Dr Elizabeth Dridge
Associate Lecturer/Associate Research Fellow

Abstract:
A bacterial process for selenium nanosphere assembly.

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.

Abstract:
Quinol-cytochrome c oxidoreductase and cytochrome c4 mediate electron transfer during selenate respiration in Thauera selenatis.

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.

Abstract:
Investigation of the redox centres of periplasmic selenate reductase from Thauera selenatis by EPR spectroscopy.

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

Abstract:
Developing structure-based models to predict substrate specificity of D-group (Type II) molybdenum enzymes: application to a molybdo-enzyme of unknown function from Archaeoglobus fulgidus.

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 (NO(3)(-)) rather than tetrahedral (SeO(4)(2-) and ClO(4)(-)) substrates.

Abstract:
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.