the F₄₂₀H₂ dehydrogenase and by membrane-bound hydrogenases with F₄₂₀H₂ and H₂ as the electron donors, respectively. Furthermore, the membrane-bound heterodisulfide reductase was able to use reduced 2-hydroxyphenazine as the electron donor for the reduction of CoB-S-S-CoM.

TABLE 2.

Reactivity of 2-hydroxyphenazine with components of the electron transport chain of M. mazei Gö1

Enzyme	Electron donor (concn [μM])a	Electron acceptor (concn [μM])b	Sp act (U/mg of protein)
Membrane-bound heterodisulfide reductase	2-OH-phenazinered (100)	CoB-S-S-CoM (180)	3.5
Membrane-bound F420H2 dehydrogenase	F420H2 (20)	2-OH-phenazineox (100)	0.2
Membrane-bound hydrogenase	H2	2-OH-phenazineox (100)	2.0

^ared, reduced. 

^box, oxidized. 

The kinetics of the H₂-dependent 2-hydroxyphenazine reduction as catalyzed by washed membranes from M. mazei Gö1 is shown in Fig. 5. The reaction started immediately after addition of the electron acceptor, indicating that an activation of the hydrogenase was not necessary. The specific activity within the first 2 min after substrate addition was 1.7 U/mg of protein. A total amount of 120 nmol of 2-hydroxyphenazine was added; this amount was almost completely reduced within 10 min. After replacing the hydrogen atmosphere with N₂, 180 nmol of heterodisulfide was added. 2-Hydroxyphenazine was reoxidized at a rate of 2.0 U/mg of protein. These results revealed that the cofactor can act as an intermediate electron carrier between hydrogenase and heterodisulfide reductase in the electron transport chain.

FIG. 5.

Coupling of 2-hydroxyphenazine reduction by H₂ (A) and its oxidation by heterodisulfide under N₂ (B) as catalyzed by washed membranes from M. mazei Gö1. Washed membranes (12 μg of protein) were suspended in 1 ml of 25 mM MOPS buffer (pH 7) containing 2 mM dithioerythritol and 1 mg of resazurin per liter under a hydrogen atmosphere. The reaction was started by the addition of 6 μl of a 2-hydroxyphenazine solution (20 mM in ethanol-acetic acid [1:1, vol/vol]). After 15 min, the cuvette was flushed with nitrogen for 30 min and 2 μl of CoB-S-S-CoM (90 mM) was added. The reaction was monitored at 425 nm as described in Materials and Methods.

DISCUSSION

In recent years, the mechanism of energy conservation in methylotrophic methanogenic archaea and the components which are involved in this process have been analyzed by using M. mazei Gö1 as a model (8). It was found that the organism contains two electron transfer systems, termed F₄₂₀H₂:heterodisulfide oxidoreductase and H₂:heterodisulfide oxidoreductase, which catalyze electron transport from H₂ and F₄₂₀H₂ to CoB-S-S-CoM, respectively (Fig. 6). It was shown that electron flow is coupled to the generation of an electrochemical proton gradient which is the driving force for ATP synthesis (4, 5). During methanogenesis from H₂ + CO₂, a membrane-bound hydrogenase channels electrons into the respiratory chain which are transferred to the heterodisulfide reductase. It is known that both proteins are connected to b-type cytochromes which are referred to as cytochromes b₁ and b₂ (7, 13, 14). Key enzymes of the F₄₂₀H₂-dependent system that is involved in methanol degradation are the F₄₂₀H₂ dehydrogenase (1, 11) and the heterodisulfide reductase (14). The question arises as to which electron carriers mediate the electron transfer from membrane-bound hydrogenase and F₄₂₀H₂ dehydrogenase to the heterodisulfide reductase. In this study, it was shown that phenazine derivatives might be involved in this process. A small, hydrophobic, redox-active cofactor referred to as methanophenazine was extracted from the cytoplasmic membrane and purified to homogeneity. The elucidation of the chemical structure indicated that methanophenazine is composed of a 2-hydroxyphenazine moiety connected via an ether bridge to a hydrophobic side chain with a molecular formula of C₂₅H₄₃. The cofactor revealed a strongly hydrophobic character and was soluble only in organic solvents such as petroleum ether or isopentane. Since enzyme activity has to be measured in aqueous solutions, it is most possible that the proteins could not react with methanophenazine because of its low solubility in water. Similar results were obtained for the assay of NADH:coenzyme Q oxidoreductase activity and PQQ-dependent glucose dehydrogenase activity that requires the use of artificial acceptors because the physiological quinones such as ubiquinone-10 are too insoluble in aqueous buffer systems to be added as substrates (9, 20). The most commonly used acceptors are short-chain coenzyme Q homologs.

FIG. 6.

Tentative scheme of membrane-bound electron transport in M. mazei Gö1. Cytb₁ and Cytb₂, cytochromes b₁ and b₂, respectively.

These results suggested that phenazine derivatives should replace methanophenazine as the electron carrier. It was found that 2-hydroxyphenazine can act as the electron acceptor of the purified F₄₂₀H₂ dehydrogenase and of the membrane-bound hydrogenases. Furthermore, the reduced form of this analog was used as the electron donor for heterodisulfide reduction (Fig. 6). It is still questionable whether phenazines interact directly with heterodisulfide reductase and F₄₂₀-nonreactive hydrogenase, since activities have not yet been checked by using purified proteins.

The participation of phenazines in electron transport is also supported by their midpoint potentials. The redox potential of 2-hydroxyphenazine, which is a potential precursor of methanophenazine, was determined to be −255 mV (19, 21). With the assumption that the redox potential of methanophenazine is similar to this, this cofactor is sufficiently positive to act as an effective oxidant of the F₄₂₀/F₄₂₀H₂ redox couple (−360 mV) as well as for the H₂/2H⁺ redox couple (−420 mV). Since most disulfides exhibit redox potentials of about −200 mV, it has been assumed that the midpoint potential of the CoB-S-S-CoM/CoM-SH + CoB-SH couple is in the same range (12). Hence, reduced methanophenazine could transfer electrons to the heterodisulfide reductase. These findings clearly indicate that phenazine derivatives can react with enzymes which participate in the energy-conserving systems of M. mazei Gö1. It is therefore reasonable to assume that, in vivo, methanophenazine plays an important role in membrane-bound electron transport.

The natural occurrence of phenazine pigments has been known for a long time. These nitrogen-containing heterocyclic molecules show broad-spectrum antibiotic activity. They are produced by different bacteria, e.g., by species of the genera Pseudomonas, Actinomyces, Brevibacterium, and Streptomyces (16). Although many phenazines are redox active, there is no evidence that any phenazine functions physiologically in the respiratory chain of bacteria (26). Methanophenazine is the first example of a phenazine chromophore produced by archaea. Moreover, evidence that phenazines function as redox-active hydrogen carriers in membrane-bound electron transport and are involved in the mechanism of energy conservation of M. mazei Gö1 is provided in this report.