Ann Reilly

Sequence analysis of subunits of the membrane‐bound nitrate reductase from a denitrifying bacterium: the integral membrane subunit provides a prototype for the dihaem electron‐carrying arm of a redox loop

Three genes, narH, narJ and narl of the membrane‐bound nitrate reductase operon of the denitrifying bacterium Thiosphaera pantotropha have been identified and sequenced. The derived gene products show high sequence similarity to the equivalent (β, putative δ and γ) subunits of the two membrane‐bound nitrate reductases of the enteric bacterium Escherichia coli. AU iron‐sulphur cluster ligands proposed for the E. coliβ subunits are conserved in T. pantotropha NarH. Secondary structure analysis of NarJ suggests that this protein has a predominantly α‐helical structure. Comparison of T. pantotropha Narl wilh the b‐haembinding integral membrane subunits of the E. coli enzymes allows assignment of His‐53, His‐63, His‐186 and His‐204 (T. pantotropha Narl numbering) as b‐haem axial ligands and the construction of a three‐dimensional model of this subunit. This model, in which the two b‐haems are in different halves of the membrane bilayer, is consistent with a mechanism of energy conservation whereby electrons are moved from the periplasmic to the cytoplasmic side of the membrane via the haems. Similar movement of electrons is required in the membrane‐bound uptake hydrogenases and membrane‐bound formate dehydrogenases. We have identified two pairs of conserved histidine residues in the integral membrane subunits of these enzymes that are appropriately positioned to bind one haem towards each side of the membrane bilayer. One subunit of a hydrogenase complex involved in transfer of electrons across the cytoplasmic membrane of sulphate‐reducing bacteria has structural resemblance to Narl.

Purification of hydroxylamine oxidase from Thiosphaera pantotropha. Identification of electron acceptors that couple heterotrophic nitrification to aerobic denitrification.

Thiosphaera pantotropha, a Gram‐negative heterotrophic nitrifying bacterium, expresses a soluble 20 kDa monomeric periplasmic hydroxylamine oxidase that differs markedly from the hydroxylamine oxidase found in autotrophic bacteria. This enzyme can use the periplasmic redox proteins, cytochrome C 551 and pseudoazurin as electron acceptors, both of which can also donate electrons to denitrification enzymes. A model of electron transfer is proposed, that suggests a coupling of nitrification to denitrification and provides a mechanism by which nitrification can play a role in dissipating reductant.

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

Electron paramagnetic resonance spectroscopy signals attributable to low‐spin haem c in the oxidised protein and [4Fe–4S]1+ in the dithionite‐reduced protein were identified, at low temperature, in Thiosphaera pantotropha periplasmic nitrate reductase. Spin integration of these signals as well as elemental analysis suggest a stoichiometry of 1.3–1.6 c‐haem and 1 [4Fe–4S] cluster per enzyme molecule. The E m (at pH 7.4) of the [4F–4S]2+,1+ couple, −160 mV, means that it is unlikely to be physiologically reducible. Peptide sequences from the 90 kDa subunit indicate that the enzyme is a member of the family of molybdopterin guanine dinucleotide‐binding polypeptides, the majority of which possess a putative [4Fe–4S] cluster binding sequence and thus may also bind a (low potential) iron—sulphur cluster.

Structural dynamics of the membrane translocation domain of colicin E9 and its interaction with TolB

In order for the 61 kDa colicin E9 protein toxin to enter the cytoplasm of susceptible cells and kill them by hydrolysing their DNA, the colicin must interact with the outer membrane BtuB receptor and Tol translocation pathway of target cells. The translocation function is located in the N-terminal domain of the colicin molecule. (1)H, (1)H-(1)H-(15)N and (1)H-(13)C-(15)N NMR studies of intact colicin E9, its DNase domain, minimal receptor-binding domain and two N-terminal constructs containing the translocation domain showed that the region of the translocation domain that governs the interaction of colicin E9 with TolB is largely unstructured and highly flexible. Of the expected 80 backbone NH resonances of the first 83 residues of intact colicin E9, 61 were identified, with 43 of them being assigned specifically. The absence of secondary structure for these was shown through chemical shift analyses and the lack of long-range NOEs in (1)H-(1)H-(15)N NOESY spectra (tau(m)=200 ms). The enhanced flexibility of the region of the translocation domain containing the TolB box compared to the overall tumbling rate of the protein was identified from the relatively large values of backbone and tryptophan indole (15)N spin-spin relaxation times, and from the negative (1)H-(15)N NOEs of the backbone NH resonances. Variable flexibility of the N-terminal region was revealed by the (15)N T(1)/T(2) ratios, which showed that the C-terminal end of the TolB box and the region immediately following it was motionally constrained compared to other parts of the N terminus. This, together with the observation of inter-residue NOEs involving Ile54, indicated that there was some structural ordering, resulting most probably from the interactions of side-chains. Conformational heterogeneity of parts of the translocation domain was evident from a multiplicity of signals for some of the residues. Im9 binding to colicin E9 had no effect on the chemical shifts or other NMR characteristics of the region of colicin E9 containing the TolB recognition sequence, though the interaction of TolB with intact colicin E9 bound to Im9 did affect resonances from this region. The flexibility of the translocation domain of colicin E9 may be connected with its need to recognise protein partners that assist it in crossing the outer membrane and in the translocation event itself.

NMR detection of slow conformational dynamics in an endonuclease toxin.

The cytotoxic activity of the secreted bacterial toxin colicin E9 is due to a non-specific DNase housed in the C-terminus of the protein. Double-resonance and triple-resonance NMR studies of the 134-amino acid15 N- and 13C/15N-labelled DNase domain are presented. Extensive conformational heterogeneity was evident from the presence of far more resonances than expected based on the amino acid sequence of the DNase, and from the appearance of chemical exchange cross-peaks in TOCSY and NOESY spectra. EXSY spectra were recorded to confirm that slow chemical exchange was occurring. Unambiguous sequence-specific resonance assignments are presented for one region of the protein, Pro65-Asn72, which exists in two slowly exchanging conformers based on the identification of chemical exchange cross-peaks in 3D 1H-1H-15N EXSY-HSQC, NOESY-HSQC and TOCSY-HSQC spectra, together with Cα and Cβ chemical shifts measured in triple-resonance spectra and sequential NH NOEs. The rates of conformational exchange for backbone amide resonances in this stretch of amino acids, and for the indole NH of either Trp22 or Trp58, were determined from the intensity variation of the appropriate diagonal and chemical exchange cross-peaks recorded in 3D1 H-1H-15N NOESY-HSQC spectra. The data fitted a model in which this region of the DNase has two conformers, NA and NB, which interchange at 15 °C with a forward rate constant of 1.61 ± 0.5 s-1 and a backward rate constant of 1.05 ± 0.5 s-1. Demonstration of this conformational equilibrium has led to a reappraisal of a previously proposed kinetic scheme describing the interaction of E9 DNase with immunity proteins [Wallis et al. (1995) Biochemistry, 34, 13743–13750 and 13751–13759]. The revised scheme is consistent with the specific inhibitor protein for the E9 DNase, Im9, associating with both the NA and NB conformers of the DNase and with binding only to the NB conformer detected because the rate of dissociation of the complex of Im9 and the NA conformer, NAI, is extremely rapid. In this model stoichiometric amounts of Im9 convert, the E9 DNase is converted wholly into the NBI form. The possibility that cis–trans isomerisation of peptide bonds preceding proline residues is the cause of the conformational heterogeneity is discussed. E9 DNase contains 10 prolines, with two bracketing the stretch of amino acids that have allowed the NA ⇋ NB interconversion to be identified, Pro65 and Pro73. The model assumes that one or both of these can exist in either the cis or trans form with strong Im9 binding possible to only one form.

Microbial reduction of selenate and nitrate: common themes and variations.

A number of biochemically distinct systems have been characterized for the microbial reduction of the oxyanions, selenate (SeO(4)(2-)) and nitrate (NO(3)(-)). Two classes of molybdenum-dependent nitrate reductase catalyse the respiratory-linked reduction of nitrate (NO(3)(-)) to nitrite (NO(2)(-)). The main respiratory nitrate reductase (NAR) is membrane-anchored, with its active site facing the cytoplasmic compartment. The other enzyme (NAP) is water-soluble and located in the periplasm. In recent years, our understanding of each of these enzyme systems has increased significantly. The crystal structures of both NAR and NAP have now been solved and they provide new insight into the structure, function and evolution of these respiratory complexes. In contrast, our understanding of microbial selenate (SeO(4)(2-)) reduction and respiration is at an early stage; however, similarities to the nitrate reductase systems are emerging. This review will consider some of the common themes and variations between the different classes of nitrate and selenate reductases.

Reductive activation of nitrate reductases

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.

Crystallization and preliminary X-ray crystallographic analysis of a periplasmic tetrahaem flavocytochrome c3 from Shewanella frigidimarina NCIMB400 which has fumarate reductase activity.

The fumarate reductase of Escherichia coli and other bacteria is a membrane-bound enzyme consisting of four subunits. A soluble periplasmic 64 kDa tetrahaem flavocytochrome c3 from Shewanella frigidimarina NCIMB400 which possesses a catalytic fumarate reductase activity has been crystallized. The crystals belong to space group P212121 with unit-cell parameters a = 72.4, b = 110.1, c = 230.2 A. Assuming a molecular dimer in the asymmetric unit, the crystals contain 65% solvent and, when cryocooled to 100 K, the crystals diffract to at least 3.0 A resolution. The crystals, however, display an inherent lack of isomorphism and the plausibility of a MAD phasing experiment has therefore been investigated by measuring the iron K absorption edge from a single crystal.

Hybrid formation in the shikimate pathway enzyme dehydroquinase

Abstract The guanidine hydrochloride-induced denaturation of unmodified and product-linked forms of the dimeric shikimate pathway enzyme 3-dehydroquinase are shown, by fluorescence spectroscopy, to be reversible. Mixing the unfolded polypeptides prior to renaturation yields a hybrid dimer, which has been identified by isoelectric focussing and its relative yield quantitated by laser densitometry.