Acetate and Acetyl-CoA Metabolism of ANME-2 Anaerobic Archaeal Methanotrophs.

Acetyl-CoA synthetase (ACS) and acetate ligase (ACD) are widespread among microorganisms, including archaea, and play an important role in their carbon metabolism, although only a few of these enzymes have been characterized. Anaerobic methanotrophs (ANMEs) have been reported to convert methane anaerobically into CO2, polyhydroxyalkanoate, and acetate. Furthermore, it has been suggested that they might be able to use acetate for anabolism or aceticlastic methanogenesis. To better understand the potential acetate metabolism of ANMEs, we characterized an ACS from ANME-2a as well as an ACS and an ACD from ANME-2d. The conversion of acetate into acetyl-CoA (Vmax of 8.4 µmol mg-1 min-1 and Km of 0.7 mM acetate) by the monomeric 73.8-kDa ACS enzyme from ANME-2a was more favorable than the formation of acetate from acetyl-CoA (Vmax of 0.4 µmol mg-1 min-1 and Km of 0.2 mM acetyl-CoA). The monomeric 73.4-kDa ACS enzyme from ANME-2d had similar Vmax values for both directions (Vmax,acetate of 0.9 µmol mg-1 min-1 versus Vmax,acetyl-CoA of 0.3 µmol mg-1 min-1). The heterotetrameric ACD enzyme from ANME-2d was active solely in the acetate-producing direction. Batch incubations of an enrichment culture dominated by ANME-2d fed with 13C2-labeled acetate produced 3 µmol of [13C]methane in 7 days, suggesting that this anaerobic methanotroph might have the potential to reverse its metabolism and perform aceticlastic methanogenesis using ACS to activate acetate albeit at low rates (2 nmol g [dry weight]-1 min-1). Together, these results show that ANMEs may have the potential to use acetate for assimilation as well as to use part of the surplus acetate for methane production. IMPORTANCE Acetyl-CoA plays a key role in carbon metabolism and is found at the junction of many anabolic and catabolic reactions. This work describes the biochemical properties of ACS and ACD enzymes from ANME-2 archaea. This adds to our knowledge of archaeal ACS and ACD enzymes, only a few of which have been characterized to date. Furthermore, we validated the in situ activity of ACS in ANME-2d, showing the conversion of acetate into methane by an enrichment culture dominated by ANME-2d.

Bioelectrocatalytic CO2 Reduction by Mo-Dependent Formylmethanofuran Dehydrogenase.

Massive efforts are invested in developing innovative CO2 -sequestration strategies to counter climate change and transform CO2 into higher-value products. CO2 -capture by reduction is a chemical challenge, and attention is turned toward biological systems that selectively and efficiently catalyse this reaction under mild conditions and in aqueous solvents. While a few reports have evaluated the effectiveness of isolated bacterial formate dehydrogenases as catalysts for the reversible electrochemical reduction of CO2 , it is imperative to explore other enzymes among the natural reservoir of potential models that might exhibit higher turnover rates or preferential directionality for the reductive reaction. Here, we present electroenzymatic catalysis of formylmethanofuran dehydrogenase, a CO2 -reducing-and-fixing biomachinery isolated from a thermophilic methanogen, which was deposited on a graphite rod electrode to enable direct electron transfer for electroenzymatic CO2 reduction. The gas is reduced with a high Faradaic efficiency (109±1 %), where a low affinity for formate prevents its electrochemical reoxidation and favours formate accumulation. These properties make the enzyme an excellent tool for electroenzymatic CO2 -fixation and inspiration for protein engineering that would be beneficial for biotechnological purposes to convert the greenhouse gas into stable formate that can subsequently be safely stored, transported, and used for power generation without energy loss.

Reaction of Thiosulfate Dehydrogenase with a Substrate Mimic Induces Dissociation of the Cysteine Heme Ligand Giving Insights into the Mechanism of Oxidative Catalysis.

Thiosulfate dehydrogenases are bacterial cytochromes that contribute to the oxidation of inorganic sulfur. The active sites of these enzymes contain low-spin c-type heme with Cys-/His axial ligation. However, the reduction potentials of these hemes are several hundred mV more negative than that of the thiosulfate/tetrathionate couple (Em, +198 mV), making it difficult to rationalize the thiosulfate oxidizing capability. Here, we describe the reaction of Campylobacter jejuni thiosulfate dehydrogenase (TsdA) with sulfite, an analogue of thiosulfate. The reaction leads to stoichiometric conversion of the active site Cys to cysteinyl sulfonate (Cα-CH2-S-SO3-) such that the protein exists in a form closely resembling a proposed intermediate in the pathway for thiosulfate oxidation that carries a cysteinyl thiosulfate (Cα-CH2-S-SSO3-). The active site heme in the stable sulfonated protein displays an Em approximately 200 mV more positive than the Cys-/His-ligated state. This can explain the thiosulfate oxidizing activity of the enzyme and allows us to propose a catalytic mechanism for thiosulfate oxidation. Substrate-driven release of the Cys heme ligand allows that side chain to provide the site of substrate binding and redox transformation; the neighboring heme then simply provides a site for electron relay to an appropriate partner. This chemistry is distinct from that displayed by the Cys-ligated hemes found in gas-sensing hemoproteins and in enzymes such as the cytochromes P450. Thus, a further class of thiolate-ligated hemes is proposed, as exemplified by the TsdA centers that have evolved to catalyze the controlled redox transformations of inorganic oxo anions of sulfur.

A novel methoxydotrophic metabolism discovered in the hyperthermophilic archaeon Archaeoglobus fulgidus.

Methoxylated aromatic compounds (MACs) are important components of lignin found in significant amounts in the subsurface. Recently, the methanogenic archaeon Methermicoccus shengliensis was shown to be able to use a variety of MACs during methoxydotrophic growth. After a molecular survey, we found that the hyperthermophilic non-methanogenic archaeon Archaeoglobus fulgidus also encodes genes for a bacterial-like demethoxylation system. In this study, we performed growth and metabolite analysis, and used transcriptomics to investigate the response of A. fulgidus during growth on MACs in comparison to growth on lactate. We observed that A. fulgidus converts MACs to their hydroxylated derivatives with CO2 as the main product and sulfate as electron acceptor. Furthermore, we could show that MACs improve the growth of A. fulgidus in the presence of organic substrates such as lactate. We also found evidence that other archaea such as Bathyarchaeota, Lokiarchaeota, Verstraetearchaeota, Korarchaeota, Helarchaeota and Nezhaarchaeota encode a demethoxylation system. In summary, we here describe the first non-methanogenic archaeon with the ability to grow on MACs indicating that methoxydotrophic archaea might play a so far underestimated role in the global carbon cycle.

Heme ligation and redox chemistry in two bacterial thiosulfate dehydrogenase (TsdA) enzymes.

Thiosulfate dehydrogenases (TsdAs) are bidirectional bacterial di-heme enzymes that catalyze the interconversion of tetrathionate and thiosulfate at measurable rates in both directions. In contrast to our knowledge of TsdA activities, information on the redox properties in the absence of substrates is rather scant. To address this deficit, we combined magnetic CD (MCD) spectroscopy and protein film electrochemistry (PFE) in a study to resolve heme ligation and redox chemistry in two representative TsdAs. We examined the TsdAs from Campylobacter jejuni, a microaerobic human pathogen, and from the purple sulfur bacterium Allochromatium vinosum In these organisms, the enzyme functions as a tetrathionate reductase and a thiosulfate oxidase, respectively. The active site Heme 1 in both enzymes has His/Cys ligation in the ferric and ferrous states and the midpoint potentials (Em ) of the corresponding redox transformations are similar, -185 mV versus standard hydrogen electrode (SHE). However, fundamental differences are observed in the properties of the second, electron transferring, Heme 2. In C. jejuni, TsdA Heme 2 has His/Met ligation and an Em of +172 mV. In A. vinosum TsdA, Heme 2 reduction triggers a switch from His/Lys ligation (Em , -129 mV) to His/Met (Em , +266 mV), but the rates of interconversion are such that His/Lys ligation would be retained during turnover. In summary, our findings have unambiguously assigned Em values to defined axial ligand sets in TsdAs, specified the rates of Heme 2 ligand exchange in the A. vinosum enzyme, and provided information relevant to describing their catalytic mechanism(s).

Several ways one goal-methanogenesis from unconventional substrates.

Methane is the second most important greenhouse gas on earth. It is produced by methanogenic archaea, which play an important role in the global carbon cycle. Three main methanogenesis pathways are known: in the hydrogenotrophic pathway H2 and carbon dioxide are used for methane production, whereas in the methylotrophic pathway small methylated carbon compounds like methanol and methylated amines are used. In the aceticlastic pathway, acetate is disproportionated to methane and carbon dioxide. However, next to these conventional substrates, further methanogenic substrates and pathways have been discovered. Several phylogenetically distinct methanogenic lineages (Methanosphaera, Methanimicrococcus, Methanomassiliicoccus, Methanonatronarchaeum) have evolved hydrogen-dependent methylotrophic methanogenesis without the ability to perform either hydrogenotrophic or methylotrophic methanogenesis. Genome analysis of the deep branching Methanonatronarchaeum revealed an interesting membrane-bound hydrogenase complex affiliated with the hardly described class 4 g of multisubunit hydrogenases possibly providing reducing equivalents for anabolism. Furthermore, methylated sulfur compounds such as methanethiol, dimethyl sulfide, and methylmercaptopropionate were described to be converted into adapted methylotrophic methanogenesis pathways of Methanosarcinales strains. Moreover, recently it has been shown that the methanogen Methermicoccus shengliensis can use methoxylated aromatic compounds in methanogenesis. Also, tertiary amines like choline (N,N,N-trimethylethanolamine) or betaine (N,N,N-trimethylglycine) have been described as substrates for methane production in Methanococcoides and Methanolobus strains. This review article will provide in-depth information on genome-guided metabolic reconstructions, physiology, and biochemistry of these unusual methanogenesis pathways. KEY POINTS: • Newly discovered methanogenic substrates and pathways are reviewed for the first time. • The review provides an in-depth analysis of unusual methanogenesis pathways. • The hydrogenase complex of the deep branching Methanonatronarchaeum is analyzed.

Electron Accepting Units of the Diheme Cytochrome c TsdA, a Bifunctional Thiosulfate Dehydrogenase/Tetrathionate Reductase.

The enzymes of the thiosulfate dehydrogenase (TsdA) family are wide-spread diheme c-type cytochromes. Here, redox carriers were studied mediating the flow of electrons arising from thiosulfate oxidation into respiratory or photosynthetic electron chains. In a number of organisms, including Thiomonas intermedia and Sideroxydans lithotrophicus, the tsdA gene is immediately preceded by tsdB encoding for another diheme cytochrome. Spectrophotometric experiments in combination with enzymatic assays in solution showed that TsdB acts as an effective electron acceptor of TsdA in vitro when TsdA and TsdB originate from the same source organism. Although TsdA covers a range from -300 to +150 mV, TsdB is redox active between -100 and +300 mV, thus enabling electron transfer between these hemoproteins. The three-dimensional structure of the TsdB-TsdA fusion protein from the purple sulfur bacterium Marichromatium purpuratum was solved by X-ray crystallography to 2.75 Å resolution providing insights into internal electron transfer. In the oxidized state, this tetraheme cytochrome c contains three hemes with axial His/Met ligation, whereas heme 3 exhibits the His/Cys coordination typical for TsdA active sites. Interestingly, thiosulfate is covalently bound to Cys330 on heme 3. In several bacteria, including Allochromatium vinosum, TsdB is not present, precluding a general and essential role for electron flow. Both AvTsdA and the MpTsdBA fusion react efficiently in vitro with high potential iron-sulfur protein from A. vinosum (Em +350 mV). High potential iron-sulfur protein not only acts as direct electron donor to the reaction center in anoxygenic phototrophs but can also be involved in aerobic respiratory chains.

Catalytic Protein Film Electrochemistry Provides a Direct Measure of the Tetrathionate/Thiosulfate Reduction Potential.

The tetrathionate/thiosulfate interconversion is a two-electron process: S4O6(2-) + 2 e(-) ↔ 2 S2O3(2-). Both transformations can support bacterial growth since S2O3(2-) provides an energy source, while S4O6(2-) serves as respiratory electron acceptor. Interest in the corresponding S2O3(2-) oxidation also arises from its widespread use in volumetric analysis of oxidizing agents and bleach neutralization during water treatment. Here we report protein film electrochemistry that defines the reduction potential of the S4O6(2-)/S2O3(2-) couple. The relevant interconversion is not reversible at inert electrodes. However, facile reduction of S4O6(2-) to S2O3(2-) and the reverse reaction are catalyzed by enzymes of the thiosulfate dehydrogenase, TsdA, family adsorbed on graphite electrodes. Zero-current potentials measured with different enzymes, at three pH values, and multiple S4O6(2-) and S2O3(2-) concentrations together with the relevant Nernst equation resolved the tetrathionate/thiosulfate reduction potential as +198 ± 4 mV versus SHE. This potential lies in the ∼250 mV window encompassing previously reported values calculated from parameters including the free energy of formation. However, the value is considerably more positive than widely used in discussions of bacterial bioenergetics. As a consequence anaerobic respiration by tetrathionate reduction is likely to be more prevalent than presently thought in tetrathionate-containing environments such as marine sediments and the human gut.

Differences in regulation mechanisms of glutamine synthetases from methanogenic archaea unveiled by structural investigations.

Glutamine synthetases (GS) catalyze the ATP-dependent ammonium assimilation, the initial step of nitrogen acquisition that must be under tight control to fit cellular needs. While their catalytic mechanisms and regulations are well-characterized in bacteria and eukaryotes, only limited knowledge exists in archaea. Here, we solved two archaeal GS structures and unveiled unexpected differences in their regulatory mechanisms. GS from Methanothermococcus thermolithotrophicus is inactive in its resting state and switched on by 2-oxoglutarate, a sensor of cellular nitrogen deficiency. The enzyme activation overlays remarkably well with the reported cellular concentration for 2-oxoglutarate. Its binding to an allosteric pocket reconfigures the active site through long-range conformational changes. The homolog from Methermicoccus shengliensis does not harbor the 2-oxoglutarate binding motif and, consequently, is 2-oxoglutarate insensitive. Instead, it is directly feedback-inhibited through glutamine recognition by the catalytic Asp50'-loop, a mechanism common to bacterial homologs, but absent in M. thermolithotrophicus due to residue substitution. Analyses of residue conservation in archaeal GS suggest that both regulations are widespread and not mutually exclusive. While the effectors and their binding sites are surprisingly different, the molecular mechanisms underlying their mode of action on GS activity operate on the same molecular determinants in the active site.

A novel mechanism for dissimilatory nitrate reduction to ammonium in Acididesulfobacillus acetoxydans.

The biological route of nitrate reduction has important implications for the bioavailability of nitrogen within ecosystems. Nitrate reduction via nitrite, either to ammonium (ammonification) or to nitrous oxide or dinitrogen (denitrification), determines whether nitrogen is retained within the system or lost as a gas. The acidophilic sulfate-reducing bacterium (aSRB) Acididesulfobacillus acetoxydans can perform dissimilatory nitrate reduction to ammonium (DNRA). While encoding a Nar-type nitrate reductase, A. acetoxydans lacks recognized nitrite reductase genes. In this study, A. acetoxydans was cultivated under conditions conducive to DNRA. During cultivations, we monitored the production of potential nitrogen intermediates (nitrate, nitrite, nitric oxide, hydroxylamine, and ammonium). Resting cell experiments were performed with nitrate, nitrite, and hydroxylamine to confirm their reduction to ammonium, and formed intermediates were tracked. To identify the enzymes involved in DNRA, comparative transcriptomics and proteomics were performed with A. acetoxydans growing under nitrate- and sulfate-reducing conditions. Nitrite is likely reduced to ammonia by the previously undescribed nitrite reductase activity of the NADH-linked sulfite reductase AsrABC, or by a putatively ferredoxin-dependent homolog of the nitrite reductase NirA (DEACI_1836), or both. We identified enzymes and intermediates not previously associated with DNRA and nitrosative stress in aSRB. This increases our knowledge about the metabolism of this type of bacteria and helps the interpretation of (meta)genome data from various ecosystems on their DNRA potential and the nitrogen cycle.IMPORTANCENitrogen is crucial to any ecosystem, and its bioavailability depends on microbial nitrogen-transforming reactions. Over the recent years, various new nitrogen-transforming reactions and pathways have been identified, expanding our view on the nitrogen cycle and metabolic versatility. In this study, we elucidate a novel mechanism employed by Acididesulfobacillus acetoxydans, an acidophilic sulfate-reducing bacterium, to reduce nitrate to ammonium. This finding underscores the diverse physiological nature of dissimilatory reduction to ammonium (DNRA). A. acetoxydans was isolated from acid mine drainage, an extremely acidic environment where nitrogen metabolism is poorly studied. Our findings will contribute to understanding DNRA potential and variations in extremely acidic environments.

Metabolic potential of anaerobic methane oxidizing archaea for a broad spectrum of electron acceptors.

Methane (CH4) is a potent greenhouse gas significantly contributing to the climate warming we are currently facing. Microorganisms play an important role in the global CH4 cycle that is controlled by the balance between anaerobic production via methanogenesis and CH4 removal via methanotrophic oxidation. Research in recent decades advanced our understanding of CH4 oxidation, which until 1976 was believed to be a strictly aerobic process. Anaerobic oxidation of methane (AOM) coupled to sulfate reduction is now known to be an important sink of CH4 in marine ecosystems. Furthermore, in 2006 it was discovered that anaerobic CH4 oxidation can also be coupled to nitrate reduction (N-DAMO), demonstrating that AOM may be much more versatile than previously thought and linked to other electron acceptors. In consequence, an increasing number of studies in recent years showed or suggested that alternative electron acceptors can be used in the AOM process including FeIII, MnIV, AsV, CrVI, SeVI, SbV, VV, and BrV. In addition, humic substances as well as biochar and perchlorate (ClO4-) were suggested to mediate AOM. Anaerobic methanotrophic archaea, the so-called ANME archaea, are key players in the AOM process, yet we are still lacking deeper understanding of their metabolism, electron acceptor preferences and their interaction with other microbial community members. It is still not clear whether ANME archaea can oxidize CH4 and reduce metallic electron acceptors independently or via electron transfer to syntrophic partners, interspecies electron transfer, nanowires or conductive pili. Therefore, the aim of this review is to summarize and discuss the current state of knowledge about ANME archaea, focusing on their physiology, metabolic flexibility and potential to use various electron acceptors.

Unraveling Nitrogen, Sulfur, and Carbon Metabolic Pathways and Microbial Community Transcriptional Responses to Substrate Deprivation and Toxicity Stresses in a Bioreactor Mimicking Anoxic Brackish Coastal Sediment Conditions.

Microbial communities are key drivers of carbon, sulfur, and nitrogen cycling in coastal ecosystems, where they are subjected to dynamic shifts in substrate availability and exposure to toxic compounds. However, how these shifts affect microbial interactions and function is poorly understood. Unraveling such microbial community responses is key to understand their environmental distribution and resilience under current and future disturbances. Here, we used metagenomics and metatranscriptomics to investigate microbial community structure and transcriptional responses to prolonged ammonium deprivation, and sulfide and nitric oxide toxicity stresses in a controlled bioreactor system mimicking coastal sediment conditions. Ca. Nitrobium versatile, identified in this study as a sulfide-oxidizing denitrifier, became a rare community member upon ammonium removal. The ANaerobic Methanotroph (ANME) Ca. Methanoperedens nitroreducens showed remarkable resilience to both experimental conditions, dominating transcriptional activity of dissimilatory nitrate reduction to ammonium (DNRA). During the ammonium removal experiment, increased DNRA was unable to sustain anaerobic ammonium oxidation (anammox) activity. After ammonium was reintroduced, a novel anaerobic bacterial methanotroph species that we have named Ca. Methylomirabilis tolerans outcompeted Ca. Methylomirabilis lanthanidiphila, while the anammox Ca. Kuenenia stuttgartiensis outcompeted Ca. Scalindua rubra. At the end of the sulfide and nitric oxide experiment, a gammaproteobacterium affiliated to the family Thiohalobacteraceae was enriched and dominated transcriptional activity of sulfide:quinone oxidoreductases. Our results indicate that some community members could be more resilient to the tested experimental conditions than others, and that some community functions such as methane and sulfur oxidation coupled to denitrification can remain stable despite large shifts in microbial community structure. Further studies on complex bioreactor enrichments are required to elucidate coastal ecosystem responses to future disturbances.

Methanogenic archaea use a bacteria-like methyltransferase system to demethoxylate aromatic compounds.

Methane-generating archaea drive the final step in anaerobic organic compound mineralization and dictate the carbon flow of Earth's diverse anoxic ecosystems in the absence of inorganic electron acceptors. Although such Archaea were presumed to be restricted to life on simple compounds like hydrogen (H2), acetate or methanol, an archaeon, Methermicoccus shengliensis, was recently found to convert methoxylated aromatic compounds to methane. Methoxylated aromatic compounds are important components of lignin and coal, and are present in most subsurface sediments. Despite the novelty of such a methoxydotrophic archaeon its metabolism has not yet been explored. In this study, transcriptomics and proteomics reveal that under methoxydotrophic growth M. shengliensis expresses an O-demethylation/methyltransferase system related to the one used by acetogenic bacteria. Enzymatic assays provide evidence for a two step-mechanisms in which the methyl-group from the methoxy compound is (1) transferred on cobalamin and (2) further transferred on the C1-carrier tetrahydromethanopterin, a mechanism distinct from conventional methanogenic methyl-transfer systems which use coenzyme M as final acceptor. We further hypothesize that this likely leads to an atypical use of the methanogenesis pathway that derives cellular energy from methyl transfer (Mtr) rather than electron transfer (F420H2 re-oxidation) as found for methylotrophic methanogenesis.

Anaerobic methanotrophic archaea of the ANME-2d clade feature lipid composition that differs from other ANME archaea.

The anaerobic oxidation of methane (AOM) is a microbial process present in marine and freshwater environments. AOM is important for reducing the emission of the second most important greenhouse gas methane. In marine environments anaerobic methanotrophic archaea (ANME) are involved in sulfate-reducing AOM. In contrast, Ca. Methanoperedens of the ANME-2d cluster carries out nitrate AOM in freshwater ecosystems. Despite the importance of those organisms for AOM in non-marine environments little is known about their lipid composition or carbon sources. To close this gap, we analysed the lipid composition of ANME-2d archaea and found that they mainly synthesise archaeol and hydroxyarchaeol as well as different (hydroxy-) glycerol dialkyl glycerol tetraethers, albeit in much lower amounts. Abundant lipid headgroups were dihexose, monomethyl-phosphatidyl ethanolamine and phosphatidyl hexose. Moreover, a monopentose was detected as a lipid headgroup that is rare among microorganisms. Batch incubations with 13C labelled bicarbonate and methane showed that methane is the main carbon source of ANME-2d archaea varying from ANME-1 archaea that primarily assimilate dissolved inorganic carbon (DIC). ANME-2d archaea also assimilate DIC, but to a lower extent than methane. The lipid characterisation and analysis of the carbon source of Ca. Methanoperedens facilitates distinction between ANME-2d and other ANMEs.

TsdC, a unique lipoprotein from Wolinella succinogenes that enhances tetrathionate reductase activity of TsdA.

The diheme cytochromes c of the widespread TsdA family are bifunctional thiosulfate dehydrogenase/tetrathionate reductases. Here, biochemical information was collected about TsdA from the Epsilonproteobacterium Wolinella succinogenes (WsTsdA). The situation in W. succinogenes is unique since TsdA is closely associated with the unprecedented lipoprotein TsdC encoded immediately downstream of tsdA in the same direction of transcription. WsTsdA purified from Escherichia coli catalyzed both thiosulfate oxidation and tetrathionate reduction. After co-production of TsdC and WsTsdA in E. coli, TsdC was found to mediate membrane attachment of TsdA and to ensure its full catalytic activity. This effect was much stronger in the tetrathionate-reducing than in the thiosulfate-oxidizing direction. It is concluded that the TsdAC complex predominantly acts as a tetrathionate reductase in vivo.

Influence of haem environment on the catalytic properties of the tetrathionate reductase TsdA from Campylobacter jejuni.

Bifunctional dihaem cytochrome c thiosulfate dehydrogenases/tetrathionate reductases (TsdA) exhibit different catalytic properties depending on the source organism. In the human food-borne intestinal pathogen Campylobacter jejuni, TsdA functions as a tetrathionate reductase enabling respiration with tetrathionate as an alternative electron acceptor. In the present study, evidence is provided that Cys138 and Met255 serve as the sixth ligands of Haem 1 and Haem 2 respectively, in the oxidized CjTsdA wt protein. Replacement of Cys138 resulted in a virtually inactive enzyme, confirming Haem 1 as the active site haem. Significantly, TsdA variants carrying amino acid exchanges in the vicinity of the electron-transferring Haem 2 (Met255, Asn254 and Lys252) exhibited markedly altered catalytic properties of the enzyme, showing these residues play a key role in the physiological function of TsdA. The growth phenotypes and tetrathionate reductase activities of a series of ΔtsdA/*tsdA complementation strains constructed in the original host C. jejuni 81116, showed that in vivo, the TsdA variants exhibited the same catalytic properties as the pure, recombinantly produced enzymes. However, variants that catalysed tetrathionate reduction more effectively than the wild-type enzyme did not allow better growth.