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.

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.

Department of Chemistry, The Johns Hopkins University, Charles and 34th Streets, Baltimore, Maryland 21218, Kluyver Laboratory for Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands, Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering at OHSU, Beaverton, Oregon 97006, and Department of Microbiology and Molecular Genetics, University of California, Los Angeles, California 90095

Nitric Oxide in Biological Denitrification: Fe/Cu Metalloenzyme and Metal Complex NOx Redox Chemistry

Nitric oxide reductase (NOR) is an iron-containing enzyme that catalyzes the reduction of nitric oxide (NO) to generate a major greenhouse gas, nitrous oxide (N(2)O). Here, we report the crystal structure of NOR from Pseudomonas aeruginosa at 2.7 angstrom resolution. The structure reveals details of the catalytic binuclear center. The non-heme iron (Fe(B)) is coordinated by three His and one Glu ligands, but a His-Tyr covalent linkage common in cytochrome oxidases (COX) is absent. This structural characteristic is crucial for NOR reaction. Although the overall structure of NOR is closely related to COX, neither the D- nor K-proton pathway, which connect the COX active center to the intracellular space, was observed. Protons required for the NOR reaction are probably provided from the extracellular side.

https://science.sciencemag.org/content/sci/330/6011/1666.full.pdf

Structural Basis of Biological N2O Generation by Bacterial Nitric Oxide Reductase

The overall goal of this study was to determine the molecular and metabolic responses of chemostat cultures of model nitrifying bacteria to imposition of and recovery from transient anoxic conditio...

Petra Girsch

Papers

Purification and initial kinetic and spectroscopic characterization of NO reductase from Paracoccus denitrificans