Biomedical subjects

J R Guest

Publications and source records attributed to J R Guest.

At least 37 records · Page 2Linked to original sources

PubMed1999

Inactivation and regulation of the aerobic C(4)-dicarboxylate transport (dctA) gene of Escherichia coli.

The gene (dctA) encoding the aerobic C(4)-dicarboxylate transporter (DctA) of Escherichia coli was previously mapped to the 79-min region of the linkage map. The nucleotide sequence of this region reveals two candidates for the dctA gene: f428 at 79.3 min and the o157a-o424-o328 (or orfQMP) operon at 79.9 min. The f428 gene encodes a homologue of the Sinorhizobium meliloti and Rhizobium leguminosarum H(+)/C(4)-dicarboxylate symporter, DctA, whereas the orfQMP operon encodes homologues of the aerobic periplasmic-binding protein- dependent C(4)-dicarboxylate transport system (DctQ, DctM, and DctP) of Rhodobacter capsulatus. To determine which, if either, of these loci specify the E. coli DctA system, the chromosomal f428 and orfM genes were inactivated by inserting Sp(r) or Ap(r) cassettes, respectively. The resulting f428 mutant was unable to grow aerobically with fumarate or malate as the sole carbon source and grew poorly with succinate. Furthermore, fumarate uptake was abolished in the f428 mutant and succinate transport was approximately 10-fold lower than that of the wild type. The growth and fumarate transport deficiencies of the f428 mutant were complemented by transformation with an f428-containing plasmid. No growth defect was found for the orfM mutant. In combination, the above findings confirm that f428 corresponds to the dctA gene and indicate that the orfQMP products play no role in C(4)-dicarboxylate transport. Regulation studies with a dctA-lacZ (f428-lacZ) transcriptional fusion showed that dctA is subject to cyclic AMP receptor protein (CRP)-dependent catabolite repression and ArcA-mediated anaerobic repression and is weakly induced by the DcuS-DcuR system in response to C(4)-dicarboxylates and citrate. Interestingly, in a dctA mutant, expression of dctA is constitutive with respect to C(4)-dicarboxylate induction, suggesting that DctA regulates its own synthesis. Northern blot analysis revealed a single, monocistronic dctA transcript and confirmed that dctA is subject to regulation by catabolite repression and CRP. Reverse transcriptase-mediated primer extension indicated a single transcriptional start site centered 81 bp downstream of a strongly predicted CRP-binding site.

Aerobiosis↗

PubMed1999

Identification and characterization of a two-component sensor-kinase and response-regulator system (DcuS-DcuR) controlling gene expression in response to C4-dicarboxylates in Escherichia coli.

The dcuB gene of Escherichia coli encodes an anaerobic C4-dicarboxylate transporter that is induced anaerobically by FNR, activated by the cyclic AMP receptor protein, and repressed in the presence of nitrate by NarL. In addition, dcuB expression is strongly induced by C4-dicarboxylates, suggesting the presence of a novel C4-dicarboxylate-responsive regulator in E. coli. This paper describes the isolation of a Tn10 mutant in which the 160-fold induction of dcuB expression by C4-dicarboxylates is absent. The corresponding Tn10 mutation resides in the yjdH gene, which is adjacent to the yjdG gene and close to the dcuB gene at approximately 93.5 min in the E. coli chromosome. The yjdHG genes (redesignated dcuSR) appear to constitute an operon encoding a two-component sensor-regulator system (DcuS-DcuR). A plasmid carrying the dcuSR operon restored the C4-dicarboxylate inducibility of dcuB expression in the dcuS mutant to levels exceeding those of the dcuS+ strain by approximately 1.8-fold. The dcuS mutation affected the expression of other genes with roles in C4-dicarboxylate transport or metabolism. Expression of the fumarate reductase (frdABCD) operon and the aerobic C4-dicarboxylate transporter (dctA) gene were induced 22- and 4-fold, respectively, by the DcuS-DcuR system in the presence of C4-dicarboxylates. Surprisingly, anaerobic fumarate respiratory growth of the dcuS mutant was normal. However, under aerobic conditions with C4-dicarboxylates as sole carbon sources, the mutant exhibited a growth defect resembling that of a dctA mutant. Studies employing a dcuA dcuB dcuC triple mutant unable to transport C4-dicarboxylates anaerobically revealed that C4-dicarboxylate transport is not required for C4-dicarboxylate-responsive gene regulation. This suggests that the DcuS-DcuR system responds to external substrates. Accordingly, topology studies using 14 DcuS-BlaM fusions showed that DcuS contains two putative transmembrane helices flanking a approximately 140-residue N-terminal domain apparently located in the periplasm. This topology strongly suggests that the periplasmic loop of DcuS serves as a C4-dicarboxylate sensor. The cytosolic region of DcuS (residues 203 to 543) contains two domains: a central PAS domain possibly acting as a second sensory domain and a C-terminal transmitter domain. Database searches showed that DcuS and DcuR are closely related to a subgroup of two-component sensor-regulators that includes the citrate-responsive CitA-CitB system of Klebsiella pneumoniae. DcuS is not closely related to the C4-dicarboxylate-sensing DctS or DctB protein of Rhodobacter capsulatus or rhizobial species, respectively. Although all three proteins have similar topologies and functions, and all are members of the two-component sensor-kinase family, their periplasmic domains appear to have evolved independently.

Amino Acid Sequence↗

PubMed1999

Ferritin mutants of Escherichia coli are iron deficient and growth impaired, and fur mutants are iron deficient.

Escherichia coli contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes (ftnA and bfr) were inactivated by replacing the chromosomal ftnA and bfr genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants (ftnA::spc and bfr::kan) and a double mutant (ftnA::spc bfr::kan) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the bfr mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the ftnA and ftnA bfr mutants remained unchanged. The ftnA and ftnA bfr mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation (fur) mutants also had very low iron contents (2.5-fold less iron than Fur+ strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the ftnA and fur mutants were confirmed by Mössbauer spectroscopy, which further showed that the low iron contents of ftnA mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the fur mutation, ftnA and bfr mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of "reactive ferrous iron." It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling the expression of iron-containing proteins. The role of Bfr remains unclear.

Aerobiosis↗

PubMed1998

Co-regulation of lipoamide dehydrogenase and 2-oxoglutarate dehydrogenase synthesis in Escherichia coli: characterisation of an ArcA binding site in the lpd promoter.

The lipoamide dehydrogenase gene (lpdA) encoding the E3 subunits of both the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes of Escherichia coli, is expressed from the upstream pdh and internal lpd promoters of the pdh operon (pdhR-aceEF-lpdA). Under aerobic conditions, the specific components of the 2-oxoglutarate dehydrogenase complex encoded by the sucAB genes in the sdhCDAB-sucABCD operon are expressed from the sdh promoter. The provision of lipoamide dehydrogenase subunits for assembly into the 2-oxoglutarate dehydrogenase complex could thus be controlled by co-regulation of the lpd promoter with the sdh promoter. Here, the transcription start point of the lpd promoter was defined by primer extension analysis, and an ArcA binding site, TGTTAACAAT, overlapping the lpd promoter and matching the consensus at 8 out of 10 positions, was identified by in vitro footprint analysis. PdhR was not bound to the lpd promoter nor was ArcA bound specifically to the pdh promoter. These results support the view that co-regulation of the lpd and sdh promoters is mediated primarily by ArcA.

Aerobiosis↗

PubMed1998

Altering the anaerobic transcription factor FNR confers a hemolytic phenotype on Escherichia coli K12.

The recent outbreaks of Escherichia coli O157-associated food poisoning have focused attention on the virulence determinants of E. coli. Here, it is reported that single base substitutions in the fnr gene encoding the oxygen-responsive transcription regulator FNR (fumarate and nitrate reduction regulator) are sufficient to confer a hemolytic phenotype on E. coli K12, the widely used laboratory strain. The mechanism involves enhancing the expression of a normally dormant hemolysin gene (hlyE) located in the E. coli chromosome. The mutations direct single amino acid substitutions in the activating regions (AR1 and AR3) of FNR that contact RNA polymerase. It is concluded that altering a resident transcription regulator, or acquisition of a competent heterologous regulator, could generate a pool of hemolytic, and therefore more virulent, strains of E. coli in nature.

Amino Acid Sequence↗

PubMed1998

How the presence of three iron binding sites affects the iron storage function of the ferritin (EcFtnA) of Escherichia coli.

The iron storage proteins, ferritins, are found in all organisms which use iron. Here iron storage processes in the Escherichia coli ferritin (EcFtnA) are compared with those in human H-type ferritin (HuHF). Both proteins contain dinuclear iron centres that enable the rapid oxidation of 2 Fe(II) by O2. The presence of a third iron binding site in EcFtnA, although not essential for fast oxidation, causes the O2/Fe ratio to increase from 2 to 3-4. In EcFtnA the rate of iron oxidation falls markedly after the oxidation of 48 Fe(II) atoms/molecule probably because some of it remains at the oxidation site. However a compensatory physiological advantage is conferred because this iron is more readily available to meet the cell's needs.

Amino Acid Sequence↗

PubMed1998

Regulation of thiamin diphosphate-dependent 2-oxo acid decarboxylases by substrate and thiamin diphosphate.Mg(II) - evidence for tertiary and quaternary interactions.

The regulatory mechanism of substrate activation in yeast pyruvate decarboxylase is triggered by the interaction of pyruvic acid with C221 located on the beta domain at >20 A from the thiamin diphosphate (ThDP). To trace the putative information transfer pathway, substitutions were made at H92 on the alpha domain, across the domain divide from C221, at E91, next to H92 and hydrogen bonded to W412, the latter being intimately involved in the coenzyme binding locus. Additional substitutions were made at D28, E51, H114, H115, I415 and E477, all near the active center. The pH-dependent steady-state kinetic parameters, including the Hill coefficient, provide useful insight to this effort. In addition to C221, the residues H92, E91, E51 and H114 and H115 together appear to have a critical impact on the Hill coefficient, providing a pathway for information transfer. To study the activation by ThDP.Mg(II), variants at G231 (of the conserved GDG triplet) and at N258 and C259 (all three being part of the putative ThDP fold) of the E1 component of the Escherichia coli pyruvate dehydrogenase multienzyme complex were studied. Kinetic and spectroscopic evidence suggests that the Mg(II) ligands are very important to activation of the enzymes by cofactors.

Binding Sites↗

PubMed1998

Environmental control of pyruvate dehydrogenase complex expression in Escherichia coli.

The effects of changing environmental conditions on expression of the pdh operon were studied in strains containing pyruvate dehydrogenase (PDH) complexes having either one or three lipoyl domains per lipoate acetyltransferase chain. The expression of the pdh operon was lowered during growth on reduced carbon sources and when the mode of energy generation was changed from aerobic respiration to anaerobic respiration and fermentation. In contrast, growth at non-optimal pH increased expression. Operon expression was generally higher in the 1 lip strain compared to the 3 lip strain. Expression of the pdh operon was shown to be tightly controlled in response to environmental stimuli, consistent with its importance in defining metabolic flux.

Carbon↗

PubMed1998

Systematic study of the six cysteines of the E1 subunit of the pyruvate dehydrogenase multienzyme complex from Escherichia coli: none is essential for activity.

Variants of the Escherichia coli 1-lip pyruvate dehydrogenase multienzyme complex (1-lip PDHc) with the C259N and C259S substitutions in the putative thiamin diphosphate-(ThDP-) binding motif of the pyruvate dehydrogenase component (E1, EC 1.2.4.1) were characterized. Single substitutions were made at the five remaining cysteines of the E1 component, creating the C120A, C575A, C610A, C654A, and C770S variants to test the hypothesis that the activity loss that accompanies exposure of the enzyme to fluoropyruvate, bromopyruvate, and 2-oxo-3-butynoic acid is the result of the modification of approximately one cysteine residue per E1 monomer. Surprisingly, all single cysteine E1 variants could be reconstituted with E2-E3 subcomplex and showed PDHc activity ranging from 74% to 96% that of the parental enzyme. The specific activities of C259N and C259S variants of 1-lip PDHc were 58% and 27% relative to that of the parental 1-lip PDHc. All five single cysteine E1 variants, along with the C259N and C259S variants of 1-lip PDHc, could also (1) be inactivated with fluoropyruvate and 2-oxo-3-butynoic acid, (2) were subject to inactivation by the monoclonal antibody 18A9 reported from one of our laboratories, and (3) were subject to regulation by pyruvate and acetyl-CoA. It was therefore concluded that none of the six cysteine residues is essential for the activity of the E1 component or of the complex. When tested with the putative transition-state analogue, thiamin 2-thiothiazolone diphosphate, all but the C259S and C259N variants were very potently inhibited, the stoichiometry for parental E1 being about 1.6 mol of inhibitor/mol of E1 subunit. The C259S and C259N E1 variants required at least 25-fold greater inhibitor concentration to achieve the same level of inhibition. C259 is located in the putative thiamin diphosphate-binding motif of the enzyme [more exactly, it is adjacent to a ligand to the Mg(II) ion]. It is therefore concluded that thiamin 2-thiothiazolone diphosphate is not a transition-state analogue; rather, it is a potent inhibitor of the complex because of a specific interaction with the C259 residue.

Acetyl Coenzyme A↗

PubMed1998

Topological analysis of DcuA, an anaerobic C4-dicarboxylate transporter of Escherichia coli.

Escherichia coli possesses three independent anaerobic C4-dicarboxylate transport systems encoded by the dcuA, dcuB, and dcuC genes. The dcuA and dcuB genes encode related integral inner-membrane proteins, DcuA and DcuB (433 and 446 amino acid residues), which have 36% amino acid sequence identity. A previous amino acid sequence-based analysis predicted that DcuA and DcuB contain either 12 or 14 transmembrane helices, with the N and C termini located in the cytoplasm or periplasm (S. Six, S. C. Andrews, G. Unden, and J. R. Guest, J. Bacteriol. 176:6470-6478, 1994). These predictions were tested by constructing and analyzing 66 DcuA-BlaM fusions in which C terminally truncated forms of DcuA are fused to a beta-lactamase protein lacking the N-terminal signal peptide. The resulting topological model differs from those previously predicted. It has just 10 transmembrane helices and a central, 80-residue cytoplasmic loop between helices 5 and 6. The N and C termini are located in the periplasm and the predicted orientation is consistent with the "positive-inside rule." Two highly hydrophobic segments are not membrane spanning: one is in the cytoplasmic loop; the other is in the C-terminal periplasmic region. The topological model obtained for DcuA can be applied to DcuA homologues in other bacteria as well as to DcuB. Overproduction of DcuA to 15% of inner-membrane protein was obtained with the lacUV5-promoter-based plasmid, pYZ4.

Amino Acid Sequence↗

PubMed1998

Transcriptional regulation and organization of the dcuA and dcuB genes, encoding homologous anaerobic C4-dicarboxylate transporters in Escherichia coli.

The dcuA and dcuB genes of Escherichia coli encode homologous proteins that appear to function as independent and mutually redundant C4-dicarboxylate transporters during anaerobiosis. The dcuA gene is 117 bp downstream of, and has the same polarity as, the aspartase gene (aspA), while dcuB is 77 bp upstream of, and has the same polarity as, the anaerobic fumarase gene (fumB). To learn more about the respective roles of the dcu genes, the environmental and regulatory factors influencing their expression were investigated by generating and analyzing single-copy dcuA- and dcuB-lacZ transcriptional fusions. The results show that dcuA is constitutively expressed whereas dcuB expression is highly regulated. The dcuB gene is strongly activated anaerobically by FNR, repressed in the presence of nitrate by NarL, and subject to cyclic AMP receptor protein (CRP)-mediated catabolite repression. In addition, dcuB is strongly induced by C4-dicarboxylates, suggesting that dcuB is under the control of an uncharacterized C4-dicarboxylate-responsive gene regulator. Northern blotting confirmed that dcuA (and aspA) is expressed under both aerobic and anaerobic conditions and that dcuB (and fumB) is induced anaerobically. Major monocistronic transcripts were identified for aspA and dcuA, as well as a minor species possibly corresponding to an aspA-dcuA cotranscript. Five major transcripts were observed for dcuB and fumB: monocistronic transcripts for both fumB and dcuB; a dcuB-fumB cotranscript; and two transcripts, possibly corresponding to dcuB-fumB and fumB mRNA degradation products. Primer extension analysis revealed independent promoters for aspA, dcuA, and dcuB, but surprisingly no primer extension product could be detected for fumB. The expression of dcuB is entirely consistent with a primary role for DcuB in mediating C4-dicarboxylate transport during anaerobic fumarate respiration. The precise physiological purpose of DcuA remains unclear.

Amino Acid Sequence↗

PubMed1997

FNR is a direct oxygen sensor having a biphasic response curve.

FNR is a transcription regulator that controls the expression of target genes in response to anoxia. Anaerobiosis is accompanied by the acquisition of two [4Fe-4S]2+ clusters per FNR dimer and the ability to bind DNA site-specifically. Oxidation of the [4Fe-4S]2+ form of FNR by O2 produced a non-DNA-binding, transcriptionally inactive form which also contains an iron-sulfur cluster, recently identified by Mossbauer spectroscopy as a [2Fe-2S] cluster (Khoroshilova et al., 1997, PNAS. 94, 6078). Complete conversion needed at least 2.5-3.0 molecules of O2 per [4Fe-4S]2+ cluster. Using sub-stoicheiometric amounts of air-saturated buffer, stable equilibria were established in which the [4Fe-4S]2+ and [2Fe-2S]2+ forms co-exist and no EPR detectable free ferric ions were released. In contrast, a 20-fold molar excess K3Fe(CN)6 was required to oxidise the [4Fe-4S]2+ cluster and in this case, ferric ions were released. FNR is therefore a sensitive O2 sensor.

Anaerobiosis↗

PubMed1997

2-Oxo-3-alkynoic acids, universal mechanism-based inactivators of thiamin diphosphate-dependent decarboxylases: synthesis and evidence for potent inactivation of the pyruvate dehydrogenase multienzyme complex.

A new class of compounds, the 2-oxo-3-alkynoic acids with a phenyl substituent at carbon 4 was reported by the authors as potent irreversible and mechanism-based inhibitors of the thiamin diphosphate- (ThDP-) dependent enzyme pyruvate decarboxylase [Chiu, C.-F., & Jordan, F. (1994) J. Org. Chem. 59, 5763-5766]. The method has been successfully extended to the synthesis of the 4-, 5-, and 7-carbon aliphatic members of this family of compounds. These three compounds were then tested on three ThDP-dependent pyruvate decarboxylases: the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) and its E1 (ThDP-dependent) component, pyruvate oxidase (POX, phosphorylating; from Lactobacillus plantarum),and pyruvate decarboxylase (PDC) from Saccharomycescerevisiae. All three enzymes were irreversibly inhibited by the new compounds. The 4-carbon acid is the best substrate-analog inactivator known to date for PDHc, more potent than either fluoropyruvate or bromopyruvate. The following conclusions were drawn from extensive studies with PDHc: (a) The kinetics of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is time- and concentration-dependent. (b) The 4-carbon acid has a Ki 2 orders of magnitude stronger than the 5-carbon acid, clearly demonstrating the substrate specificity of PDHc. (c) The rate of inactivation of PDH complexes and of resolved E1 by 2-oxo-3-alkynoic acids is enhanced by the addition of ThDP and MgCl2. (d) Pyruvate completely protects E1 and partially protects PDHc from inactivation by 2-oxo-3-butynoic acid. (e) E1 but not E2-E3 is the target of inactivation by 2-oxo-3-butynoic acid. (f) Inactivation of E1 by 2-oxo-3-butynoic acid is accompanied by modification of 1.3 cysteines/E1 monomer. The order of reactivity with the 4-carbon acid was PDHc > POX > PDC. While the order of reactivity with PDHc and POX was 2-oxo-3-butynoic acid > 2-oxo-3-pentynoic acid > 2-oxo-3-heptynoic acid, the order of reactivity was reversed with PDC.

Bacterial Proteins↗

PubMed1997

Dinuclear center of ferritin: studies of iron binding and oxidation show differences in the two iron sites.

The ferroxidase activity of human ferritin has previously been associated with a diiron site situated centrally within the four-helix bundle of H-type chains (HuHF). However, direct information about the site of Fe(II) binding has been lacking, and events between Fe(II) binding and its oxidation have not previously been studied. A sequential stopped-flow assay has now been developed to enable the dissection of binding and oxidation. It depends on the ability of 1,10-phenanthroline to complex protein-bound Fe(II) and to distinguish it from the more immediately available free Fe(II). This approach, aided by the use of site-directed variants, indicates that in HuHF and the non-heme ferritin of Escherichia coli the first 48 Fe(II) atoms/molecule added are bound and oxidized at the dinuclear centers. At a constant iron concentration, the rate of Fe(II) oxidation was maximal for additions of 2 Fe(II) atoms/subunit, consistent with a two-electron oxidation of the Fe(II) pair. Although, at low Fe(II)/protein ratios, no cooperativity in Fe(II) binding was observed; a preferred order of binding was deduced [Fe(II) binding first at site A and then at site B]. Binding of Fe(II) at both sites was essential for fast oxidation. Modification of site A ligands resulted in slow iron binding and slow oxidation. Modification of site B did not prevent Fe(II) binding at site A but greatly reduced its oxidation rate. These differences may mean that dioxygen is initially bound to Fe(II) at site B.

Binding Sites↗

PubMed1997

The aconitase family: three structural variations on a common theme.

The aconitase family contains a diverse group of iron-sulphur (Fe-S) isomerases and two types of iron regulatory protein (IRP). Structural comparisons have revealed three architecturally distinct variants in which one of the four structural domains is covalently linked at either the amino- or carboxy-terminal end of a single polypeptide or else this domain exists as an independent subunit.

Aconitate Hydratase↗

PubMed1996

Effect of substitutions in the thiamin diphosphate-magnesium fold on the activation of the pyruvate dehydrogenase complex from Escherichia coli by cofactors and substrate.

The homotropic regulation of the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) by its coenzyme thiamin diphosphate and its substrate pyruvate was re-examined with complexes containing three and one lipoyl domains per E2 chain, and several variants of the latter, containing substitutions in the putative thiamin diphosphate fold of E1 (G231A, G231S, C259S, C259N, and N258Q). It was found that all of the E1 variants had significantly reduced specific activities, as reported elsewhere (Russell, G. C., Machado, R. S., and Guest, J. R. (1992) Biochem. J. 287, 611-619). In addition, extensive kinetic studies were performed in an attempt to determine the effects of the amino acid substitutions on the Hill coefficients with respect to thiamin diphosphate and pyruvate. All but one of the variants were incapable of being saturated with thiamin diphosphate, even at concentrations > 5 mM. Most importantly, the striking activation lag phase lasting for many seconds in the parental complexes containing three and one lipoyl domains per E2 chain was totally eliminated in the variants. Furthermore, activation by the coenzyme was localized to the E1 subunit, because resolved E1 exhibits virtually the same behavior during the activation lag phase as does the complex. In the parental complexes two distinct lag phases could be resolved, the duration of both decreases with increasing ThDP concentration. A mechanism that is consistent with all of the kinetic data on the parental complexes involves rapid equilibration of the first ThDP with the E1 dimer, followed by a slow conformational equilibration, that in turn is followed by slow addition of the second ThDP to form the fully activated dimer. When the diphosphate site is badly impaired, the binding affinity is very much reduced, this perhaps eliminates the slow step leading to the activated dimer form of the E1.

Acetyltransferases↗

PubMed1996

Spectroscopic and voltammetric characterisation of the bacterioferritin-associated ferredoxin of Escherichia coli.

The bacterioferritin-associated ferredoxin (Bfd) of Escherichia coli is a 64-residue polypeptide encoded by the bfd gene located upstream of the gene (bfr) encoding the iron-storage haemoprotein, bacterioferritin. The Bfd sequence resembles those of the approximately 60-residue domains found in NifU proteins (required for metallocluster assembly), nitrite reductases, and Klebsiella pneumoniae nitrate reductase. These related-domains contain four well-conserved cysteine residues, which are thought to function as ligands to a [2Fe-2S] cluster. The Bfd protein was over-produced, purified, and characterised. Bfd was found to be a positively-charged monomer containing two iron atoms and two labile sulphides. Ultraviolet-visible, EPR, variable-temperature magnetic-circular dichroism and resonance Raman spectroscopies, together with cyclic voltogram measurements, revealed the presence of a [2Fe-2S]2+,+ centre (E1/2 = -254 mV) having remarkably similar properties to the Fe-S cluster of NifU. Bfd may thus be a 2Fe ferredoxin participating either in release/delivery of iron from/to bacterioferritin (or other iron complexes), or in iron-dependent regulation of bfr expression.

Amino Acid Sequence↗

PubMed1996

Reconstitution of the [4Fe-4S] cluster in FNR and demonstration of the aerobic-anaerobic transcription switch in vitro.

The FNR protein of Escherichia coli is a redox-responsive transcription regulator that activates and represses a family of genes required for anaerobic and aerobic metabolism. Reconstitution of wild-type FNR by anaerobic treatment with ferrous ions, cysteine and the NifS protein of Azotobacter vinelandii leads to the incorporation of two [4Fe-4S]2+ clusters per FNR dimer. The UV-visible spectrum of reconstituted FNR has a broad absorbance at 420 nm. The clusters are EPR silent under anaerobic conditions but are degraded to [3Fe-4S]+ by limited oxidation with air, and completely lost on prolonged air exposure. The association of FNR with the iron-sulphur clusters is confirmed by CD spectroscopy. Incorporation of the [4Fe-4S]2+ clusters increases site-specific DNA binding about 7-fold compared with apo-FNR. Anaerobic transcription activation and repression in vitro likewise depends on the presence of the iron-sulphur cluster, and its inactivation under aerobic conditions provides a demonstration in vitro of the FNR-mediated aerobic-anaerobic transcriptional switch.

Aerobiosis↗