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...

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...

It is becoming recognized that leghaemoglobin constitutes an important buffer for the cytotoxic nitric oxide radical (NO N ) in root nodules, although the sources of this NO N within nodules are unclear. In Bradyrhizobium japonicum bacteroids, NO N can be produced through the denitrification process, during which nitrate is reduced to nitrite by the periplasmic nitrate reductase Nap, and nitrite is reduced to NO N by the respiratory nitrite reductase NirK. To assess the contribution of bacteroidal denitrification to the NO N within nitrate-treated soybean nodules, electron paramagnetic resonance and UV-visible spectroscopy were employed to study the presence of nitrosylleghaemoglobin (LbNO) within nodules from plants inoculated with wild-type, napA or nirK B. japonicum strains. Since it has been found that hypoxia induces NO N production in plant root tissue, and that plant roots can be subjected to hypoxic stress during drought and flooding, the effect of hypoxic stress on the formation of LbNO complexes within nodules was also investigated. Maximal levels of LbNO were observed in nodules from plants treated with nitrate and subjected to hypoxic conditions. It is shown that, in the presence of nitrate, all of the LbNO within normoxic nodules arises from nitrate reduction by the bacteroidal periplasmic nitrate reductase, whereas Nap activity is only responsible for half of the LbNO within hypoxic nodules. In contrast to Nap, NirK is not essential for LbNO formation under any condition tested.

Protein film voltammetry of Paracoccus pantotrophus respiratory nitrate reductase (NarGH) and Synechococcus elongatus assimilatory nitrate reductase (NarB) shows that reductive activation of these enzymes may be required before steady state catalysis is observed. For NarGH complementary spectroscopic studies suggest a structural context for the activation. Catalytic protein film voltammetry at a range of temperatures has allowed quantitation of the activation energies for nitrate reduction. For NarGH with an operating potential of ca. 0.05 V the activation energy of ca. 35 kJ mol-1 is over twice that measured for NarB whose operating potential is ca. -0.35 V.

Bacterial nitrate reductases can be classified into at least three groups according to their localization and function, namely membrane-bound (NAR) or periplasmic (NAP) respiratory and cytoplasmic assimilatory (NAS) enzymes. Monomeric NASs are the simplest of the soluble nitrate reductases, although heterodimeric NASs exist, and a common structural arrangement of NAP is that of a NapAB heterodimer. Using bioinformatic analysis of published genomes, we have identified more representatives of a monomeric class of NAP, which is the evolutionary link between the monomeric NASs and the heterodimeric NAPs. This has further established the monomeric structural clade of NAP. The operons of the monomeric NAP do not contain NapB and suggest that other redox partners are employed by these enzymes, including NapM or NapG predicted proteins. A structural alignment and comparison of the monomeric and heterodimeric NAPs suggests that a difference in surface polarity is related to the interaction of the respective catalytic subunit and redox partner.

Periplasmic SER (selenate reductase) from Thauera selenatis is classified as a member of the Tat (twin-arginine translocase)-translocated (Type II) molybdoenzymes and comprises three subunits each containing redox cofactors. Variable-temperature X-band EPR spectra of the purified SER complex showed features attributable to centres [3Fe-4S]1+, [4Fe-4S]1+, Mo(V) and haem-b. EPR-monitored redox-potentiometric titration of the SerABC complex (SerA-SerB-SerC, a hetero-trimetric complex of alphabetagamma subunits) revealed that the [3Fe-4S] cluster (FS4, iron-sulfur cluster 4) titrated as n=1 Nernstian component with a midpoint redox potential (E(m)) of +118+/-10 mV for the [3Fe-4S]1+/0 couple. A [4Fe-4S]1+ cluster EPR signal developed over a range of potentials between 300 and -200 mV and was best fitted to two sequential Nernstian n=1 curves with midpoint redox potentials of +183+/-10 mV (FS1) and -51+/-10 mV (FS3) for the two [4Fe-4S]1+/2+ cluster couples. Upon further reduction, the observed signal intensity of the [4Fe-4S]1+ cluster decreases. This change in intensity can again be fitted to an n=1 Nernstian component with a midpoint potential (E(m)) of about -356 mV (FS2). It is considered likely that, at low redox potential (E(m) less than -300 mV), the remaining oxidized cluster is reduced (spin S=1/2) and strongly spin-couples to a neighbouring [4Fe-4S]1+ cluster rendering both centres EPR-silent. The involvement of both [3Fe-4S] and [4Fe-4S] clusters in electron transfer to the active site of the periplasmic SER was demonstrated by the re-oxidation of the clusters under anaerobic selenate turnover conditions. Attempts to detect a high-spin [4Fe-4S] cluster (FS0) in SerA at low temperature (5 K) and high power (100 mW) were unsuccessful. The Mo(V) EPR recorded at 60 K, in samples poised at pH 6.0, displays principal g values of g3 approximately 1.999, g2 approximately 1.996 and g1 approximately 1.965 (g(av) 1.9867). The dominant features at g2 and g3 are not split, but hyperfine splitting is observed in the g1 region of the spectrum and can be best simulated as arising from a single proton with a coupling constant of A1 (1H)=1.014 mT. The presence of the haem-b moiety in SerC was demonstrated by the detection of a signal at g approximately 3.33 and is consistent with haem co-ordinated by methionine and lysine axial ligands. The combined evidence from EPR analysis and sequence alignments supports the assignment of the periplasmic SER as a member of the Type II molybdoenzymes and provides the first spectro-potentiometric insight into an enzyme that catalyses a key reductive reaction in the biogeochemical selenium cycle.

Paracoccus pantotrophus grown anaerobically under denitrifying conditions expressed similar levels of the periplasmic nitrate reductase (NAP) when cultured in molybdate-or tungstate-containing media. A native PAGE gel stained for nitrate reductase activity revealed that only NapA from molybdate-grown cells displayed readily detectable nitrate reductase activity. Further kinetic analysis showed that the periplasmic fraction from cells grown on molybdate (3 WM) reduced nitrate at a rate of V max = 3.41 þ 0.16 Wmol [NO ] min31 mg 31 and K m = 3.91 þ 0.45 mM) and was labile during prolonged incubation at s 20 ‡C. Nitratedependent growth of Escherichia coli strains expressing only nitrate reductase A was inhibited by sub-mM concentrations of tungstate in the medium. In contrast, a strain expressing only NAP was only partially inhibited by 10 mM tungstate. However, none of the above experimental approaches revealed evidence that tungsten could replace molybdenum at the active site of E. coli NapA. The combined data show that tungsten can function at the active site of some, but not all, molybdoenzymes from mesophilic bacteria.

Bacterial nitrate reductases can be classified into at least three groups according to their localization and function, namely membrane-bound (NAR) or periplasmic (NAP) respiratory and cytoplasmic assimilatory (NAS) enzymes. Monomeric NASs are the simplest of the soluble nitrate reductases, although heterodimeric NASs exist, and a common structural arrangement of NAP is that of a NapAB heterodimer. Using bioinformatic analysis of published genomes, we have identified more representatives of a monomeric class of NAP, which is the evolutionary link between the monomeric NASs and the heterodimeric NAPs. This has further established the monomeric structural clade of NAP. The operons of the monomeric NAP do not contain NapB and suggest that other redox partners are employed by these enzymes, including NapM or NapG predicted proteins. A structural alignment and comparison of the monomeric and heterodimeric NAPs suggests that a difference in surface polarity is related to the interaction of the respective catalytic subunit and redox partner.