https://zenodo.org/record/1258475/files/article.pdf

Electron transfers in chemistry and biology

Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

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Cell biology and molecular basis of denitrification.

Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions

The respiration of oxygen is fundamental to the life of higher animals and plants. The basic respiratory process in the mitochondria of these organisms involves the donation of electrons by low-redox-potential electron donors such as NADH. This is followed by electron transfer through a range of redox cofactors, bound to integral membrane or membrane-associated protein complexes. The process terminates in the reduction of the high-redox-potential electron acceptor, oxygen (Fig. 1). The free energy released during this electrontransfer process is used to drive the translocation of protons across the mitochondrial membrane to generate a trans-membrane proton electrochemical gradient or protonmotive force (∆p) that can drive the synthesis of ATP (Fig. 1). The respiratory flexibility of the mammalian mitochondrion is rather poor. There is some flexibility at the level of electron input (Fig. 1), but none at the level of electron output where cytochrome aa $ oxidase provides the only means of oxygen reduction. In the case of plant mitochondria, a slightly greater degree of respiratory flexibility is encountered with a number of alternative NADH dehydrogenases and two oxidases being apparent. This respiratory flexibility affords plant mitochondria with the capacity to contribute to processes other than the generation of ATP. For example, electron transfer from the alternative NADH dehydrogenase to the alternative oxidase is not coupled to the generation of ∆p and instead serves to release energy as heat, which can volatilize insect attractants to aid pollination. In the American skunk cabbage this same mechanism for heat production serves to permit growth at subzero temperatures (Nicholls & Ferguson, 1992). There is also some respiratory flexibility in the mitochondria of yeast, filamentous fungi and ancient protozoa, but it is amongst the Bacteria and Archaea that respiratory flexibility can be found at its most extreme. In these organisms, a diverse range of electron acceptors can be utilized including elemental sulphur and sulphur oxyanions (Hamilton, 1998), organic sulphoxides and sulphonates (Lie et al., 1999; McAlpine et al., 1998), nitrogen oxy-anions and nitrogen oxides (Berks et al., 1995), organic N-oxides (Czjzek et al., 1998), halogenated organics (Dolfing, 1990; Louie & Mohn, 1999; van de Pas et al., 1999), metalloid oxy-anions such as selenate and arsenate (Krafft & Macy, 1998; Macy et al., 1996, 1993; Schroder et al., 1997), transition metals such as Fe(III) and Mn(IV) (Lovley, 1991), and radionuclides such as U(VI) (Lovley & Phillips, 1992) and Tc(VII) (Lloyd et al., 1999). This respiratory diversity can be found amongst pyschrophiles, mesophiles and hyperthermophiles and contributes to the ability of prokaryotes to colonize many of Earth’s most hostile microoxic and anoxic environments.

Bacterial respiration: a flexible process for a changing environment.

Nitrogen is a basic element for life because it is a component of the two preeminent biological macromolecules: proteins and nucleic acids. Nitrogen exists in the biosphere in several oxidation states, from N(V) to N(−III). Interconversions of these nitrogen species constitute the global

Prokaryotic Nitrate Reduction: Molecular Properties and Functional Distinction among Bacterial Nitrate Reductases

Recent discoveries relating to pathways of anaerobic electron transport in the Rhodospirillaceae are reviewed. The main emphasis is on the organism Rhodobacter capsulatus ∗∗ but comparisons are made with Rhodobacter sphaeroides ∗∗ f. sp. denitrificans and Rhodopseudomonas palustris. The known electron acceptors for anaerobic respiration in Rhodobacter capsulatus are trimethylamine-N-oxide (TMAO), dimethyl sulphoxide (DMSO), nitrate and nitrous oxide. In each case respiration generates a proton electrochemical gradient and in some cases can support growth on non-fermentable carbon sources. However, the principal objective of this review is to discuss the possibility that, apart from a role in energy conservation, anaerobic respiration in the photosynthetic bacteria may have a special function in maintaining redox balance during photosynthetic metabolism. Thus the electron acceptors mentioned above may serve as auxiliary oxidants: (a) to maintain an optimal redox poise of the photosynthetic electron transport chain; (b) to provide a sink for electrons during phototrophic growth on highly reduced carbon substrates.

Molecular properties of the nitrate reductase, nitrous oxide reductase and a single enzyme responsible for reduction of TMAO and DMSO are discussed. These enzymes are all located in the periplasm. Electrons destined for all three enzymes can originate from the rotenone-sensitive NADH dehydrogenase but do not proceed through the antimycin- and myxothiazol-sensitive cytochrome bc1 complex. It is likely, therefor, that the pathways of anaerobic respiration overlap with the cyclic photosynthetic electron transport chain only at the level of the ubiquinone pool. Redox components which might be involved in the terminal branches of anaerobic respiration are discussed.

Anaerobic respiration in the Rhodospirillaceae: characterisation of pathways and evaluation of roles in redox balancing during photosynthesis

Phototrophic growth of Rhodobacter capsulatus (formerly Rhodopseudomonas capsulata) under anaerobic conditions with either butyrate or propionate as carbonsource was dependent on the presence of either CO2 or an auxiliary oxidant. NO
 -
 3
 , N2O, trimethylamine-N-oxide (TMAO) or dimethylsulphoxide (DMSO) were effective provided the appropriate anaerobic respiratory pathway was present. NO
 -
 3
 was reduced extensively to NO
 -
 3
 , TMAO to trimethylamine and DMSO to dimethylsulphide under these conditions. Analysis of culture fluids by nuclear magnetic resonance showed that two moles of TMAO or DMSO were reduced per mole of butyrate utilized and one mole of either oxidant was reduced per mole of propionate consumed. The growth rate of Rb. capsulatus on succinate or malate as carbon source was enhanced by TMAO in cultures at low light intensity but not at high light intensities. A new function for anaerobic respiration during photosynthesis is proposed: it permits reducing equivalents from reduced substrates to pass to auxiliary oxidants present in the medium. The use of CO2 or auxiliary oxidants under phototrophic conditions may be influence by the availability of energy from light. It is suggested that the nuclear magnetic resonance methodology developed could have further applications in studies of bacterial physiology.

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The role of auxiliary oxidants in maintaining redox balance during phototrophic growth of Rhodobacter capsulatus on propionate or butyrate

1.

The properties of nitrate reductase activities have been compared in several strains of Rhodopseudomonas capsulata grown phototrophically in the presence of nitrate as sole nitrogen source.




2.

Strains AD2 and BK5 resemble the spontaneous mutant N22DNAR+ (described by McEwan et al. 1982 FEBS Lett. 150, 277\2-280) in that reduction of nitrate was inhibited by either illumination or oxygen but not by NH 4 + , and that electron flow to nitrate under dark anaerobic conditions generated a cytoplasmic membrane potential (as judged by an electrochromic shift in the absorbance spectrum of endogenous carotenoid pigments). In contrast disappearance of nitrate from suspensions of strains N22 and St. Louis was dependent upon illumination and was inhibited by NH 4 + . Membrane potentials were not generated by addition of nitrate in the dark to N22, St. Louis or strain Kbl.




3.

Nitrate reductase was shown to be located in the periplasmic space of both strain AD2 and mutant N22DNAR+. The nitrate reductase activity in cells of AD2 and N22DNAR+ was relatively insensitive to azide, with 0.5mM azide required for 50% inhibition. The nitrate reductase of strain BK5 was more strongly associated with the cytoplasmic membrane and no conclusion could be reached about whether it was located on the periplasmic or cytoplasmic surface. In BK5 cells nitrate reductase activity was sensitive to low concentrations of azide (50% inhibition with 2 \gmM azide). It is proposed that functionally the nitrate reductase activity in strains AD2, BK5 and N22DNAR+ has identical roles. These roles are suggested to include:




(i)

The first step in the assimilation of nitrate.




(ii).

Provision of an alternative electron acceptor to oxygen for generating a membrane potential.




(iii).

A mechanism for disposing of excess reducing equivalents in the maintenance of balanced growth.

This type of nitrate reductase, especially in AD2 and N22DNAR+, appears to resemble that described in a denitrifying strain of Rps. sphaeroides, but to differ markedly from its membrane-bound counterpart in other bacteria including the denitrifying Paracoccus denitrificans and Escherichia coli.




4.

In other strains of Rps. capsulata including St. Louis, N22 and Kbl, only an assimilatory nitrate reductase, whose activity in intact cells is relatively sensitive to azide, is present in anaerobic, phototrophic cultures grown with nitrate as nitrogen source. As this reductase cannot be detected after breakage of cells, no conclusion can be made as to its location in the cell.

Rationalization of properties of nitrate reductases in Rhodopseudomonas capsulata

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/0014-5793%2878%2980218-X

The charging capacitance of the chromatophore membrane

Under dark and essentially anaerobic conditions electron flow to either dimethylsulphoxide or trimethylamine-N-oxide in cells of Rhodopseudomonas capsulata has been shown to generate a membrane potential. This conclusion is based on the observation of a red shift in the carotenoid absorption band which is a well characterised indicator of membrane potential in this bacterium. The magnitude of the dimethylsulphoxide- or trimethylamine-N-oxide-dependent membrane potential was reduced either by a protonophore uncoupler of oxidative phosphorylation or synergistically by a combination of a protonophore plus rotenone, an inhibitor of electron flow from NADH dehydrogenase. These findings, together with the observation that venturicidin, an inhibitor of the proton translocating ATPase, did not reduce the membrane potential, show that electron flow to dimethylsulphoxide or trimethylamine-N-oxide is coupled to proton translocation. Thus contrary to some previous proposals dark and anaerobic growth of Rps. capsulata in the presence of dimethylsulphoxide or trimethylamine-N-oxide cannot be regarded as purely fermentative.

J. Barry Jackson

Papers

Anaerobic respiration in the Rhodospirillaceae: characterisation of pathways and evaluation of roles in redox balancing during photosynthesis

The role of auxiliary oxidants in maintaining redox balance during phototrophic growth of Rhodobacter capsulatus on propionate or butyrate

Rationalization of properties of nitrate reductases in Rhodopseudomonas capsulata

The charging capacitance of the chromatophore membrane

Electron flow to dimethylsulphoxide or trimethylamine-N-oxide generates a membrane potential in Rhodopseudomonas capsulata.