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.

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 N and O 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 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.

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.

Nitric oxide (NO) and nitrous oxide (N2O) 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 (ClO2−). 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.