Enzyme-catalysed nitrate reduction—themes and variations as revealed by protein film voltammetry

Butt, Julea N.; Anderson, Lee J.; Rubio, Luis M.; Richardson, David J.; Flores, Enrique; Herrero, Antonia

doi:10.1016/s1567-5394(02)00049-x

Cited by 11 publications

(14 citation statements)

References 4 publications

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“…Protein Film Voltammetry-Protein film voltammetry was performed with a three-electrode cell configuration housed in an anaerobic chamber as described previously (27). All potentials are reported with respect to the standard hydrogen electrode.…”

Section: Methodsmentioning

confidence: 99%

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: Methodsmentioning

confidence: 99%

Tuning a Nitrate Reductase for Function

Jepson¹,

Anderson

Rubio

et al. 2004

Journal of Biological Chemistry

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“…Nitrate reductases have extensively been studied by direct immobilisation of the enzyme on an electrode 30,116 , which has provided much insight into the catalytic mechanism of 110 selected nitrate reductases [117][118][119][120][121] . NO 3 − binds to Mo(IV), which is oxidised to Mo(VI) upon ligation; the cycle is closed by two ensuing one-electron steps (thus involving Mo(V)) leading to NO 2 − and Mo(IV) 122 .…”

Section: Molecular Catalysts: Macrocycles and Enzymesmentioning

confidence: 99%

Powering denitrification: the perspectives of electrocatalytic nitrate reduction

Duca

Koper

2012

Energy Environ. Sci.

535

439

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“…For example, in the periplasmic enzymes, cysteine provides a thiol ligand to the molybdenum ion, whereas aspartate provides one or two oxygen ligands to the molybdenum ion in the membrane-bound enzymes (3, 4, 8 -10). In evolutionary terms the periplasmic and cytoplasmic assimilatory enzymes are most closely related, and this is reflected in similar spectroscopic properties between the two enzymes, although the redox properties of the molybdenum centers have become tuned for their different metabolic functions (2,(11)(12)(13).…”

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confidence: 99%

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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Enzyme-catalysed nitrate reduction—themes and variations as revealed by protein film voltammetry

Cited by 11 publications

References 4 publications

Tuning a Nitrate Reductase for Function

Tuning a Nitrate Reductase for Function

Powering denitrification: the perspectives of electrocatalytic nitrate reduction

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

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