Characterization of the paramagnetic iron‐containing redox centres of <i>Thiosphaera pantotropha</i> periplasmic nitrate reductase

Bretón, J.; Berks, Ben C.; Reilly, Ann; Thomson, Andrew J.; Ferguson, Stuart J.; Richardson, David J.

doi:10.1016/0014-5793(94)00445-5

Cited by 56 publications

(57 citation statements)

References 52 publications

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“…The similarity in the NarB and periplasmic nitrate reductase iron-sulfur clusters is also reflected in rather similar redox properties, with the NarB cluster having a midpoint redox potential of Ϫ190 mV that compares with Ϫ160 mV for the equivalent cluster in the periplasmic nitrate reductase of Paracoccus denitrificans (7).…”

Section: Discussionmentioning

confidence: 95%

“…Periplasmic nitrate reductases are also linked to quinol oxidation in respiratory electron transport chains, but they have a range of different functions including the disposal of reducing equivalents during aerobic growth and nitrate respiration in nitrate-limited environments (5,6). In periplasmic nitrate reductases, electrons from quinol are generally passed through one or two cytochrome c-containing proteins (NapC and NapB) to the catalytic subunit, NapA, that contains a bis-Mo-MGD cofactor and a [4Fe-4S] cluster (7)(8)(9)(10). As with the membrane-bound nitrate reductases, synthesis of the periplasmic nitrate reductase is insensitive to ammonium, but the enzyme can be expressed either anaerobically or aerobically depending on the organism (2).…”

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confidence: 99%

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Tuning a Nitrate Reductase for Function

Jepson¹,

Anderson

Rubio

et al. 2004

Journal of Biological Chemistry

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Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. In the present study the ferredoxindependent nitrate reductase NarB of the cyanobacterium Synechococcus sp. PCC 7942 was analyzed by spectropotentiometry and protein film voltammetry. Metal and acid-labile sulfide analysis revealed nearest integer values of 4:4:1 (iron/sulfur/molybdenum)/molecule of NarB. Analysis of dithionite-reduced enzyme by low temperature EPR revealed at 10 K the presence of a signal that is characteristic of a [4Fe-4S] 1؉ cluster. EPRmonitored potentiometric titration of NarB revealed that this cluster titrated as an n ‫؍‬ 1 Nernstian component with a midpoint redox potential (E m ) of ؊190 mV. EPR spectra collected at 60 K revealed a Mo(V) signal termed "very high g" with g av ‫؍‬ 2.0047 in air-oxidized enzyme that accounted for only 10 -20% of the total molybdenum. This signal disappeared upon reduction with dithionite, and a new "high g" species (g av ‫؍‬ 1.9897) was observed. In potentiometric titrations the high g Mo(V) signal developed over the potential range of ؊100 to ؊350 mV (E m Mo 6؉/5؉ ‫؍‬ ؊150 mV), and when fully developed, it accounted for 1 mol of Mo(V)/mol of enzyme. Protein film voltammetry of NarB revealed that activity is turned on at potentials below ؊200 mV, where the cofactors are predominantly [4Fe-4S] 1؉ and Mo 5؉ . The data suggests that during the catalytic cycle nitrate will bind to the Mo 5؉ state of NarB in which the enzyme is minimally two-electron-reduced. Comparison of the spectral properties of NarB with those of the membranebound and periplasmic respiratory nitrate reductases reveals that it is closely related to the periplasmic enzyme, but the potential of the molybdenum center of NarB is tuned to operate at lower potentials, consistent with the coupling of NarB to low potential ferredoxins in the cell cytoplasm.Nitrate is a widely used and readily available source of inorganic nitrogen for plants and microorganisms (1). Fixed inorganic nitrogen is mainly supplied to natural environments either from human agricultural or industrial activities or from biological nitrogen fixation. Most of it is converted to nitrate by nitrifying bacteria, and the nitrate then serves as a nitrogen source for assimilation or as a respiratory electron acceptor. Bacterial nitrate reductases are molybdoenzymes that can catalyze the two-electron reduction of nitrate to nitrite and can be classified into three groups according to their localization and function (2). Respiratory membrane-bound nitrate reductases are generally integral membrane protein complexes with the active site located on the cytoplasmic face of the cytoplasmic membrane and are constituted by subunits (e.g. NarI and NarH) that mediate electron transfer from the quinol pool to the catalytic subunit, NarG, which contains a bismolybdopterin guanine dinucleotide (bis-Mo-MGD) 1 cofactor and a [4Fe-4S] cluster (3, 4). These membrane-bound nitrate reductases couple quinol oxidation b...

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Section: Discussionmentioning

confidence: 95%

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confidence: 99%

Tuning a Nitrate Reductase for Function

Jepson¹,

Anderson

Rubio

et al. 2004

Journal of Biological Chemistry

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“…The latter enzyme is soluble and appears to consist of two subunits, one with a c-type heine and one with a molybdenum center (Breton et al, 1994). The enzyme has been suggested to be essential for aerobic denitrification.…”

Section: The Anaerobic Respiratory Networkmentioning

confidence: 99%

Regulation of oxidative phosphorylation: The flexible respiratory network ofParacoccus denitrificans

Spanning

Boer

Reijnders

et al. 1995

J Bioenerg Biomembr

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General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Download date: 12 May 2018Journal of Bioenergetics and Biomembranes, Vol. 27, No. 5, 1995 Received May 29, 1995 Paracoccus denitrificans is a facultative anaerobic bacterium that has the capacity to adjust its metabolic infrastructure, quantitatively and/or qualitatively, to the prevailing growth condition. In this bacterium the relative activity of distinct catabolic pathways is subject to a hierarchical control. In the presence of oxygen the aerobic respiration, the most efficient way of electron transfer-linked phosphorylation, has priority. At high oxygen tensions P. denitrificans synthesizes an oxidase with a relatively low affinity for oxygen, whereas under oxygen limitation a high-affinity oxidase appears specifically induced. During anaerobiosis, the pathways with lower free energy-transducing efficiency are induced. In the presence of nitrate, the expression of a number of dehydrogenases ensures the continuation of oxidative pbosphorylation via denitrification. After identification of the structural components that are involved in both the aerobic and the anaerobic respiratory networks of P. denitrificans, the intriguing next challenge is to get insight in its regulation. Two transcription regulators have recently been demonstrated to be involved in the expression of a number of aerobic and/or anaerobic respiratory complexes in P. denitrificans. Understanding of the regulation machinery is beginning to emerge and promises much excitement in discovery.KEY WORDS: Respiratory network; multiple oxidases; denitrification; gene regulation; FNR; Paracoccus denitrificans; Escherichia coli. ~TRODUCTIONThe ultimate goal of living organisms is to contribute to a continued existence of their species by means of survival and reproduction. In unicellular organisms, the ability to survive depends on the cell's potential to adapt its metabolism to the available carbon and free-energy sources in its natural habitat, ensuring maintenance and growth. The more such an environment is subject to fluctuations in the supply of these substrates, the higher the demands that are made upon the potential of the cell to adjust its metabolic properties. A profound example of this flexibility is the process of bacterial respiration...

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“…Thus Nap systems have a range of physiological functions that include the disposal of reducing equivalents during aerobic growth on reduced carbon substrates and anaerobic nitrate respiration as part of bacterial ammonification or denitrification pathways (2,5,6). In Nap systems electrons from quinol are generally, but not always, passed through one or two cytochrome c-containing proteins (NapC and NapB) to the catalytic subunit, NapA, that contains a bis-Mo-MGD cofactor and a [4Fe-4S] cluster (7)(8)(9)(10). The periplasmic and membrane-bound nitrate reductases are structurally quite distinct.…”

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Spectropotentiometric and Structural Analysis of the Periplasmic Nitrate Reductase from Escherichia coli

Jepson¹,

Mohan

Clarke³

et al. 2007

Journal of Biological Chemistry

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105

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The Escherichia coli NapA (periplasmic nitrate reductase) contains a [4Fe-4S] cluster and a Mo-bis-molybdopterin guanine dinucleotide cofactor. The NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). These proteins have been purified and studied by spectropotentiometry, and the structure of NapA has been determined. In contrast to the well characterized heterodimeric NapAB systems of ␣-proteobacteria, such as Rhodobacter sphaeroides and Paracoccus pantotrophus, the ␥-proteobacterial E. coli NapA and NapB proteins purify independently and not as a tight heterodimeric complex. This relatively weak interaction is reflected in dissociation constants of 15 and 32 M determined for oxidized and reduced NapAB complexes, respectively. The surface electrostatic potential of E. coli NapA in the apparent NapB binding region is markedly less polar and anionic than that of the ␣-proteobacterial NapA, which may underlie the weaker binding of NapB. The molybdenum ion coordination sphere of E. coli NapA includes two molybdopterin guanine dinucleotide dithiolenes, a protein-derived cysteinyl ligand and an oxygen atom. The Mo-O bond length is 2.6 Å , which is indicative of a water ligand. The potential range over which the Mo 6؉ state is reduced to the Mo 5؉ state in either NapA (between ؉100 and ؊100 mV) or the NapAB complex (؊150 to ؊350 mV) is much lower than that reported for R. sphaeroides NapA (midpoint potential Mo 6؉/5؉ > ؉350 mV), and the form of the Mo 5؉ EPR signal is quite distinct. In E. coli NapA or NapAB, the Mo 5؉ state could not be further reduced to Mo 4؉. We then propose a catalytic cycle for E. coli NapA in which nitrate binds to the Mo 5؉ ion and where a stable des-oxo Mo 6؉ species may participate.Bacterial nitrate reductases are molybdoenzymes that catalyze the two-electron reduction of nitrate to nitrite. They can be classified into three groups according to their localization and function, namely membrane-bound respiratory, periplasmic respiratory, or cytoplasmic assimilatory enzymes (1, 2). Bacterial respiratory membrane-bound nitrate reductases, such as Escherichia coli NarGHI, are generally integral membrane protein complexes that have an active site on the cytoplasmic face of the membrane and couple quinol oxidation by nitrate to the generation of a transmembrane proton electrochemical gradient (2). The catalytic subunit, NarG, contains a Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) 3 cofactor and a [4Fe-4S] cluster (3, 4). Periplasmic nitrate reductases (Nap) are also linked to quinol oxidation in respiratory electron transport chains, but do not conserve the free energy of the QH 2 -nitrate couple. Nitrate reduction via Nap can be coupled to energy conservation if the primary quinone reductase, for example NADH dehydrogenase or formate dehydrogenase, generates a proton electrochemical gradient. Thus Nap systems have a range of physiological functions that include the disposal of reducing equivalents during aerobic growth on reduced carbon substrates and anaerob...

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Characterization of the paramagnetic iron‐containing redox centres of Thiosphaera pantotropha periplasmic nitrate reductase

Cited by 56 publications

References 52 publications

Tuning a Nitrate Reductase for Function

Tuning a Nitrate Reductase for Function

Regulation of oxidative phosphorylation: The flexible respiratory network ofParacoccus denitrificans

Spectropotentiometric and Structural Analysis of the Periplasmic Nitrate Reductase from Escherichia coli

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