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Nicholas J Watmough - One of the best experts on this subject based on the ideXlab platform.

  • unexpected weak magnetic exchange coupling between haem and non haem iron in the catalytic site of nitric oxide Reductase norbc from paracoccus denitrificans
    Biochemical Journal, 2013
    Co-Authors: Jessica H Van Wonderen, D J Richardson, Nicholas J Watmough, Andrew J. Thomson, Vasily S Oganesyan, Myles R. Cheesman
    Abstract:

    Bacterial NOR (nitric oxide Reductase) is a major source of the powerful greenhouse gas N 2 O. NorBC from Paracoccus denitrificans is a heterodimeric multi-haem transmembrane complex. The active site, in NorB, comprises high-spin haem b 3 in close proximity with non-haem iron, Fe B . In oxidized NorBC, the active site is EPR-silent owing to exchange coupling between Fe III haem b 3 and Fe B III (both S =5/2). On the basis of resonance Raman studies [Moenne-Loccoz, Richter, Huang, Wasser, Ghiladi, Karlin and de Vries (2000) J. Am. Chem. Soc. 122 , 9344–9345], it has been assumed that the coupling is mediated by an oxo-bridge and subsequent studies have been interpreted on the basis of this model. In the present study we report a VFVT (variable-field variable-temperature) MCD (magnetic circular dichroism) study that determines an isotropic value of J =−1.7 cm −1 for the coupling. This is two orders of magnitude smaller than that encountered for oxo-bridged diferric systems, thus ruling out this configuration. Instead, it is proposed that weak coupling is mediated by a conserved glutamate residue.

  • Exploring the terminal region of the proton pathway in the bacterial nitric oxide Reductase.
    Journal of inorganic biochemistry, 2009
    Co-Authors: Ulrika Flock, Nicholas J Watmough, Joachim Reimann, Peter Lachmann, Pia Ädelroth
    Abstract:

    The c-type nitric oxide Reductase (cNOR) from Paracoccus (P.) denitrificans is an integral membrane protein that catalyzes NO reduction; 2NO+2e(-)+2H(+)-->N(2)O+H(2)O. It is also capable of catalyzing the reduction of oxygen to water, albeit more slowly than NO reduction. cNORs are divergent members of the heme-copper oxidase superfamily (HCuOs) which reduce NO, do not pump protons, and the reaction they catalyse is non-electrogenic. All known cNORs have been shown to have five conserved glutamates (E) in the catalytic subunit, by P. denitrificans numbering, the E122, E125, E198, E202 and E267. The E122 and E125 are presumed to face the periplasm and the E198, E202 and E267 are located in the interior of the membrane, close to the catalytic site. We recently showed that the E122 and E125 define the entry point of the proton pathway leading from the periplasm into the active site [U. Flock, F.H. Thorndycroft, A.D. Matorin, D.J. Richardson, N.J. Watmough, P. Adelroth, J. Biol. Chem. 283 (2008) 3839-3845]. Here we present results from the reaction between fully reduced NOR and oxygen on the alanine variants of the E198, E202 and E267. The initial binding of O(2) to the active site was unaffected by these mutations. In contrast, proton uptake to the bound O(2) was significantly inhibited in both the E198A and E267A variants, whilst the E202A NOR behaved essentially as wildtype. We propose that the E198 and E267 are involved in terminating the proton pathway in the region close to the active site in NOR.

  • The bacterial respiratory nitric oxide Reductase.
    Biochemical Society transactions, 2009
    Co-Authors: Nicholas J Watmough, Sarah J Field, Ross J.l. Hughes, David J. Richardson
    Abstract:

    The two-subunit cytochrome bc complex (NorBC) isolated from membranes of the model denitrifying soil bacterium Paracoccus denitrificans is the best-characterized example of the bacterial respiratory nitric oxide Reductases. These are members of the super-family of haem-copper oxidases and are characterized by the elemental composition of their active site, which contains non-haem iron rather than copper, at which the reductive coupling of two molecules of nitric oxide to form nitrous oxide is catalysed. The reaction requires the presence of two substrate molecules at the active site along with the controlled input of two electrons and two protons from the same side of the membrane. In the present paper, we consider progress towards understanding the pathways of electron and proton transfer in NOR and how this information can be integrated with evidence for the likely modes of substrate binding at the active site to propose a revised and experimentally testable reaction mechanism.

  • the respiratory nitric oxide Reductase norbc from paracoccus denitrificans
    Methods in Enzymology, 2008
    Co-Authors: Sarah J Field, D J Richardson, Faye H Thorndycroft, Andrey D Matorin, Nicholas J Watmough
    Abstract:

    The two subunit cytochrome bc complex (NorBC) isolated from membranes of the model denitrifying soil bacterium Paracoccus denitrificans is the best characterized example of the bacterial respiratory nitric oxide Reductases. These are members of the superfamily of heme-copper oxidases and are characterized by the elemental composition of their active site, which contains nonheme iron rather than copper, at which the reductive coupling of two molecules of nitric oxide to form nitrous oxide is catalyzed. This chapter describes methods for the purification and characterization of both native nitric oxide Reductase from P. denitrificans and a recombinant form of the enzyme expressed in Escherichia coli, which enables site-directed mutagenesis of the catalytic subunit NorB. Examples are given of electronic absorption and electron paramagnetic resonance spectra that characterize the enzyme in a number of redox states, along with a method for the routine assay of the complex using its natural electron donor pseudoazurin.

  • Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide Reductase
    Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008
    Co-Authors: Sofia Kapetanaki, Ursula Liebl, Sarah J Field, Nicholas J Watmough, Ross J.l. Hughes, Marten H. Vos
    Abstract:

    AbstractThe active site of nitric oxide Reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in ~13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site

Yoshitsugu Shiro - One of the best experts on this subject based on the ideXlab platform.

  • Molecular dynamics simulations reveal proton transfer pathways in cytochrome C-dependent nitric oxide Reductase.
    PLoS computational biology, 2012
    Co-Authors: Andrei V. Pisliakov, Tomoya Hino, Yoshitsugu Shiro, Yuji Sugita
    Abstract:

    Nitric oxide Reductases (NORs) are membrane proteins that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N2O), which is a critical step of the nitrate respiration process in denitrifying bacteria. Using the recently determined first crystal structure of the cytochrome c-dependent NOR (cNOR) [Hino T, Matsumoto Y, Nagano S, Sugimoto H, Fukumori Y, et al. (2010) Structural basis of biological N2O generation by bacterial nitric oxide Reductase. Science 330: 1666–70.], we performed extensive all-atom molecular dynamics (MD) simulations of cNOR within an explicit membrane/solvent environment to fully characterize water distribution and dynamics as well as hydrogen-bonded networks inside the protein, yielding the atomic details of functionally important proton channels. Simulations reveal two possible proton transfer pathways leading from the periplasm to the active site, while no pathways from the cytoplasmic side were found, consistently with the experimental observations that cNOR is not a proton pump. One of the pathways, which was newly identified in the MD simulation, is blocked in the crystal structure and requires small structural rearrangements to allow for water channel formation. That pathway is equivalent to the functional periplasmic cavity postulated in cbb3 oxidase, which illustrates that the two enzymes share some elements of the proton transfer mechanisms and confirms a close evolutionary relation between NORs and C-type oxidases. Several mechanisms of the critical proton transfer steps near the catalytic center are proposed.

  • Structural basis for nitrous oxide generation by bacterial nitric oxide Reductases
    Philosophical transactions of the Royal Society of London. Series B Biological sciences, 2012
    Co-Authors: Yoshitsugu Shiro, Hiroshi Sugimoto, Shingo Nagano, Takehiko Tosha, Tomoya Hino
    Abstract:

    The crystal structure of the bacterial nitric oxide Reductase (cNOR) from Pseudomonas aeruginosa is reported. Its overall structure is similar to those of the main subunit of aerobic and micro-aerobic cytochrome oxidases (COXs), in agreement with the hypothesis that all these enzymes are members of the haem-copper oxidase superfamily. However, substantial structural differences between cNOR and COX are observed in the catalytic centre and the delivery pathway of the catalytic protons, which should be reflected in functional differences between these respiratory enzymes. On the basis of the cNOR structure, we propose a possible reaction mechanism of nitric oxide reduction to nitrous oxide as a working hypothesis.

  • Crystal structure of quinol-dependent nitric oxide Reductase from Geobacillus stearothermophilus.
    Nature structural & molecular biology, 2012
    Co-Authors: Yushi Matsumoto, Tomoya Hino, Shingo Nagano, Hiroshi Sugimoto, Takehiko Tosha, Andrei V. Pisliakov, Yuji Sugita, Yoshitsugu Shiro
    Abstract:

    The structure of quinol-dependent nitric oxide Reductase (qNOR) from G. stearothermophilus, which catalyzes the reduction of NO to produce the major ozone-depleting gas N(2)O, has been characterized at 2.5 A resolution. The overall fold of qNOR is similar to that of cytochrome c-dependent NOR (cNOR), and some structural features that are characteristic of cNOR, such as the calcium binding site and hydrophilic cytochrome c domain, are observed in qNOR, even though it harbors no heme c. In contrast to cNOR, structure-based mutagenesis and molecular dynamics simulation studies of qNOR suggest that a water channel from the cytoplasm can serve as a proton transfer pathway for the catalytic reaction. Further structural comparison of qNOR with cNOR and aerobic and microaerobic respiratory oxidases elucidates their evolutionary relationship and possible functional conversions.

  • Molecular structure and function of bacterial nitric oxide Reductase
    Biochimica et biophysica acta, 2011
    Co-Authors: Tomoya Hino, Shingo Nagano, Hiroshi Sugimoto, Takehiko Tosha, Yoshitsugu Shiro
    Abstract:

    Abstract The crystal structure of the membrane-integrated nitric oxide Reductase cNOR from Pseudomonas aeruginosa was determined. The smaller NorC subunit of cNOR is comprised of 1 trans-membrane helix and a hydrophilic domain, where the heme c is located, while the larger NorB subunit consists of 12 trans-membrane helices, which contain heme b and the catalytically active binuclear center (heme b 3 and non-heme Fe B ). The roles of the 5 well-conserved glutamates in NOR are discussed, based on the recently solved structure. Glu211 and Glu280 appear to play an important role in the catalytic reduction of NO at the binuclear center by functioning as a terminal proton donor, while Glu215 probably contributes to the electro-negative environment of the catalytic center. Glu135, a ligand for Ca 2+ sandwiched between two heme propionates from heme b and b 3 , and the nearby Glu138 appears to function as a structural factor in maintaining a protein conformation that is suitable for electron-coupled proton transfer from the periplasmic region to the active site. On the basis of these observations, the possible molecular mechanism for the reduction of NO by cNOR is discussed. This article is part of a Special Issue entitled: Respiratory Oxidases.

  • Structural Basis of Biological N2O Generation by Bacterial Nitric Oxide Reductase
    Science (New York N.Y.), 2010
    Co-Authors: Tomoya Hino, Shingo Nagano, Hiroshi Sugimoto, Yushi Matsumoto, Yoshihiro Fukumori, Takeshi Murata, So Iwata, Yoshitsugu Shiro
    Abstract:

    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.

Constantinos Varotsis - One of the best experts on this subject based on the ideXlab platform.

  • Structure and properties of the catalytic site of nitric oxide Reductase at ambient temperature.
    Biochimica et biophysica acta, 2015
    Co-Authors: Vangelis Daskalakis, Takehiro Ohta, Teizo Kitagawa, Constantinos Varotsis
    Abstract:

    Abstract Nitric oxide Reductase (Nor) is the third of the four enzymes of bacterial denitrification responsible for the catalytic formation of laughing gas (N 2 O). Here we report the detection of the hyponitrite (HO–N = N–O − ) species (ν N–N  = 1332 cm − 1 ) in the heme b 3 Fe–Fe B dinuclear center of Nor from Paracoccus denitrificans . We have also applied density functional theory (DFT) to characterize the bimetallic-bridging hyponitrite species in the reduction of NO to N 2 O by Nor and compare the present results with those recently reported for the N–N bond formation in the ba 3 and caa 3 oxidoReductases from Thermus thermophilus .

  • Nitric oxide activation and reduction by heme-copper oxidoReductases and nitric oxide Reductase.
    Journal of inorganic biochemistry, 2008
    Co-Authors: Eftychia Pinakoulaki, Constantinos Varotsis
    Abstract:

    The understanding of the dynamics and conformational control involved in the interplay between structure and function of nitric oxide Reductase (Nor) and heme-copper oxidoReductases in their function to convert nitric oxide (NO) to nitrous oxide (N2O) is of fundamental importance in bioenergetics. We have applied resonance Raman spectroscopy to investigate the NO ligation/deligation reactions and the extent of communication between the metal centers at the heme a3-CuB site of heme-copper oxidases and of the heme Fe-non-heme Fe in Nor. The present study provides information of the electronic and vibrational structure of intermediates, and thus, it forms the basis for an atomic-level description of the key steps in the N-N bond formation and the N-O bond cleavage mechanism. The present experiments provide evidence as to the validity of the proposed hypothesis of the common evolutionary origin of aerobic respiration and bacterial denitrification.

  • Nitric oxide activation and reduction by heme–copper oxidoReductases and nitric oxide Reductase
    Journal of Inorganic Biochemistry, 2008
    Co-Authors: Eftychia Pinakoulaki, Constantinos Varotsis
    Abstract:

    Abstract The understanding of the dynamics and conformational control involved in the interplay between structure and function of nitric oxide Reductase (Nor) and heme–copper oxidoReductases in their function to convert nitric oxide (NO) to nitrous oxide (N2O) is of fundamental importance in bioenergetics. We have applied resonance Raman spectroscopy to investigate the NO ligation/deligation reactions and the extent of communication between the metal centers at the heme a3–CuB site of heme–copper oxidases and of the heme Fe–non-heme Fe in Nor. The present study provides information of the electronic and vibrational structure of intermediates, and thus, it forms the basis for an atomic-level description of the key steps in the N–N bond formation and the N–O bond cleavage mechanism. The present experiments provide evidence as to the validity of the proposed hypothesis of the common evolutionary origin of aerobic respiration and bacterial denitrification.

  • Resonance Raman Spectroscopy of Nitric Oxide Reductase and cbb3 Heme-Copper Oxidase
    The journal of physical chemistry. B, 2008
    Co-Authors: Eftychia Pinakoulaki, Constantinos Varotsis
    Abstract:

    Elucidating the structure and properties of the active sites in cbb 3 heme-copper oxidase and in nitric oxide Reductase (Nor) is crucial in understanding the reaction mechanisms of oxygen and nitric oxide reduction by both enzymes. In the work here, we have applied resonance Raman (RR) spectroscopy to investigate the structure and properties of the binuclear heme b 3 -Cu B center of cbb 3 heme-copper oxidase from Pseudomonas stutzeri and the dinuclear heme b 3 -Fe B center of Nor from Paracoccus denitrificans in the ligand-free and CO-bound forms and in the reactions with O 2 and NO. The RR data demonstrate that in the Nor/NO reaction, the formation of the N-N bond occurs with the His-Fe heme b 3 bond intact, and reformation of the heme b 3 -O-Fe B dinuclear center causes the rupture of the proximal His-Fe heme b 3 bond. In the reactions of Nor and cbb 3 with O 2 , distinct oxidized heme b 3 species, which differ from the as-isolated oxidized forms, have been characterized. The activation and reduction of O 2 and NO by cbb 3 oxidase and nitric oxide Reductase are compared and discussed.

  • Nitric-Oxide Reductase STRUCTURE AND PROPERTIES OF THE CATALYTIC SITE FROM RESONANCE RAMAN SCATTERING
    The Journal of biological chemistry, 2002
    Co-Authors: Eftychia Pinakoulaki, Sabine Gemeinhardt, Matti Saraste, Constantinos Varotsis
    Abstract:

    Abstract We have applied resonance Raman spectroscopy to investigate the properties of the dinuclear center of oxidized, reduced, and NO-bound Nitric-Oxide Reductase from Paracoccus denitrificans. The spectra of the oxidized enzyme show two distinct νas(Fe-O-Fe) modes at 815 and 833 cm−1 of the heme/non-heme diiron center. The splitting of the Fe-O-Fe mode suggests that two different conformations (open and closed) are present in the catalytic site of the enzyme. We find evidence from deuterium exchange experiments that in the dominant conformation (833 cm−1mode, closed), the Fe-O-Fe unit is hydrogen-bonded to distal residue(s). The ferric nitrosyl complex of Nitric-Oxide Reductase exhibits the ν(Fe3+-NO) and ν(N-O) at 594 and 1904 cm−1, respectively. The nitrosyl species we detect is photolabile and can be photolyzed to generate a new form of oxidized enzyme in which the proximal histidine is ligated to hemeb 3, in contrast to the resting form. Photodissociation of the NO ligand yields a five-coordinate high-spin heme b 3. Based on the findings reported here, the structure and properties of the dinuclear center of nitric- oxide Reductase in the oxidized, reduced, and NO-bound form as well as its photoproduct can be described with certainty.

Sarah J Field - One of the best experts on this subject based on the ideXlab platform.

  • The bacterial respiratory nitric oxide Reductase.
    Biochemical Society transactions, 2009
    Co-Authors: Nicholas J Watmough, Sarah J Field, Ross J.l. Hughes, David J. Richardson
    Abstract:

    The two-subunit cytochrome bc complex (NorBC) isolated from membranes of the model denitrifying soil bacterium Paracoccus denitrificans is the best-characterized example of the bacterial respiratory nitric oxide Reductases. These are members of the super-family of haem-copper oxidases and are characterized by the elemental composition of their active site, which contains non-haem iron rather than copper, at which the reductive coupling of two molecules of nitric oxide to form nitrous oxide is catalysed. The reaction requires the presence of two substrate molecules at the active site along with the controlled input of two electrons and two protons from the same side of the membrane. In the present paper, we consider progress towards understanding the pathways of electron and proton transfer in NOR and how this information can be integrated with evidence for the likely modes of substrate binding at the active site to propose a revised and experimentally testable reaction mechanism.

  • the respiratory nitric oxide Reductase norbc from paracoccus denitrificans
    Methods in Enzymology, 2008
    Co-Authors: Sarah J Field, D J Richardson, Faye H Thorndycroft, Andrey D Matorin, Nicholas J Watmough
    Abstract:

    The two subunit cytochrome bc complex (NorBC) isolated from membranes of the model denitrifying soil bacterium Paracoccus denitrificans is the best characterized example of the bacterial respiratory nitric oxide Reductases. These are members of the superfamily of heme-copper oxidases and are characterized by the elemental composition of their active site, which contains nonheme iron rather than copper, at which the reductive coupling of two molecules of nitric oxide to form nitrous oxide is catalyzed. This chapter describes methods for the purification and characterization of both native nitric oxide Reductase from P. denitrificans and a recombinant form of the enzyme expressed in Escherichia coli, which enables site-directed mutagenesis of the catalytic subunit NorB. Examples are given of electronic absorption and electron paramagnetic resonance spectra that characterize the enzyme in a number of redox states, along with a method for the routine assay of the complex using its natural electron donor pseudoazurin.

  • Ultrafast ligand binding dynamics in the active site of native bacterial nitric oxide Reductase
    Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2008
    Co-Authors: Sofia Kapetanaki, Ursula Liebl, Sarah J Field, Nicholas J Watmough, Ross J.l. Hughes, Marten H. Vos
    Abstract:

    AbstractThe active site of nitric oxide Reductase from Paracoccus denitrificans contains heme and non-heme iron and is evolutionarily related to heme-copper oxidases. The CO and NO dynamics in the active site were investigated using ultrafast transient absorption spectroscopy. We find that, upon photodissociation from the active site heme, 20% of the CO rebinds in 170 ps, suggesting that not all the CO transiently binds to the non-heme iron. The remaining 80% does not rebind within 4 ns and likely migrates out of the active site without transient binding to the non-heme iron. Rebinding of NO to ferrous heme takes place in ~13 ps. Our results reveal that heme-ligand recombination in this enzyme is considerably faster than in heme-copper oxidases and are consistent with a more confined configuration of the active site

  • Spectral properties of bacterial Nitric-Oxide Reductase: resolution of pH-dependent forms of the active site heme b3.
    The Journal of biological chemistry, 2002
    Co-Authors: Sarah J Field, Nicholas J Watmough, Louise Prior, M. Dolores Roldán, Myles R. Cheesman, Andrew J. Thomson, Stephen Spiro, Julea N. Butt, David J. Richardson
    Abstract:

    Abstract Bacterial Nitric-Oxide Reductase catalyzes the two electron reduction of nitric oxide to nitrous oxide. In the oxidized form the active site non-heme FeB and high spin heme b 3 are μ-oxo bridged. The hemeb 3 has a ligand-to-metal charge transfer band centered at 595 nm, which is insensitive to pH over the range of 6.0–8.5. Partial reduction of Nitric-Oxide Reductase yields a three electron-reduced state where only the hemeb 3 remains oxidized. This results in a shift of the heme b 3 charge transfer band λmax to longer wavelengths. At pH 6.0 the charge transfer band λmax is 605 nm, whereas at pH 8.5 it is 635 nm. At pH 6.5 and 7.5 the Nitric-Oxide Reductase ferric hemeb 3 population is a mixture of both 605- and 635-nm forms. Magnetic circular dichroism spectroscopy suggests that at all pH values examined the proximal ligand to the ferric hemeb 3 in the three electron-reduced form is histidine. At pH 8.5 the distal ligand is hydroxide, whereas at pH 6.0, when the enzyme is most active, it is water.

James P. Shapleigh - One of the best experts on this subject based on the ideXlab platform.

  • Site-directed mutagenesis of NnrR: a transcriptional regulator of nitrite and nitric oxide Reductase in Rhodobacter sphaeroides
    FEMS microbiology letters, 2003
    Co-Authors: William P. Laratta, James P. Shapleigh
    Abstract:

    NnrR, a transcriptional activator and member of the CRP/FNR family of regulators, is responsible for controlling the expression of a number of denitrification genes in Rhodobacter sphaeroides 2.4.3. The apparent effector for NnrR is nitric oxide, and in its presence NnrR activates expression of the nirK gene and the nor operon, encoding nitrite Reductase and nitric oxide Reductase, respectively. Whether nitric oxide directly interacts with NnrR to activate transcription is unknown. Other denitrifiers carry putative orthologs of NnrR. To gain insight into NnrR function, a number of conserved residues were mutagenized. The impact of these changes on NnrR function was assessed by monitoring expression of a nirK-lacZ fusion. In this way a region spanning from Tyr93 to Cys103 that contains residues critical for NnrR activity was identified.

  • Characterization of the nitric oxide Reductase-encoding region in Rhodobacter sphaeroides 2.4.3.
    Journal of bacteriology, 1997
    Co-Authors: Thomas B. Bartnikas, William P. Laratta, I E Tosques, J Shi, James P. Shapleigh
    Abstract:

    A gene cluster which includes genes required for the expression of nitric oxide Reductase in Rhodobacter sphaeroides 2.4.3 has been isolated and characterized. Sequence analysis indicates that the two proximal genes in the cluster are the Nor structural genes. These two genes and four distal genes apparently constitute an operon. Mutational analysis indicates that the two structural genes, norC and norB, and the genes immediately downstream, norQ and norD, are required for expression of an active Nor complex. The remaining two genes, nnrT and nnrU, are required for expression of both Nir and Nor. The products of norCBQD have significant identity with products from other denitrifiers, whereas the predicted nnrT and nnrU gene products have no similarity with products corresponding to other sequences in the database. Mutational analysis and functional complementation studies indicate that the nnrT and nnrU genes can be expressed from an internal promoter. Deletion analysis of the regulatory region upstream of norC indicated that a sequence motif which has identity to a motif in the gene encoding nitrite Reductase in strain 2.4.3 is critical for nor operon expression. Regulatory studies demonstrated that the first four genes, norCBQD, are expressed only when the oxygen concentration is low and nitrate is present but that the two distal genes, nnrTU, are expressed constitutively.