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Contains fulltext : 209065.pdf (Publisher’s version ) (Open Access) Anaerobic ammonium oxidation (anammox) is a microbial process responsible for significant nitrogen loss from the oceans and other ecosystems. The redox reactions at the heart of anammox are catalyzed by large multiheme enzyme complexes that rely on small cytochrome c proteins for electron shuttling. Among the most highly abundant of these cytochromes is a unique heterodimeric complex composed of class I and class II c-type cytochrome called NaxLS, which has distinctive biochemical and spectroscopic properties. Here, we present the 1.7 Å resolution crystal structure of this complex from the anammox organism Kuenenia stuttgartiensis (KsNaxLS). The structure reveals that the heme irons in each subunit exhibit a rare His/Cys ligation, which, as we show by substitution, causes the observed unusual spectral properties. Unlike its individual subunits, the KsNaxLS complex binds nitric oxide (NO) only at the distal heme side, forming 6cNO adducts. This is likely due to steric immobilization of the proximal heme binding motifs upon complex formation, a finding that may be of functional relevance, since NO is an intermediate in the central anammox metabolism. Pulldown experiments with K. stuttgartiensis cell-free extract showed that the KsNaxLS complex binds specifically to one of the central anammox enzyme complexes, hydrazine synthase, which uses NO as one of its substrates. It is therefore possible that the KsNaxLS complex plays a role in binding the volatile NO to retain it in the cell for transfer to hydrazine synthase. Alternatively, we propose that KsNaxLS may shuttle electrons to this enzyme complex. 22 september 2019

Methanotrophs play a prominent role in the global carbon cycle, by oxidizing the potent greenhouse gas methane to CO2. Methane is first converted into methanol by methane monooxygenase. This methanol is subsequently oxidized by either a calcium-dependent MxaF-type or a lanthanide-dependent XoxF-type methanol dehydrogenase (MDH). Electrons from methanol oxidation are shuttled to a cytochrome redox partner, termed cytochrome cL. Here, the cytochrome cL homolog from the thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV was characterized. SolV cytochrome cGJ is a fusion of a XoxG cytochrome and a periplasmic binding protein XoxJ. Here we show that XoxGJ functions as the direct electron acceptor of its corresponding XoxF-type MDH and can sustain methanol turnover, when a secondary cytochrome is present as final electron acceptor. SolV cytochrome cGJ (XoxGJ) further displays a unique, red-shifted absorbance spectrum, with a Soret and Q bands at 440, 553 and 595 nm in the reduced state, respectively. VTVH-MCD spectroscopy revealed the presence of a low spin iron heme and the data further shows that the heme group exhibits minimal ruffling. The midpoint potential Em,pH7 of +240 mV is similar to other cytochrome cL type proteins but remarkably, the midpoint potential of cytochrome cGJ was not influenced by lowering the pH. Cytochrome cGJ represents the first example of a cytochrome from a strictly lanthanide-dependent methylotrophic microorganism.

The hydroxylamine oxidoreductase (HAO) family consists of octaheme proteins that harbor seven bis-His ligated electron-transferring hemes and one 5-coordinate catalytic heme with His axial ligation. Oxidative HAOs have a homotrimeric configuration with the monomers covalently attached to each other via a unique double cross-link between a Tyr residue and the catalytic heme moiety of an adjacent subunit. This cross-linked active site heme, termed the P460 cofactor, has been hypothesized to modulate enzyme reactivity toward oxidative catalysis. Conversely, the absence of this cross-link is predicted to favor reductive catalysis. However, this prediction has not been directly tested. In this study, an HAO homolog that lacks the heme-Tyr cross-link (HAOr) was purified to homogeneity from the nitrite-dependent anaerobic ammonium-oxidizing (anammox) bacterium Kuenenia stuttgartiensis, and its catalytic and spectroscopic properties were assessed. We show that HAOr reduced nitrite to nitric oxide and also reduced nitric oxide and hydroxylamine as nonphysiological substrates. In contrast, HAOr was not able to oxidize hydroxylamine or hydrazine supporting the notion that cross-link-deficient HAO enzymes are reductases. Compared with oxidative HAOs, we found that HAOr harbors an active site heme with a higher (at least 80 mV) midpoint potential and a much lower degree of porphyrin ruffling. Based on the physiology of anammox bacteria and our results, we propose that HAOr reduces nitrite to nitric oxide in vivo, providing anammox bacteria with NO, which they use to activate ammonium in the absence of oxygen.

In recent years, the externalization of electrons as part of respiratory metabolic processes has been discovered in many different bacteria and some archaea. Microbial extracellular electron transfer (EET) plays an important role in many anoxic natural or engineered ecosystems. In this study, an anaerobic methane-converting microbial community was investigated with regard to its potential to perform EET. At this point, it is not well-known if or how EET confers a competitive advantage to certain species in methane-converting communities. EET was investigated in a two-chamber electrochemical system, sparged with methane and with an applied potential of +400 mV versus standard hydrogen electrode. A biofilm developed on the working electrode and stable low-density current was produced, confirming that EET indeed did occur. The appearance and presence of redox centers at −140 to −160 mV and at −230 mV in the biofilm was confirmed by cyclic voltammetry scans. Metagenomic analysis and fluorescence in situ hybridization of the biofilm showed that the anaerobic methanotroph ‘Candidatus Methanoperedens BLZ2’ was a significant member of the biofilm community, but its relative abundance did not increase compared to the inoculum. On the contrary, the relative abundance of other members of the microbial community significantly increased (up to 720-fold, 7.2% of mapped reads), placing these microorganisms among the dominant species in the bioanode community. This group included Zoogloea sp., Dechloromonas sp., two members of the Bacteroidetes phylum, and the spirochete Leptonema sp. Genes encoding proteins putatively involved in EET were identified in Zoogloea sp., Dechloromonas sp. and one member of the Bacteroidetes phylum. We suggest that instead of methane, alternative carbon sources such as acetate were the substrate for EET. Hence, EET in a methane-driven chemolithoautotrophic microbial community seems a complex process in which interactions within the microbial community are driving extracellular electron transfer to the electrode.

Nitrate is an abundant nutrient and electron acceptor throughout Earth’s biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein’s electron transport chain, and elucidate the electron transfer pathways within the complex., The oxidoreductase (NXR) multiprotein complex is a key enzyme in the nitrogen cycle. A detailed structural and biochemical characterization of NXR from the anammox bacterium Kuenenia stuttgartiensis shows that this complex is a filament-forming protein that catalysers both nitrite oxidation and nitrate reduction, and elucidates the mechanisms governing complex assembly and function.

Aerobic and nitrite-dependent methanotrophs make a living from oxidizing methane via methanol to carbon dioxide. In addition, these microorganisms cometabolize ammonia due to its structural similarities to methane. The first step in both of these processes is catalyzed by methane monooxygenase, which converts methane or ammonia into methanol or hydroxylamine, respectively. Methanotrophs use methanol for energy conservation, whereas toxic hydroxylamine is a potent inhibitor that needs to be rapidly removed. It is suggested that many methanotrophs encode a hydroxylamine oxidoreductase (mHAO) in their genome to remove hydroxylamine, although biochemical evidence for this is lacking. HAOs also play a crucial role in the metabolism of aerobic and anaerobic ammonia oxidizers by converting hydroxylamine to nitric oxide (NO). Here, we purified an HAO from the thermophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum SoIV and characterized its kinetic properties. This mHAO possesses the characteristic P-460 chromophore and is active up to at least 80 degrees C. It catalyzes the rapid oxidation of hydroxylamine to NO. In methanotrophs, mHAO efficiently removes hydroxylamine, which severely inhibits calcium-dependent, and as we show here, lanthanidedependent methanol dehydrogenases, which are more prevalent in the environment. Our results indicate that mHAO allows methanotrophs to thrive under high ammonia concentrations in natural and engineered ecosystems, such as those observed in rice paddy fields, landfills, or volcanic mud pots, by preventing the accumulation of inhibitory hydroxylamine. Under oxic conditions, methanotrophs mainly oxidize ammonia to nitrite, whereas in hypoxic and anoxic environments reduction of both ammonia-derived nitrite and NO could lead to nitrous oxide (N2O) production.

Anaerobic ammonium oxidation (anammox) bacteria contribute significantly to the global nitrogen cycle and play a major role in sustainable wastewater treatment. Anammox bacteria convert ammonium (NH4+) to dinitrogen gas (N2) using intracellular electron acceptors such as nitrite (NO2−) or nitric oxide (NO). However, it is still unknown whether anammox bacteria have extracellular electron transfer (EET) capability with transfer of electrons to insoluble extracellular electron acceptors. Here we show that freshwater and marine anammox bacteria couple the oxidation of NH4+ with transfer of electrons to insoluble extracellular electron acceptors such as graphene oxide or electrodes in microbial electrolysis cells. 15N-labeling experiments revealed that NH4+ was oxidized to N2 via hydroxylamine (NH2OH) as intermediate, and comparative transcriptomics analysis revealed an alternative pathway for NH4+ oxidation with electrode as electron acceptor. Complete NH4+ oxidation to N2 without accumulation of NO2− and NO3− was achieved in EET-dependent anammox. These findings are promising in the context of implementing EET-dependent anammox process for energy-efficient treatment of nitrogen., Bacteria capable of anaerobic ammonium oxidation (anammox) produce half of the nitrogen gas in the atmosphere, but much of their physiology is still unknown. Here the authors show that anammox bacteria are capable of a novel mechanism of ammonium oxidation using extracellular electron transfer.

International audience; The most abundant transition metal in biological systems is iron. It is incorporated into protein cofactors and serves either catalytic, redox or regulatory purposes. Anaerobic ammonium oxidizing (anammox) bacteria rely heavily on iron-containing proteins – especially cytochromes – for their energy conservation, which occurs within a unique organelle, the anammoxosome. Both their anaerobic lifestyle and the presence of an additional cellular compartment challenge our understanding of iron processing. Here, we combine existing concepts of iron uptake, utilization and metabolism, and cellular fate with genomic and still limited biochemical and physiological data on anammox bacteria to propose pathways these bacteria may employ.

Anaerobic ammonium oxidation (anammox) is a microbial process responsible for significant nitrogen loss from the oceans and other ecosystems. The redox reactions at the heart of anammox are catalyzed by large multiheme enzyme complexes that rely on small cytochrome c proteins for electron shuttling. Among the most highly abundant of these cytochromes is a unique heterodimeric complex composed of class I and class II c-type cytochromes called NaxLS, which has distinctive biochemical and spectroscopic properties. Here, we present the 1.7 Å resolution crystal structure of this complex from the anammox organism Kuenenia stuttgartiensis (KsNaxLS). The structure reveals that the heme irons in each subunit exhibit a rare His/Cys ligation, which, as we show by substitution, causes the observed unusual spectral properties. Unlike its individual subunits, the KsNaxLS complex binds nitric oxide (NO) only at the distal heme side, forming 6cNO adducts. This is likely due to steric immobilization of the proximal heme-binding motifs upon complex formation, a finding that may be of functional relevance, because NO is an intermediate in the central anammox metabolism. Pulldown experiments with K. stuttgartiensis cell-free extract showed that the KsNaxLS complex binds specifically to one of the central anammox enzyme complexes, hydrazine synthase, which uses NO as one of its substrates. It is therefore possible that the KsNaxLS complex plays a role in binding the volatile NO to retain it in the cell for transfer to hydrazine synthase. Alternatively, we propose that KsNaxLS may shuttle electrons to this enzyme complex.

A protein complex key to the global nitrogen cycle has an unprecedented electron transport network of 192 heme groups., Anaerobic ammonium oxidation (anammox) is a major process in the biogeochemical nitrogen cycle in which nitrite and ammonium are converted to dinitrogen gas and water through the highly reactive intermediate hydrazine. So far, it is unknown how anammox organisms convert the toxic hydrazine into nitrogen and harvest the extremely low potential electrons (−750 mV) released in this process. We report the crystal structure and cryo electron microscopy structures of the responsible enzyme, hydrazine dehydrogenase, which is a 1.7 MDa multiprotein complex containing an extended electron transfer network of 192 heme groups spanning the entire complex. This unique molecular arrangement suggests a way in which the protein stores and releases the electrons obtained from hydrazine conversion, the final step in the globally important anammox process.

Methanotrophs play a prominent role in the global carbon cycle, by oxidizing the potent greenhouse gas methane to CO

The atmospheric concentration of the potent greenhouse gases methane and nitrous oxide (N2O) has increased drastically during the last century. Methylomirabilis bacteria can play an important role in controlling the emission of these two gases from natural ecosystems, by oxidizing methane to CO2 and reducing nitrite to N2 without producing N2O. These bacteria have an anaerobic metabolism, but are proposed to possess an oxygen-dependent pathway for methane activation. Methylomirabilis bacteria reduce nitrite to NO, and are proposed to dismutate NO into O2 and N2 by a putative NO dismutase (NO-D). The O2 produced in the cell can then be used to activate methane by a particulate methane monooxygenase. So far, the metabolic model of Methylomirabilis bacteria was based mainly on (meta)genomics and physiological experiments. Here we applied a complexome profiling approach to determine which of the proposed enzymes are actually expressed in Methylomirabilis lanthanidiphila. To validate the proposed metabolic model, we focused on enzymes involved in respiration, as well as nitrogen and carbon transformation. All complexes suggested to be involved in nitrite-dependent methane oxidation, were identified in M. lanthanidiphila, including the putative NO-D. Furthermore, several complexes involved in nitrate reduction/nitrite oxidation and NO reduction were detected, which likely play a role in detoxification and redox homeostasis. In conclusion, complexome profiling validated the expression and composition of enzymes hypothesized to be involved in the energy, methane and nitrogen metabolism of M. lanthanidiphila, thereby further corroborating their unique metabolism involved in the environmentally relevant process of nitrite-dependent methane oxidation.

International audience; Anaerobic ammonium-oxidizing (anammox) bacteria convert nitrite and ammonium via nitric oxide (NO) and hydrazine into dinitrogen gas by using a diverse array of proteins, including numerous c-type cytochromes. Many new catalytic and spectroscopic properties of c-type cytochromes have been unraveled by studies on the biochemical pathways underlying the anammox process. The unique anammox intermediate hydrazine is produced by a multiheme cytochrome c protein, hydrazine synthase, through the comproportionation of ammonium and NO and the input of three electrons. It is unclear how these electrons are delivered to hydrazine synthase. Here, we report the discovery of a functional tetraheme c-type cytochrome from the anammox bacterium Kuenenia stuttgartiensis with a naturally occurring contracted Cys–Lys–Cys–His (CKCH) heme-binding motif, which is encoded in the hydrazine synthase gene cluster. The purified tetraheme protein (named here KsTH) exchanged electrons with hydrazine synthase. Complementary spectroscopic techniques revealed that this protein harbors four low-spin hexa-coordinated hemes with His/Lys (heme 1), His/Cys (heme 2), and two His/His ligations (hemes 3 and 4). A genomic database search revealed that c-type cytochromes with a contracted CXCH heme-binding motif are present throughout the bacterial and archaeal domains in the tree of life, suggesting that this heme recognition site may be employed by many different groups of microorganisms.

Microbial methane oxidation is an important process to reduce the emission of the greenhouse gas methane. Anaerobic microorganisms couple the oxidation of methane to the reduction of sulfate, nitrate and nitrite, and possibly oxidized iron and manganese minerals. In this article, we review the recent finding of the intriguing nitrate- and nitrite-dependent anaerobic oxidation of methane (AOM). Nitrate-dependent AOM is catalyzed by anaerobic archaea belonging to the ANME-2d clade closely related to Methanosarcina methanogens. They were named 'Candidatus Methanoperedens nitroreducens' and use reverse methanogenesis with the key enzyme methyl-coenzyme M (methyl-CoM) reductase for methane activation. Their major end product is nitrite which can be taken up by nitrite-dependent methanotrophs. Nitrite-dependent AOM is performed by the NC10 bacterium 'Candidatus Methylomirabilis oxyfera' that probably utilizes an intra-aerobic pathway through the dismutation of NO to N2 and O2 for aerobic methane activation by methane monooxygenase, yet being a strictly anaerobic microbe. Environmental distribution, physiological and biochemical aspects are discussed in this article as well as the cooperation of the microorganisms involved.

Anaerobic ammonium-oxidizing (anammox) bacteria derive their energy for growth from the oxidation of ammonium with nitrite as the electron acceptor. N2, the end product of this metabolism, is produced from the oxidation of the intermediate, hydrazine (N2H4). Previously, we identified N2-producing hydrazine dehydrogenase (KsHDH) from the anammox organism Kuenenia stuttgartiensis as the gene product of kustc0694 and determined some of its catalytic properties. In the genome of K. stuttgartiensis, kustc0694 is one out of ten paralogs related to octaheme hydroxylamine (NH2OH) oxidoreductase (HAO). Here, we characterized KsHDH as a covalently cross-linked homotrimeric octaheme protein as found for HAO and HAOrelated hydroxylamine-oxidizing enzyme kustc1061 (KsHOX) from K. stuttgartiensis. Interestingly, the HDH trimers formed octamers in solution, each octamer harbouring an amazing 192 c-type heme moieties. While HAO and KsHOX are capable of hydrazine oxidation as well, KsHDH was highly specific for this activity. To understand this specificity, we performed detailed amino acid sequence analyses and investigated the catalytic and spectroscopic (electronic absorbance, EPR) properties of KsHDH in comparison with the well-defined HAO and HOX. We conclude that HDH specificity is most likely derived from structural changes around the catalytic heme 4 (“P460”) and of the electron-wiring circuit comprising seven His/His-ligated c-type hemes in each subunit. These nuances make HDH a globally prominent N2-producing enzyme, next to nitrous oxide (N2O) reductase from denitrifying microorganisms.

Methane is a potent greenhouse gas, which can be converted by microorganism at the expense of oxygen, nitrate, nitrite, metal-oxides or sulfate. The bacterium ‘Candidatus Methylomirabilis oxyfera,’ a member of the NC10 phylum, is capable of nitrite-dependent anaerobic methane oxidation. Prolonged enrichment of ‘Ca. M. oxyfera’ with cerium added as trace element and without nitrate resulted in the shift of the dominant species. Here, we present a high quality draft genome of the new species ‘Candidatus Methylomirabilis lanthanidiphila’ and use comparative genomics to analyze its metabolic potential in both nitrogen and carbon cycling. To distinguish between gene content specific for the ‘Ca. Methylomirabilis’ genus and the NC10 phylum, the genome of a distantly related NC10 phylum member, CSP1-5, an aerobic methylotroph, is included in the analysis. All genes for the conversion of nitrite to N2 identified in ‘Ca. M. oxyfera’ are conserved in ‘Ca. M. lanthanidiphila,’ including the two putative genes for NO dismutase. In addition both species have several heme-copper oxidases potentially involved in NO and O2 respiration. For the oxidation of methane ‘Ca. Methylomirabilis’ species encode a membrane bound methane monooxygenase. CSP1-5 can act as a methylotroph, but lacks the ability to activate methane. In contrast to ‘Ca. M. oxyfera,’ which harbors three methanol dehydrogenases (MDH), both CSP1-5 and ‘Ca. M. lanthanidiphila’ only encode a lanthanide-dependent XoxF-type MDH, once more underlining the importance of rare earth elements for methylotrophic bacteria. The pathways for the subsequent oxidation of formaldehyde to carbon dioxide and for the Calvin–Benson–Bassham cycle are conserved in all species. Furthermore, CSP1-5 can only interconvert nitrate and nitrite, but lacks subsequent nitrite or NO reductases. Thus, it appears that although the conversion of methanol to carbon dioxide is present in several NC10 phylum bacteria, the coupling of nitrite reduction to the oxidation of methane is a trait so far unique to the genus ‘Ca. Methylomirabilis.’

Methanol dehydrogenase (MDH) catalyzes the first step in methanol use by methylotrophic bacteria and the second step in methane conversion by methanotrophs. Gram-negative bacteria possess an MDH with pyrroloquinoline quinone (PQQ) as its catalytic center. This MDH belongs to the broad class of eight-bladed β propeller quinoproteins, which comprise a range of other alcohol and aldehyde dehydrogenases. A well-investigated MDH is the heterotetrameric MxaFI-MDH, which is composed of two large catalytic subunits (MxaF) and two small subunits (MxaI). MxaFI-MDHs bind calcium as a cofactor that assists PQQ in catalysis. Genomic analyses indicated the existence of another MDH distantly related to the MxaFI-MDHs. Recently, several of these so-called XoxF-MDHs have been isolated. XoxF-MDHs described thus far are homodimeric proteins lacking the small subunit and possess a rare-earth element (REE) instead of calcium. The presence of such REE may confer XoxF-MDHs a superior catalytic efficiency. Moreover, XoxF-MDHs are able to oxidize methanol to formate, rather than to formaldehyde as MxaFI-MDHs do. While structures of MxaFI- and XoxF-MDH are conserved, also regarding the binding of PQQ, the accommodation of a REE requires the presence of a specific aspartate residue near the catalytic site. XoxF-MDHs containing such REE-binding motif are abundantly present in genomes of methylotrophic and methanotrophic microorganisms and also in organisms that hitherto are not known for such lifestyle. Moreover, sequence analyses suggest that XoxF-MDHs represent only a small part of putative REE-containing quinoproteins, together covering an unexploited potential of metabolic functions.

The NO reductase from Paracoccus denitrificans reduces NO to N2O (2NO + 2H(+) + 2e(-) → N2O + H2O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O2-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O2 reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O2. We further show that electron transfer during single-turnover reduction of O2 is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.

CytcO (cytochrome c oxidase) is a membrane-bound multisubunit protein which catalyses the reduction of O2 to H2O. The reaction is arranged topographically so that the electrons and protons are taken from opposite sides of the membrane and, in addition, it is also linked to proton pumping across the membrane. Thus the CytcO moves an equivalent of two positive charges across the membrane per electron transferred to O2. Proton transfer through CytcO must be controlled by the protein to prevent leaks, which would dissipate the proton electrochemical gradient that is maintained across the membrane. The molecular mechanism by which the protein controls the unidirectionality of proton-transfer (cf. proton diode) reactions and energetically links electron transfer to proton translocation is not known. This short review summarizes selected results from studies aimed at understanding this mechanism, and we discuss a possible mechanistic principle utilized by the oxidase to pump protons.

Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N(2)O (2NO+2e(-)+2H(+)-->N(2)O+H(2)O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O(2)-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O(2), we demonstrate that protons are indeed consumed from the 'outside'. First, multiple turnover reduction of O(2) resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O(2) shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O(2) as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O(2) show electron transfer coupled to proton uptake from outside the NOR-liposomes with a tau=15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Adelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.

Ammonium and methane are inert molecules and dedicated enzymes are required to break up the N-H and C-H bonds. Until recently, only aerobic microorganisms were known to grow by the oxidation of ammonium or methane. Apart from respiration, oxygen was specifically utilized to activate the inert substrates. The presumed obligatory need for oxygen may have resisted the search for microorganisms that are capable of the anaerobic oxidation of ammonium and of methane. However extremely slowly growing, these "impossible" organisms exist and they found other means to tackle ammonium and methane. Anaerobic ammonium-oxidizing (anammox) bacteria use the oxidative power of nitric oxide (NO) by forging this molecule to ammonium, thereby making hydrazine (N2H4). Nitrite-dependent anaerobic methane oxidizers (N-DAMO) again take advantage of NO, but now apparently disproportionating the compound into dinitrogen and dioxygen gas. This intracellularly produced dioxygen enables N-DAMO bacteria to adopt an aerobic mechanism for methane oxidation.Although our understanding is only emerging how hydrazine synthase and the NO dismutase act, it seems clear that reactions fully rely on metal-based catalyses known from other enzymes. Metal-dependent conversions not only hold for these key enzymes, but for most other reactions in the central catabolic pathways, again supported by well-studied enzymes from model organisms, but adapted to own specific needs. Remarkably, those accessory catabolic enzymes are not unique for anammox bacteria and N-DAMO. Close homologs are found in protein databases where those homologs derive from (partly) known, but in most cases unknown species that together comprise an only poorly comprehended microbial world.

Contains fulltext : 163338.pdf (Publisher’s version ) (Closed access)

The NOR (nitric oxide reductase) from Paracoccus denitrificans catalyses the two-electron reduction of NO to N2O (2NO+2H++2e−→N2O+H2O). The NOR is a divergent member of the superfamily of haem-copper oxidases, oxygen-reducing enzymes which couple the reduction of oxygen with translocation of protons across the membrane. In contrast, reduction of NO catalysed by NOR is non-electrogenic which, since electrons are supplied from the periplasmic side of the membrane, implies that the protons needed for NO reduction are also taken from the periplasm. Thus NOR must contain a proton-transfer pathway leading from the periplasmic side of the membrane into the catalytic site. The proton pathway has not been identified, and the mechanism and timing of proton transfer during NO reduction is unknown. To address these questions, we have studied the reaction between NOR and the chemically less reactive oxidant O2 [Flock, Watmough and Adelroth (2005) Biochemistry 44 , 10711–10719]. When fully reduced NOR reacts with O2, proton-coupled electron transfer occurs in a reaction that is rate-limited by internal proton transfer from a group with a p K a of 6.6. This group is presumably an amino acid residue close to the active site that acts as a proton donor also during NO reduction. The results are discussed in the framework of a structural model that identifies possible candidates for the proton donor as well as for the proton-transfer pathway. Abbreviations: HCuO, haem-copper oxidase; NOR, nitric oxide reductase

Background Anaerobic ammonium oxidizing (anammox) bacteria may contribute up to 50% to the global nitrogen production, and are, thus, key players of the global nitrogen cycle. The molecular mechanism of anammox was recently elucidated and is suggested to proceed through a branched respiratory chain. This chain involves an exceptionally high number of c-type cytochrome proteins which are localized within the anammoxosome, a unique subcellular organelle. During transport into the organelle the c-type cytochrome apoproteins need to be post-translationally processed so that heme groups become covalently attached to them, resulting in mature c-type cytochrome proteins. Results In this study, a comparative genome analysis was performed to identify the cytochrome c maturation system employed by anammox bacteria. Our results show that all available anammox genome assemblies contain a complete type II cytochrome c maturation system. Conclusions Our working model suggests that this machinery is localized at the anammoxosome membrane which is assumed to be the locus of anammox catabolism. These findings will stimulate further studies in dissecting the molecular and cellular basis of cytochrome c biogenesis in anammox bacteria.

Bacterial nitric oxide reductases (NOR) are integral membrane proteins that catalyse the reduction of nitric oxide to nitrous oxide, often as a step in the process of denitrification. Most functional data has been obtained with NORs that receive their electrons from a soluble cytochrome c in the periplasm and are hence termed cNOR. Very recently, the structure of a different type of NOR, the quinol-dependent (q)-NOR from the thermophilic bacterium Geobacillus stearothermophilus was solved to atomic resolution [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238–246]. In this study, we have investigated the reaction between this qNOR and oxygen. Our results show that, like some cNORs, the G. stearothermophilus qNOR is capable of O2 reduction with a turnover of ~3electronss−1 at 40°C. Furthermore, using the so-called flow-flash technique, we show that the fully reduced (with three available electrons) qNOR reacts with oxygen in a reaction with a time constant of 1.8ms that oxidises the low-spin heme b. This reaction is coupled to proton uptake from solution and presumably forms a ferryl intermediate at the active site. The pH dependence of the reaction is markedly different from a corresponding reaction in cNOR from Paracoccus denitrificans, indicating that possibly the proton uptake mechanism and/or pathway differs between qNOR and cNOR. This study furthermore forms the basis for investigation of the proton transfer pathway in qNOR using both variants with putative proton transfer elements modified and measurements of the vectorial nature of the proton transfer. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Nitric oxide (NO) and nitrous oxide (N(2)O) are among nature's most powerful electron acceptors. In recent years it became clear that microorganisms can take advantage of the oxidizing power of these compounds to degrade aliphatic and aromatic hydrocarbons. For two unrelated bacterial species, the "NC10" phylum bacterium "Candidatus Methylomirabilis oxyfera" and the γ-proteobacterial strain HdN1 it has been suggested that under anoxic conditions with nitrate and/or nitrite, monooxygenases are used for methane and hexadecane oxidation, respectively. No degradation was observed with nitrous oxide only. Similarly, "aerobic" pathways for hydrocarbon degradation are employed by (per)chlorate-reducing bacteria, which are known to produce oxygen from chlorite [Formula: see text]. In the anaerobic methanotroph M. oxyfera, which lacks identifiable enzymes for nitrogen formation, substrate activation in the presence of nitrite was directly associated with both oxygen and nitrogen formation. These findings strongly argue for the role of NO, or an oxygen species derived from it, in the activation reaction of methane. Although oxygen generation elegantly explains the utilization of "aerobic" pathways under anoxic conditions, the underlying mechanism is still elusive. In this perspective, we review the current knowledge about intra-aerobic pathways, their potential presence in other organisms, and identify candidate enzymes related to quinol-dependent NO reductases (qNORs) that might be involved in the formation of oxygen.

Heme–copper oxidases (HCuOs) terminate the respiratory chain in mitochondria and most bacteria. They are transmembrane proteins that catalyse the reduction of oxygen and use the liberated free energy to maintain a proton-motive force across the membrane. The HCuO superfamily has been divided into the oxygen-reducing A-, B- and C-type oxidases as well as the bacterial NO reductases (NOR), catalysing the reduction of NO in the denitrification process. Proton transfer to the catalytic site in the mitochondrial-like A family occurs through two well-defined pathways termed the D- and K-pathways. The B, C, and NOR families differ in the pathways as well as the mechanisms for proton transfer to the active site and across the membrane. Recent structural and functional investigations, focussing on proton transfer in the B, C and NOR families will be discussed in this review. This article is part of a Special Issue entitled: Respiratory Oxidases.

Cytochrome ba(3) from Thermus thermophilus is a member of the family of B-type heme-copper oxidases, which have a low degree of sequence homology to the well-studied mitochondrial-like A-type enzymes. Recently, it was suggested that the ba(3) oxidase has only one pathway for the delivery of protons to the active site and that this pathway is spatially analogous to the K-pathway in the A-type oxidases [Chang, H.-Y., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 16169-16173]. This suggested pathway includes two threonines at positions 312 and 315. In this study, we investigated the time-resolved reaction between fully reduced cytochrome ba(3) and O(2) in variants where Thr-312 and Thr-315 were modified. While in the A-type oxidases this reaction is essentially unchanged in variants with the K-pathway modified, in the Thr-312 --Ser variant in the ba(3) oxidase both reactions associated with proton uptake from solution, the P(R) --F and F --O transitions, were slowed compared to those of wild-type ba(3). The observed time constants were slowed approximately 3-fold (for P(R) --F, from 60 to approximately 170 mus in the wild type) and approximately 30-fold (for F --O, from 1.1 to approximately 40 ms). In the Thr-315 --Val variant, the F --O transition was approximately 5-fold slower (5 ms) than for the wild-type oxidase, whereas the P(R) --F transition displayed an essentially unchanged time constant. However, the uptake of protons from solution was a factor of 2 slower and decoupled from the optical P(R) --F transition. Our results thus show that proton uptake is significantly and specifically inhibited in the two variants, strongly supporting the suggested involvement of T312 and T315 in the transfer of protons to the active site during O(2) reduction in the ba(3) oxidase.

Nitric -oxide reductase (NOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N(2)O) (2NO + 2H(+) + 2e(-) -->N(2)O + H(2)O) by a poorly understood mechanism. NOR contains two low spin hemes c and b, one high spin heme b(3), and a non-heme iron Fe(B). Here, we have studied the reaction between fully reduced NOR and NO using the "flow-flash" technique. Fully (four-electron) reduced NOR is capable of two turnovers with NO. Initial binding of NO to reduced heme b(3) occurs with a time constant of approximately 1 micros at 1.5 mM NO, in agreement with earlier studies. This reaction is [NO]-dependent, ruling out an obligatory binding of NO to Fe(B) before ligation to heme b(3). Oxidation of hemes b and c occurs in a biphasic reaction with rate constants of 50 s(-1) and 3 s(-1) at 1.5 mM NO and pH 7.5. Interestingly, this oxidation is accelerated as [NO] is lowered; the rate constants are 120 s(-1) and 12 s(-1) at 75 microM NO. Protons are taken up from solution concomitantly with oxidation of the low spin hemes, leading to an acceleration at low pH. This effect is, however, counteracted by a larger degree of substrate inhibition at low pH. Our data thus show that substrate inhibition in NOR, previously observed during multiple turnovers, already occurs during a single oxidative cycle. Thus, NO must bind to its inhibitory site before electrons redistribute to the active site. The further implications of our data for the mechanism of NO reduction by NOR are discussed.

The heme-copper oxidase (HCuO) superfamily consists of integral membrane proteins that catalyze the reduction of either oxygen or nitric oxide. The HCuOs that reduce O 2 to H 2 O couple this reaction to the generation of a transmembrane proton gradient by using electrons and protons from opposite sides of the membrane and by pumping protons from inside the cell or organelle to the outside. The bacterial NO-reductases (NOR) reduce NO to N 2 O (2NO + 2e − + 2H + → N 2 O + H 2 O), a reaction as exergonic as that with O 2 . Yet, in NOR both electrons and protons are taken from the outside periplasmic solution, thus not conserving the free energy available. The cbb 3 -type HCuOs catalyze reduction of both O 2 and NO. Here, we have investigated energy conservation in the Rhodobacter sphaeroides cbb 3 oxidase during reduction of either O 2 or NO. Whereas O 2 reduction is coupled to buildup of a substantial electrochemical gradient across the membrane, NO reduction is not. This means that although the cbb 3 oxidase has all of the structural elements for uptake of substrate protons from the inside, as well as for proton pumping, during NO reduction no pumping occurs and we suggest a scenario where substrate protons are derived from the outside solution. This would occur by a reversal of the proton pathway normally used for release of pumped protons. The consequences of our results for the general pumping mechanism in all HCuOs are discussed.

Heme–copper oxidases (HCuOs) are the terminal components of the respiratory chain in the mitochondrial membrane or the cell membrane in many bacteria. These enzymes reduce oxygen to water and use the free energy from this reaction to maintain a proton-motive force across the membrane in which they are embedded. The heme–copper oxidases of the cbb3-type are only found in bacteria, often pathogenic ones since they have a low Km for O2, enabling the bacteria to colonize semi-anoxic environments. Cbb3-type (C) oxidases are highly divergent from the mitochondrial-like aa3-type (A) oxidases, and within the heme–copper oxidase family, cbb3 is the closest relative to the most divergent member, the bacterial nitric oxide reductase (NOR). Nitric oxide reductases reduce NO to N2O without coupling the reaction to the generation of any electrochemical proton gradient. The significant structural differences between A- and C-type heme–copper oxidases are manifested in the lack in cbb3 of most of the amino acids found to be important for proton pumping in the A-type, as well as in the different binding characteristics of ligands such as CO, O2 and NO. Investigations of the reasons for these differences at a molecular level have provided insights into the mechanism of O2 and NO reduction as well as the proton-pumping mechanism in all heme–copper oxidases. In this paper, we discuss results from these studies with the focus on the relationship between proton transfer and ligand binding and reduction. In addition, we present new data, which show that CO binding to one of the c-type hemes of CcoP is modulated by protein–lipid interactions in the membrane. These results show that the heme c-CO binding can be used as a probe of protein–membrane interactions in cbb3 oxidases, and possible physiological consequences for this behavior are discussed.