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The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that Campylobacter jejuni 11168H forms persister cells at a frequency of 10−3 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated C. jejuni cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.

Vibrio parahaemolyticus is the lead causative agent for seafood-borne human gastroenteritis. While its occurrence has traditionally been uncommon in Europe and the United Kingdom, rising sea surface temperatures have resulted in an increased prevalence. Here, we present the complete genome sequences of four novel V. parahaemolyticus strains isolated in the United Kingdom.

Viable but non-culturable (VBNC) cells are cells that are metabolically active, but are unable to form colonies on standard culture media. Following environmental stimuli, such as temperature upshift, some VBNC cells can ‘resuscitate’ restoring their ability to grow on media. Currently, over 80 bacterial species are reported to enter the VBNC state. The ability of VBNC cells to go undetected by conventional microbiological practices could lead to an underestimation of total viable cells in environmental and clinical samples. Furthermore, their capacity to retain virulence potential and their ability for renewed metabolic activity means the VBNC state in pathogens may pose a risk to human health and thus warrants further investigation. This research project has investigated the ability of the human pathogen Vibrio parahaemolyticus to form VBNC cells when exposed to stressful conditions. V. parahaemolyticus is a bacterium that is present in the marine environment and can be found in seawater, shellfish (such as oysters and mussels) and in crustacea (such as crab). This bacterium is the leading cause of seafood associated gastroenteritis worldwide and often results in watery/bloody diarrhea and vomiting. We have developed robust models to generate V. parahaemolyticus VBNC cells in the laboratory and report that different sub populations of VBNC cells can occur based upon their metabolic activity, cell shape and the ability to grow and cause disease in Galleria mellonella. Using mass spectrophotometry we have identified several proteins which may play roles in VBNC formation and resuscitation in V. parahaemolyticus.

Bacterial anaerobic respiration using selenium oxyanions as the sole electron acceptor primarily result in the precipitation of selenium biominerals observed as either intracellular or extracellular selenium deposits. Although a better understanding of the enzymology of bacterial selenate reduction is emerging, the processes by which the selenium nanospheres are constructed, and in some cases secreted, has remained poorly studied. Thauera selenatis is a Gram-negative betaproteobacterium that is capable of respiring selenate due to the presence of a periplasmic selenate reductase (SerABC). SerABC is a molybdoenzyme that catalyses the reduction of selenate to selenite by accepting electrons from the Q-pool via a dihaem c -type cytochrome (cyt c 4). The product selenite is presumed to be reduced in the cytoplasm, forming intracellular selenium nanospheres that are ultimately secreted into the surrounding medium. The secretion of the selenium nanospheres is accompanied by the export of a ~95 kDa protein SefA (selenium factor A). SefA has no cleavable signal peptide, suggesting that it is also exported directly for the cytoplasmic compartment. It has been suggested that SefA functions to stabilize the formation of the selenium nanospheres before secretion, possibly providing reaction sites for selenium nanosphere creation or providing a shell to prevent subsequent selenium aggregation. The present paper draws on our current knowledge of selenate respiration and selenium biomineralization in T. selenatis and other analogous systems, and extends the application of nanoparticle tracking analysis to determine the size distribution profile of the selenium nanospheres secreted. Abbreviations: NTA, nanoparticle tracking analysis; QCR, quinol–cytochrome c oxidoreductase; SefA, selenium factor A; SEM, scanning electron microscopy; Ser, periplasmic selenate reductase; TEM, transmission electron microscopy

Metalloproteins and enzymes are an essential part of all respiratory electron-transfer chains and provide a pathway for electron transfer to terminal electron acceptors. Since bacteria can utilize a wide range of respiratory substrates, this variety of potential electron acceptors has facilitated the need for many different respiratory metalloproteins. Bacterial selenate respiration requires the sequential reduction of the selenium oxyanions selenate and selenite resulting in the precipitation of elemental selenium. The initial bioenergetic processes of selenate respiration are driven by metalloproteins utilizing cofactors containing iron and molybdenum. However, the assembly of the elemental selenium into selenium nanosphere crystals has shed light on a new family of proteins involved in selenium biomineralization. This article highlights some of the recent advances in our understanding of selenate respiration in the bacterium Thauera selenatis, with particular focus on the metalloproteins involved in selenate reduction and the novel proteins that function to deal with these insoluble selenium deposits. “As mineralogy constitutes a part of chemistry, it is clear that this arrangement of minerals must derive its principles from chemistry” Jöns Jacob Berzelius 1814

Enterobacter cloacae SLD1a-1 is capable of the complete reduction of selenate to selenium and the initial reaction is catalysed by a membrane-bound selenate reductase. In the present study, continuous culture experiments were employed to investigate the possibility that selenate reduction, via the selenate reductase, might provide sufficient energy to maintain cell viability when deprived of the preferred anaerobic terminal electron acceptor nitrate. The evidence presented indicates that the selenate reductase supports slow growth that retards the wash-out of the culture when switching to nitrate-depleted selenate-rich medium, and provides a proton motive force for sustained cell maintenance. In contrast, a strain of E. cloacae (sub sp. cloacae) that does not readily reduce selenate, cannot sustain cell maintenance when switching to a selenate-rich medium. This work demonstrates for the first time that respiratory linked selenate reduction gives E. cloacae SLD1a-1 a selective advantage when inhabiting selenate-contaminated environments and highlights the suitability of utilising E. cloacae SLD1a-1 when developing selenium remediation strategies.

Many species of Bacteria and Archaea respire nitrate using a molybdenum-dependent membrane-bound respiratory system called Nar. Classically, the 'Bacterial' Nar system is oriented such that nitrate reduction takes place on the inside of this membrane. However, the active site subunit of the 'Archaeal' Nar systems has a twin arginine ('RR') motif, which is a suggestion of translocation to the outside of the cytoplasmic membrane. These 'Archaeal' type of nitrate reductases are part of a group of molybdoenzymes with an 'RR' motif that are predicted to have an aspartate ligand to the molybdenum ion. This group includes selenate reductases and possible sequence signatures are described that serve to distinguish the Nar nitrate reductases from the selenate reductases. The 'RR' sequences of nitrate reductases of Archaea and some that have recently emerged in Bacteria are also considered and it is concluded that there is good evidence for there being both Archaeal and Bacterial examples of Nar-type nitrate reductases with an active site on the outside of the cytoplasmic membrane. Finally, the bioenergetic consequences of nitrate reduction on the outside of the cytoplasmic membrane have been explored.

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 ofalpha-proteobacteria, such as Rhodobacter sphaeroides and Paracoccus pantotrophus, the gamma-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 mum 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 alpha-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 A, 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.

Recent studies have identified a positive role for nitric oxide (NO) in the regulation of pancreatic beta-cell function. The aim of this study was to determine the effects of short-term exposure to NO on beta-cell gene expression and the activity of the transcription factor PDX-1. NO stimulated the activity of the insulin gene promoter in Min6 beta-cells and endogenous insulin mRNA levels in both Min6 and isolated islets of Langerhans. Addition of wortmannin prior to NO stimulation blocked the observed increases in insulin gene promoter activity. Although NO addition stimulated the phosphorylation of p38, inhibition by SB203580 did not block the effect of NO on the insulin gene promoter. NO addition also stimulated both the nuclear accumulation and the DNA binding activity of PDX-1. This study has shown that over 24h, NO stimulates insulin gene expression, PI-3-kinase activity and the activity of the critical beta-cell transcription factor PDX-1.

The purification of small quantities of a major small c -type cytochrome from the anammox bacterium Kuenenia stuttgartiensis has recently been reported. In order to characterise this protein further we have expressed the gene encoding this cytochrome in Escherichia coli and have purified the protein to homogeneity. The protein is directed to the E. coli periplasm using its native signal sequence suggesting that it may be translocated via a Sec-type system in K. stuttgartiensis . The cytochrome has the visible spectroscopic properties typical of a low-spin c -type cytochrome, but these spectroscopic features broaden in high salt solutions. The oxidised cytochrome was able to bind the ligands NO and cyanide. A redox potential of +230 mV suggests that the protein is suitable to act as an electron carrier protein that may be involved in the respiratory chain between hydrazine oxidation and the reduction of nitrite. The predicted protein sequence for the cytochrome suggests it to be a predominantly α-helical protein, and this is supported by circular dichroism.

A putative biosynthetic mechanism for selenium nanoparticles (SeNPs) and efficient reduction of selenite (SeO32-) in the bacterial strain Stenotrophomonas maltophilia SeITE02 are addressed here on the basis of information gained by a combined approach relying on a set of physiological, chemical/biochemical, microscopy, and proteomic analyses. S. maltophilia SeITE02 is demonstrated to efficiently transform selenite into elemental selenium (Se°) by reducing 100% of 0.5mM of this toxic oxyanion to Se° nanoparticles within 48h growth, in liquid medium. Since the selenite reducing activity was detected in the cytoplasmic protein fraction, while biogenic SeNPs showed mainly extracellular localization, a releasing mechanism of SeNPs from the intracellular environment is hypothesized. SeNPs appeared spherical in shape and with size ranging from 160nm to 250nm, depending on the age of the cultures. Proteomic analysis carried out on the cytoplasmic fraction identified an alcohol dehydrogenase homolog, conceivably correlated with the biogenesis of SeNPs. Finally, by Fourier Transformed Infrared Spectrometry, protein and lipid residues were detected on the surface of biogenic SeNPs. Eventually, this strain might be efficaciously exploited for the remediation of selenite-contaminated environmental matrices due to its high SeO32- reducing efficiency. Biogenic SeNPs may also be considered for technological applications in different fields.

The membrane-bound selenate reductase of Enterobacter cloacae SLD1a-1 is purified in low yield and has relatively low activity in the pure form compared to that of other oxyanion reductases, such as the membrane-bound and periplasmic nitrate reductases. A microtiter plate assay based on the original quartz cuvette viologen assay of Jones and Garland (R.W. Jones, P.B. Garland, Biochem. J 164 (1977) 199-211) was developed specifically for analysis of such low-abundant, labile oxyanion reductases. The plate assay detects the enzyme-dependent reoxidation of reduced methyl viologen spectrophotometrically at 600 nm. The assay is quick, uses a minimal sample volume (5 microl), can simultaneously test a range of alternative substrates, and permits activity measurements on multiple samples. We demonstrate the accuracy and versatility of the microtiter plate assay by application to the kinetic analysis, inhibition, and pH optimization of the membrane-bound selenate reductase from E. cloacae SLD1a-1. Results show that the membrane-bound selenate reductase has optimum activity at pH approximately 8 and its active site is able to accommodate larger inhibitory complexes resulting in mixed-type inhibition, in the presence of selenate and potassium thiocyanate.

Enterobacter cloacaeSLD1a-1 is capable of reductive detoxification of selenate to elemental selenium under aerobic growth conditions. The initial reductive step is the two-electron reduction of selenate to selenite and is catalyzed by a molybdenum-dependent enzyme demonstrated previously to be located in the cytoplasmic membrane, with its active site facing the periplasmic compartment (C. A. Watts, H. Ridley, K. L. Condie, J. T. Leaver, D. J. Richardson, and C. S. Butler, FEMS Microbiol. Lett.228:273-279, 2003). This study describes the purification of two distinct membrane-bound enzymes that reduce either nitrate or selenate oxyanions. The nitrate reductase is typical of the NAR-type family, with α and β subunits of 140 kDa and 58 kDa, respectively. It is expressed predominantly under anaerobic conditions in the presence of nitrate, and while it readily reduces chlorate, it displays no selenate reductase activity in vitro. The selenate reductase is expressed under aerobic conditions and expressed poorly during anaerobic growth on nitrate. The enzyme is a heterotrimeric (αβγ) complex with an apparent molecular mass of ∼600 kDa. The individual subunit sizes are ∼100 kDa (α), ∼55 kDa (β), and ∼36 kDa (γ), with a predicted overall subunit composition of α3β3γ3. The selenate reductase contains molybdenum, heme, and nonheme iron as prosthetic constituents. Electronic absorption spectroscopy reveals the presence of ab-type cytochrome in the active complex. The apparentKmfor selenate was determined to be ∼2 mM, with an observedVmaxof 500 nmol SeO42−min−1mg−1(kcat, ∼5.0 s−1). The enzyme also displays activity towards chlorate and bromate but has no nitrate reductase activity. These studies report the first purification and characterization of a membrane-bound selenate reductase.

Rhodobacter capsulatus cytochrome c' (RCCP) has been overexpressed in Escherichia coli, and its spectroscopic and ligand-binding properties have been investigated. It is concluded that the heterologously expressed protein is assembled correctly, as judged by UV-vis absorption, EPR, and resonance Raman (RR) spectroscopy of the unligated protein as well as forms in which the heme is ligated by CO or NO. To probe the oligomerization state of RCCP and its potential influence on heme reactivity, we have compared the properties of wild-type RCCP with a mutant (K42E) that lacks a salt bridge at the subunit interface. Analytical ultracentrifugation indicates that wild-type and K42E proteins are both monomeric in solution, contrary to the homodimeric structure of the crystalline state. Surprisingly, the K42E mutation produces a number of changes at the heme center (nearly 20 A distant), including perturbation of the ferric spin-state equilibrium and a change in the ferrous heme-nitrosyl complex from a six-coordinate/five-coordinate mixture to a predominantly five-coordinate heme-NO species. RR spectra indicate that ferrous K42E and wild-type RCCP both have relatively high Fe-His stretching frequencies, suggesting that the more favored five-coordinate heme-nitrosyl formation in K42E is not caused by a weaker Fe2+-His bond. Nevertheless, the altered reactivity of ferrous K42E with NO, together with its modified ferric spin state, shows that structural changes originating at the dimer interface can affect the properties of the heme center, raising the exciting possibility that intermolecular encounters at the protein surface might modulate the reactivity of cytochrome c' in vivo.

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.

Over the last 10 years, during the lifetime of the nitrogen cycle meetings, structural biology, coupled with spectroscopy, has had a major impact of our understanding enzymology of the nitrogen cycle. The three-dimensional structures for many of the key enzymes have now been resolved and have provided a wealth of information regarding the architecture of redox active metal sites, as well as revealing novel structural folds. Coupled with structure-based spectroscopic analysis, this has led to new insight into the reaction mechanisms of the diverse chemical transformations that together cycle nitrogen in the biosphere. An overview of the some of the key developments in field over the last decade is presented.

Bacterial cytoplasmic assimilatory nitrate reductases are the least well characterized of all of the subgroups of nitrate reductases. In the present study the ferredoxin-dependent 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. EPR-monitored potentiometric titration of NarB revealed that this cluster titrated as an n = 1 Nernstian component with a midpoint redox potential (Em) of –190 mV. EPR spectra collected at 60 K revealed a Mo(V) signal termed “very high g” with gav = 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 (gav = 1.9897) was observed. In potentiometric titrations the high g Mo(V) signal developed over the potential range of –100 to –350 mV (Em Mo6+/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 Mo5+. The data suggests that during the catalytic cycle nitrate will bind to the Mo5+ state of NarB in which the enzyme is minimally two-electron-reduced. Comparison of the spectral properties of NarB with those of the membrane-bound 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.

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 microM) reduced nitrate at a rate of V(max)=3.41+/-0.16 micromol [NO(3)(-)] min(-1) mg(-1) with an affinity for nitrate of K(m)=0.24+/-0.05 mM and was heat-stable up to 50 degrees C. In contrast, the periplasmic fraction obtained from cells cultured in media supplemented with tungstate (100 microM) reduced nitrate at a much slower rate, with much lower affinity (V(max)=0.05+/-0.002 micromol [NO(3)(-)] min(-1) mg(-1) and K(m)=3.91+/-0.45 mM) and was labile during prolonged incubation at20 degrees C. Nitrate-dependent 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.

The first electron nuclear double resonance (ENDOR) study of a member of the Mo-bis-molybdopterin guanine dinucleotide family of molybdoenzymes is presented, using the periplasmic nitrate reductase from Paracoccus pantotrophus. Rapid freeze-quenched time-resolved EPR revealed that during turnover the intensity of a Mo(V) EPR signal known as High-g [resting] increases. This signal is split by two interacting protons that are not solvent-exchangeable. X-band proton-ENDOR analysis resolved broad symmetrical resonance features that arose from four classes of protons weakly coupled to the Mo(V). Signals from two of these were lost upon exchange into deuterated buffer, suggesting that they may originate from OH− or H2O groups. One of these signals was also lost when the enzyme was redox-cycled in the presence of azide. Since these protons are very weakly coupled OH/H2O groups, they are not likely to be ligated directly to the Mo(V). This suggests that protonation of a Mo(VI)=O group does not occur on reduction to Mo(V), but most probably accompanies reduction of Mo(V) to Mo(IV). A resonance feature from a more strongly coupled proton, that was not lost following exchange into deuterated buffer, could also be resolved at 22–24MHz. The anisotropy of this feature, determined from ENDOR spectra collected at a range of field positions, indicated a Mo—proton distance of approx. 3.2Å, consistent with this being one of the β-methylene protons of a Mo—Cys ligand.

Stenotrophomonas maltophilia strain SeITE02 was isolated from the rhizosphere of the selenium-hyperaccumulating legume Astragalus bisculcatus . In this report, we provide the 4.56-Mb draft genome sequence of S. maltophilia SeITE02, a gammaproteobacterium that can withstand high concentrations of selenite and reduce these to elemental selenium.

The periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a soluble two-subunit enzyme (NapAB) that binds two haem groups, a [4Fe-4S] cluster and a bis(molybdopterin guanine dinucleotide) (MGD) cofactor that catalyses the reduction of nitrate to nitrite. In the present study the effect of KSCN (potassium thiocyanate) as an inhibitor and Mo ligand has been investigated. Results are presented that show NAP is sensitive to SCN(-) (thiocyanate) inhibition, with SCN(-) acting as a competitive inhibitor of nitrate (K(i) approximately 4.0 mM). The formation of a novel EPR Mo(V) species with an elevated g(av) value (g(av) approximately 1.994) compared to the Mo(V) High-g (resting) species was observed upon redox cycling in the presence of SCN(-). Mo K-edge EXAFS analysis of the dithionite-reduced NAP was best fitted as a mono-oxo Mo(IV) species with three Mo-S ligands at 2.35 A (1 A=0.1 nm) and a Mo-O ligand at 2.14 A. The addition of SCN(-) to the reduced Mo(IV) NAP generated a sample that was best fitted as a mono-oxo (1.70 A) Mo(IV) species with four Mo-S ligands at 2.34 A. Taken together, the competitive nature of SCN(-) inhibition of periplasmic nitrate reductase activity, the elevated Mo(V) EPR g(av) value following redox cycling in the presence of SCN(-) and the increase in sulphur co-ordination of Mo(IV) upon SCN(-) binding, provide strong evidence for the direct binding of SCN(-) via a sulphur atom to Mo.

Cytochrome c oxidase catalyzes the reduction of oxygen to water with a concomitant conservation of energy in the form of a transmembrane proton gradient. The enzyme has a catalytic site consisting of a binuclear center of a copper ion and a heme group. The spectroscopic parameters of this center are unusual. The origin of broad electron paramagnetic resonance (EPR) signals in the oxidized state at rather low resonant field, the so-called g' = 12 signal, has been a matter of debate for over 30 years. We have studied the angular dependence of this resonance in both parallel and perpendicular mode X-band EPR in oriented multilayers containing cytochrome c oxidase to resolve the assignment. The "slow" form and compounds formed by the addition of formate and fluoride to the oxidized enzyme display these resonances, which result from transitions between states of an integer-spin multiplet arising from magnetic exchange coupling between the five unpaired electrons of high spin Fe(III) heme a(3) and the single unpaired electron of Cu(B). The first successful simulation of similar signals observed in both perpendicular and parallel mode X-band EPR spectra in frozen aqueous solution of the fluoride compound of the closely related enzyme, quinol oxidase or cytochrome bo(3), has been reported recently (Oganesyan et al., 1998, J. Am. Chem. Soc. 120:4232-4233). This suggested that the exchange interaction between the two metal ions of the binuclear center is very weak (|J| approximately 1 cm(-1)), with the axial zero-field splitting (D approximately 5 cm(-1)) of the high-spin heme dominating the form of the ground state. We show that this model accounts well for the angular dependences of the X-band EPR spectra in both perpendicular and parallel modes of oriented multilayers of cytochrome c oxidase derivatives and that the experimental results are inconsistent with earlier schemes that use exchange coupling parameters of several hundred wavenumbers.

The periplasmic nitrate reductase from Paracoccus denitrificans is a soluble two-subunit enzyme which binds two hemes (c-type), a [4Fe-4S] center, and a bis molybdopterin guanine dinucleotide cofactor (bis-MGD). A catalytic cycle for this enzyme is presented based on a study of these redox centers using electron paramagnetic resonance (EPR) and extended X-ray absorption fine structure (EXAFS) spectroscopies. The Mo(V) EPR signal of resting NAP (High g [resting]) has g(av) = 1.9898 is rhombic, exhibits low anisotropy, and is split by two weakly interacting protons which are not solvent-exchangeable. Addition of exogenous ligands to this resting state (e.g., nitrate, nitrite, azide) did not change the form of the signal. A distinct form of the High g Mo(V) signal, which has slightly lower anisotropy and higher rhombicity, was trapped during turnover of nitrate and may represent a catalytically relevant Mo(V) intermediate (High g [nitrate]). Mo K-edge EXAFS analysis was undertaken on the ferricyanide oxidized enzyme, a reduced sample frozen within 10 min of dithionite addition, and a nitrate-reoxidized form of the enzyme. The oxidized enzyme was fitted best as a di-oxo Mo(VI) species with 5 sulfur ligands (4 at 2. 43 A and 1 at 2.82 A), and the reduced form was fitted best as a mono-oxo Mo(IV) species with 3 sulfur ligands at 2.35 A. The addition of nitrate to the reduced enzyme resulted in reoxidation to a di-oxo Mo(VI) species similar to the resting enzyme. Prolonged incubation of NAP with dithionite in the absence of nitrate (i.e., nonturnover conditions) resulted in the formation of a species with a Mo(V) EPR signal that is quite distinct from the High g family and which has a g(av) = 1.973 (Low g [unsplit]). This signal resembles those of the mono-MGD xanthine oxidase family and is proposed to arise from an inactive form of the nitrate reductase in which the Mo(V) form is only coordinated by the dithiolene of one MGD. In samples of NAP that had been reduced with dithionite, treated with azide or cyanide, and then reoxidized with ferricyanide, two Mo(V) signals were detected with g(av) elevated compared to the High g signals. Kinetic analysis demonstrated that azide and cyanide displayed competitive and noncompetitive inhibition, respectively. EXAFS analysis of azide-treated samples show improvement to the fit when two nitrogens are included in the molybdenum coordination sphere at 2.52 A, suggesting that azide binds directly to Mo(IV). Based on these spectroscopic and kinetic data, models for Mo coordination during turnover have been proposed.

For the study of the dinuclear center of heme-copper oxidases cytochrome bo3 from Escherichia coli offers several advantages over the extensively characterized bovine cytochrome c oxidase. The availability of strains with enhanced levels of expression allows purification of the significant amounts of enzyme required for detailed spectroscopic studies. Cytochrome bo3 is readily prepared as the fast form, with a homogeneous dinuclear center which gives rise to characteristic broad EPR signals not seen in CcO. The absence of CuA and the incorporation of protohemes allows for a detailed interpretation of the MCD spectra arising from the dinuclear center heme o3. Careful analysis allows us to distinguish between small molecules that bind to heme o3, those which are ligands of CuB, and those which react to yield higher oxidation states of heme o3. Here we review results from our studies of the reactions of fast cytochrome bo3 with formate, fluoride, chloride, azide, cyanide, NO, and H2O2.

The reaction of hydrogen peroxide with a number of variants of sperm-whale myoglobin in which the distal pocket histidine residue (His64) had been mutated was studied with a combination of stopped-flow spectroscopy and freeze-quench EPR. The rate of the initial bimolecular reaction with hydrogen peroxide in all the proteins studied was found to depend on the polarity of the amino acid side chain at position 64. In wild-type myoglobin there were no significant optical changes subsequent to this reaction, suggesting the rapid formation of the well-characterized oxyferryl species. This conclusion was supported by freeze-quench EPR data, which were consistent with the pattern of reactivity previously reported [King and Winfield (1963) J. Biol. Chem. 238, 1520–1528]. In those myoglobins bearing a mutation at position 64, the initial bimolecular reaction with hydrogen peroxide yielded an intermediate species that subsequently decayed via a second hydrogen peroxide-dependent step leading to modification or destruction of the haem. In the mutant His64 → Gln the calculated electronic absorption spectrum of the intermediate was not that of an oxyferryl species but seemed to be that of a low-spin ferric haem. Freeze-quench EPR studies of this mutant and the apolar mutant (His64 → Val) revealed the accumulation of a novel intermediate after the first hydrogen peroxide-dependent reaction. The unusual EPR characteristics of this species are provisionally assigned to a low-spin ferric haem with bound peroxide as the distal ligand. These results are interpreted in terms of a reaction scheme in which the polarity of the distal pocket governs the rate of binding of hydrogen peroxide to the haem iron and the residue at position 64 governs both the rate of heterolytic oxygen scission and the stability of the oxyferryl product.

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.

Cytochrome c′, a c-type cytochrome with unique spectroscopic and magnetic properties, has been characterized in a variety of denitrifying and photosynthetic bacteria. Cytochrome c′ has a role in defence and/or removal of NO but the mechanism of action is not clear. To examine the function of cytochrome c′ from Neisseria meningitidis, the protein was purified after heterologous overexpression in Escherichia coli. The electronic spectra of the oxidized c′ demonstrated a pH-dependent transition (over the pH range of 6–10) typical of known c′-type cytochromes. Interestingly, the form in which NO is supplied determines the redox state of the resultant haem-nitrosyl complex. Fe(III)–NO complexes were formed when Fe(II) or Fe(III) cytochrome c′ was sparged with NO gas, whereas an Fe(II)–NO complex was generated when NO was supplied using DEA NONOate (diazeniumdiolate).

Burkholderia thailandensis is closely related to Burkholderia pseudomallei, a bacterial pathogen and the causative agent of melioidosis. B. pseudomallei can survive and persist within a hypoxic environment for up to one year and has been shown to grow anaerobically in the presence of nitrate. Currently, little is known about the role of anaerobic respiration in pathogenesis of melioidosis. Using B. thailandensis as a model, a library of 1344 transposon mutants was created to identify genes required for anaerobic nitrate respiration. One transposon mutant (CA01) was identified with an insertion in BTH_I1704 (moeA), a gene required for the molybdopterin biosynthetic pathway. This pathway is involved in the synthesis of a molybdopterin cofactor required for a variety of molybdoenzymes, including nitrate reductase. Disruption of molybdopterin biosynthesis prevented growth under anaerobic conditions, when using nitrate as the sole terminal electron acceptor. Defects in anaerobic respiration, nitrate reduction, motility and biofilm formation were observed for CA01. Mutant complementation with pDA-17:BTH_I1704 was able to restore anaerobic growth on nitrate, nitrate reductase activity and biofilm formation, but did not restore motility. This study highlights the potential importance of molybdoenzyme-dependent anaerobic respiration in the survival and virulence of B. thailandensis.

During selenate respiration by Thauera selenatis , the reduction of selenate results in the formation of intracellular selenium (Se) deposits that are ultimately secreted as Se nanospheres of approximately 150 nm in diameter. We report that the Se nanospheres are associated with a protein of approximately 95 kDa. Subsequent experiments to investigate the expression and secretion profile of this protein have demonstrated that it is up-regulated and secreted in response to increasing selenite concentrations. The protein was purified from Se nanospheres, and peptide fragments from a tryptic digest were used to identify the gene in the draft T. selenatis genome. A matched open reading frame was located, encoding a protein with a calculated mass of 94.5 kDa. N-terminal sequence analysis of the mature protein revealed no cleavable signal peptide, suggesting that the protein is exported directly from the cytoplasm. The protein has been called Se factor A (SefA), and homologues of known function have not been reported previously. The sefA gene was cloned and expressed in Escherichia coli , and the recombinant His-tagged SefA purified. In vivo experiments demonstrate that SefA forms larger (approximately 300 nm) Se nanospheres in E. coli when treated with selenite, and these are retained within the cell. In vitro assays demonstrate that the formation of Se nanospheres upon the reduction of selenite by glutathione are stabilized by the presence of SefA. The role of SefA in selenium nanosphere assembly has potential for exploitation in bionanomaterial fabrication.

Bacterial cellular metabolism is renowned for its metabolic diversity and adaptability. However, certain environments present particular challenges. Aerobic metabolism of highly reduced carbon substrates by soil bacteria such as Paracoccus pantotrophus presents one such challenge since it may result in excessive electron delivery to the respiratory redox chain when compared with the availability of terminal oxidant, O2. The level of a periplasmic ubiquinol-dependent nitrate reductase, NAP, is up-regulated in the presence of highly reduced carbon substrates. NAP oxidizes ubiquinol at the periplasmic face of the cytoplasmic membrane and reduces nitrate in the periplasm. Thus its activity counteracts the accumulation of excess reducing equivalents in ubiquinol, thereby maintaining the redox poise of the ubiquinone/ubiquinol pool without contributing to the protonmotive force across the cytoplasmic membrane. Although P. pantotrophus NapAB shows a high level of substrate specificity towards nitrate, the enzyme has also been reported to reduce selenate in spectrophotometric solution assays. This transaction draws on our current knowledge concerning the bacterial respiratory nitrate reductases and extends the application of PFE (protein film electrochemistry) to resolve and quantify the selenate reductase activity of NapAB.

Selenate reductase (SER) from Thauera selenatis is a member of a distinct class of the TAT-translocated type II molybdoenzymes and is closely related to a group of thermostable nitrate reductases (pNAR) found in hyperthermophilic archaea. In the present study the thermostable and thermo-active properties of SER, isolated with either molybdenum (Mo) or tungsten (W) at the active site, are reported. Results show that the purified Mo–SER complex is stable and active upon heat-shock incubation for 10 min at temperatures up to 60 °C. At temperatures greater than 65 °C all three subunits (SerABC) are readily denatured. The optimum temperature for maximum activity recorded was also determined to be 65 °C. T. selenatis can grow readily on a tungstate rich medium up to concentrations of 1 mM. SER isolated from periplasmic fractions from cells grown on 1 mM tungstate displayed selenate reductase activities with a 20-fold reduction in Vmax (0.01 μmol [S]/min/mg) and a 23-fold increase in substrate binding affinity (Km 0.7 μM). The thermo-stability and pH dependence of W–SER was shown to be similar to that observed for Mo–SER. By contrast, the optimum reaction temperature for W–SER exceeded the maximum temperature tested (>80 °C). The combined data from the kinetic analysis and thermal activity profiles provide evidence that W can substitute for Mo at the active site of SER and retain detectable selenate reductase activity. It is argued that despite the similarity in their catalytic and electron conducting subunits, the presence of a membrane anchor in the archaeal pNAR system appears pivotal to the enhanced hyperthermostability. The fact that Mo–SER is thermostable up to 65 °C however, could be advantageous when designing selenate contamination remediation strategies.

Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme. The enzyme comprises three subunits SerABC, where SerC is an unusual b-heme cytochrome. In the present work the spectropotentiometric characterization of the SerC component and the identification of redox partners to SER are reported. The mid-point redox potential of the b-heme was determined by optical titration (E(m) + 234 +/- 10 mV). A profile of periplasmic c-type cytochromes expressed in T. selenatis under selenate respiring conditions was undertaken. Two c-type cytochromes were purified ( approximately 24 and approximately 6 kDa), and the 24-kDa protein (cytc-Ts4) was shown to donate electrons to SerABC in vitro. Protein sequence of cytc-Ts4 was obtained by N-terminal sequencing and liquid chromatography-tandem mass spectrometry analysis, and based upon sequence similarities, was assigned as a member of cytochrome c(4) family. Redox potentiometry, combined with UV-visible spectroscopy, showed that cytc-Ts4 is a diheme cytochrome with a redox potential of +282 +/- 10 mV, and both hemes are predicted to have His-Met ligation. To identify the membrane-bound electron donors to cytc-Ts4, growth of T. selenatis in the presence of respiratory inhibitors was monitored. The specific quinol-cytochrome c oxidoreductase (QCR) inhibitors myxothiazol and antimycin A partially inhibited selenate respiration, demonstrating that some electron flux is via the QCR. Electron transfer via a QCR and a diheme cytochrome c(4) is a novel route for a member of the DMSO reductase family of molybdoenzymes.

The present study investigated the role of selenium in the regulation of pancreatic beta-cell function. Utilising the mouse beta-cell line Min6, we have shown that selenium specifically upregulates Ipf1 (insulin promoter factor 1) gene expression, activating the −2715 to −1960 section of the Ipf1 gene promoter. Selenium increased both Ipf1 and insulin mRNA levels in Min6 cells and stimulated increases in insulin content and insulin secretion in isolated primary rat islets of Langerhans. These data are the first to implicate selenium in the regulation of specific beta-cell target genes and suggest that selenium potentially promotes an overall improvement in islet function.

The AF0174–AF0176 gene cluster in Archaeoglobus fulgidus encodes a putative oxyanion reductase of the D-type (Type II) family of molybdo-enzymes. Sequence analysis reveals that the catalytic subunit AF0176 shares low identity (31–32%) and similarity (41–42%) to both NarG and SerA, the catalytic components of the respiratory nitrate and selenate reductases respectively. Consequently, predicting the oxyanion substrate selectivity of AF0176 has proved difficult based solely on sequence alignments. In the present study, we have modelled both AF0176 and SerA on the recently determined X-ray structure of the NAR (nitrate reductase) from Escherichia coli and have identified a number of key amino acid residues, conserved in all known NAR sequences, including AF0176, that we speculate may enhance selectivity towards trigonal planar (NO3−) rather than tetrahedral (SeO42− and ClO4−) substrates.

Fully oxidized cytochrome bo3 from Escherichia coli has been studied in its oxidized and several ligand-bound forms using electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopies. In each form, the spin-coupled high-spin Fe(III) heme o3 and CuB(II) ion at the active site give rise to similar fast-relaxing broad features in the dual-mode X-band EPR spectra. Simulations of dual-mode spectra are presented which show that this EPR can arise only from a dinuclear site in which the metal ions are weakly coupled by an anisotropic exchange interaction of J 1 cm-1. A variable-temperature and magnetic field (VTVF) MCD study is also presented for the cytochrome bo3 fluoride and azide derivatives. New methods are used to extract the contribution to the MCD of the spin-coupled active site in the presence of strong transitions from low-spin Fe(III) heme b. Analysis of the MCD data, independent of the EPR study, also shows that the spin-coupling within the active site is weak with J approximately 1 cm-1. These conclusions overturn a long-held view that such EPR signals in bovine cytochrome c oxidase arise from an S' = 2 ground state resulting from strong exchange coupling (J10(2) cm-1) within the active site.

The thylakoid lumen of the cyanobacterium Synechocystis PCC 6803 is supplied with copper via two copper-transporting ATPases and a metallochaperone intermediary. We show that the copper site of this metallochaperone is unusual and consists of two cysteine residues and a histidine imidazole located on structurally dynamic loops. Substitution of this histidine residue enhances bacterial two-hybrid interaction with the cytosolic copper exporter, but not the copper importer, suggesting that the interacting surfaces are distinct, with implications for metal transfer.

Enterobacter cloacae SLD1a-1 is capable of reducing selenium oxyanions to elemental selenium under both aerobic and anaerobic conditions. In this study the enzyme that catalyses the initial reduction of selenate (SeO4(2-)) to selenite (SeO3(2-)) has been localised to isolated cytoplasmic membrane fractions. Experiments with intact cells have shown that the putative selenate reductase can accept electrons more readily from membrane-impermeable methyl viologen than membrane-permeable benzyl viologen, suggesting that the location of the catalytic site is towards the periplasmic side of the cytoplasmic membrane. Enzyme activity was enhanced by growing cells in the presence of 1 mM sodium molybdate and significantly reduced in cells grown in the presence of 1 mM sodium tungstate. Non-denaturing polyacrylamide gel electrophoresis (PAGE) gels stained for selenate and nitrate reductase activity have revealed that two distinct membrane-bound enzymes catalyse the reduction of selenate and nitrate. The role of this membrane-bound molybdenum-dependent reductase in relation to selenate detoxification and energy conservation is discussed.

The reaction of nitric oxide (NO) with fast and reduced cytochrome bo(3) (cyt bo(3)) from Escherichia coli has been investigated. The stoichiometry of NO binding to cyt bo(3) was determined using an NO electrode in the [NO] range 1-14 microM. Under reducing conditions, the initial decrease in [NO] following the addition of cyt bo(3) corresponded to binding of 1 NO molecule per cyt bo(3) functional unit. After this "rapid" NO binding phase, there was a slow, but significant rate of NO consumption ( approximately 0.3molNOmol bo(3)(-1)min(-1)), indicating that cyt bo(3) possesses a low level of NO reductase activity. The binding of NO to fast pulsed enzyme was also investigated. The results show that in the [NO] range used (1-14 microM) both fast and pulsed oxidised cyt bo(3) bind NO with a stoichiometry of 1:1 with an observed dissociation constant of K(d) =5.6+/-0.6 microM and that NO binding was inhibited by the presence of Cl(- ). The binding of nitrite to the binuclear centre causes spectral changes similar to those observed upon NO binding to fast cyt bo(3). These results are discussed in relation to the model proposed by Wilson and co-workers [FEBS Lett. 414 (1997) 281] where the binding of NO to Cu(B)(II) results in the formation of the nitrosonium (Cu(B)(I)-NO(+)) complex. NO(+) then reacts with OH(-), a Cu(B) ligand, to form nitrite, which can bind at the binuclear centre. This work suggests for the first time that the binding of NO to oxidised cyt bo(3) does result in the reduction of Cu(B).

The periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a soluble two-subunit enzyme (NapAB) that binds two c-type haems, a [4Fe–4S] cluster and a bis-molybdopterin guanine dinucleotide cofactor that catalyses the reduction of nitrate to nitrite. In the present work the NapAB complex has been studied by magneto-optical spectroscopy to probe co-ordination of both the NapB haems and the NapA active site Mo. The absorption spectrum of the NapAB complex is dominated by features from the NapB c-type cytochromes. Using a combination of electron paramagnetic resonance spectroscopy and magnetic circular dichroism it was demonstrated that both haems are low-spin with bis-histidine axial ligation. In addition, a window between 600 and 800 nm was identified in which weak absorption features that may arise from Mo could be detected. The low-temperature MCD spectrum shows oppositely signed bands in this region (peak 648 nm, trough 714 nm) which have been assigned to S-to-Mo(V) charge transfer transitions.

Cytochrome bo forms complexes with chloride, bromide and iodide in which haem o remains high-spin and in which the ‘630 nm ’ charge-transfer band is red-shifted by 7–8 nm. The chloride and bromide complexes each have a characteristic set of integer-spin EPR signals arising from spin coupling between haem o and CuB. The rate and extent of chloride binding decreases as the pH increases from 5.5 to 8.5. At pH 5.5 the dissociation constant for chloride is 2 mM and the first-order rate constant for dissociation is 2×10-4 s-1. The order of rate of binding, and of affinity, at pH 5.5 is chloride (1) > bromide (0.3) > iodide (0.1). It is suggested that the halides bind in the binuclear site but, unlike fluoride, they are not direct ligands of the iron of haem o. In addition, both the stability of the halide complexes and the rate of halide binding seem to be increased by the co-binding of a proton.

The reaction of nitric oxide (NO) with fast cytochrome bo from Escherichia coli has been studied by electronic absorption, MCD, and EPR spectroscopy. Titration of the enzyme with NO showed the formation of two distinct species, consistent with NO binding stoichiometries of 1:1 and 2:1 with observed dissociation constants at pH 7.5 of approximately 2.3 x 10(-)6 and 3.3 x 10(-)5 M. Monitoring the titration by EPR spectroscopy revealed that the broad EPR signals at g approximately 7.3, 3.7, and 2.8 due to magnetic interaction between high-spin heme o (S = 5/2) and CuBII (S = 1/2) are lost. A high-spin heme o signal at g = 6.0 appears as the 1:1 complex is formed but is lost again on formation of the 2:1 complex, which is EPR silent. The absorption spectrum shows that heme o remains in the high-spin FeIII state throughout the titration. These results are consistent with the binding of up to two NO molecules at CuBII. This has been confirmed by studies with the Cl- adduct of fast cytochrome bo. MCD evidence shows that heme o remains ligated by histidine and water. Addition of excess NO to the Cl- adduct leads to the appearance of a high-spin FeIII heme EPR signal. Hence chloride ion binds to CuB, blocking the binding of a second NO molecule. These results suggest a mechanism for the reduction of NO to nitrous oxide by cytochrome bo and cytochrome c oxidase in which the binding of two cis NO molecules at CuB permits the formation of an N-N bond and the abstraction of oxygen by the heme group.

Publisher Summary The bacterial aromatic ring-hydroxylating dioxygenases represent a broad range of multi-component enzymes that possess many properties and mechanisms in common. This has enabled their classification based on structural properties. This classification has been supported by sequence comparisons based on nucleotide and primary amino-acid sequences. Comparison of the low homology sequences has also enabled the identification of highly conserved functional motifs—for example iron-sulphur ligation domains—whereas analysis of high homology sequences has allowed the characterization of amino acids involved in more specific functions, such as the control of substrate specificity. This combination of sequence analysis with structure-function studies proves to be invaluable for future protein engineering of improved enzymes for both biocatalysis and environmental remediation. The chapter focuses on the ring-hydroxylating dioxygenases, referring to ring-cleavage dioxygenases and other related systems, and also describes bacterial oxygenases and dioxygenases.

Metal ion homeostasis mechanisms in the food-borne human pathogen Campylobacter jejuni are poorly understood. The Cj1516 gene product is homologous to the multicopper oxidase CueO, which is known to contribute to copper tolerance in Escherichia coli . Here we show, by optical absorbance and electron paramagnetic resonance spectroscopy, that purified recombinant Cj1516 contains both T1 and trinuclear copper centers, which are characteristic of multicopper oxidases. Inductively coupled plasma mass spectrometry revealed that the protein contained approximately six copper atoms per polypeptide. The presence of an N-terminal “twin arginine” signal sequence suggested a periplasmic location for Cj1516, which was confirmed by the presence of p -phenylenediamine ( p -PD) oxidase activity in periplasmic fractions of wild-type but not Cj1516 mutant cells. Kinetic studies showed that the pure protein exhibited p -PD, ferroxidase, and cuprous oxidase activities and was able to oxidize an analogue of the bacterial siderophore anthrachelin (3,4-dihydroxybenzoate), although no iron uptake impairment was observed in a Cj1516 mutant. However, this mutant was very sensitive to increased copper levels in minimal media, suggesting a role in copper tolerance. This was supported by increased expression of the Cj1516 gene in copper-rich media. A mutation in a second gene, the Cj1161c gene, encoding a putative CopA homologue, was also found to result in copper hypersensitivity, and a Cj1516 Cj1161c double mutant was found to be more copper sensitive than either single mutant. These observations and the apparent lack of alternative copper tolerance systems suggest that Cj1516 (CueO) and Cj1161 (CopA) are major proteins involved in copper homeostasis in C. jejuni .