The primary structure of a protein containing a putative [6Fe-6S] prismane cluster from Desulfovibrio desulfuricans (ATCC 27774).

The gene encoding a protein containing a novel iron sulfur cluster ([6Fe-6S]) has been cloned from Desulfovibrio desulfuricans ATCC 27774 and sequenced. An open reading frame was found encoding a 545 amino acid protein (M(r) 58,496). The amino acid sequence is highly homologous with that of the corresponding protein from D. vulgaris (Hildenborough) and contains a Cys-motif that may be involved in coordination of the Fe-S cluster.

Cloning, sequencing and expression of the tetraheme cytochrome c(3) from Desulfovibrio gigas.

The gene encoding the tetraheme cytochrome c(3) from Desulfovibrio gigas was cloned and sequenced from a 2.7-kb EcoRI-PstI insert of D. gigas DNA. The derived amino acid sequence showed that the D. gigas cytochrome c(3) is synthesized as a precursor protein with an N-terminal signal peptide sequence of 25 residues and allowed the correction of the previous reported amino acid sequence (Matias et al. Protein Science 5 (1996) 1342-1354). Expression in D. vulgaris (Hildenborough) was possible by conjugal transfer of a recombinant broad-host-range vector pSUP104 containing a SmaI fragment of the D. gigas cytochrome c(3) gene. Biochemical, immunological and spectroscopic analysis of the purified protein showed that the recombinant cytochrome is identical to that isolated from D. gigas.

Overproduction of the prismane protein from Desulfovibrio desulfuricans ATCC 27774 in Desulfovibrio vulgaris (Hildenborough) and EPR spectroscopy of the [6Fe-6S] cluster in different redox states.

The Desulfovibrio desulfuricans ATCC 27774 prismane protein was isolated from a Desulfovibrio vulgaris (Hildenborough) strain that contained the gene for this protein in expression vector pSUP104. A redox titration demonstrated that the [Fe-S] cluster in this protein may attain four different redox states, indicated as +3, +4, +5 and +6, with midpoint potentials for the transitions of approx. -220, +50/-25 and +370 mV, respectively. EPR spectra of the protein in the various redox states are reminiscent of those of the D. vulgaris prismane protein (Pierik et al. (1992) Eur. J. Biochem. 206, 705-719), but differ in details. In the +5-state, virtually all the iron is in a S = 9/2 spin state, indicative for a cluster that is more complex than common [4Fe-4S] or [2Fe-2S] clusters. Similarity of the EPR spectrum of the protein in the +3-state with those of inorganic [6Fe-6S] model compounds suggests that the cluster in the protein is also [6Fe-6S]. In the +4-state of the protein a broad signal due to an integer-spin system can be detected with normal-mode EPR. A dramatic sharpening-up and increase of intensity of this band (g = 14.7) is observed with parallel-mode EPR. In accordance with the chemically determined iron content of the protein (6.0 +/- 0.45 moles of iron/mole of protein), the spectroscopic data indicate one [6Fe-6S] cluster in this protein. We did not find evidence for a previous claim (Moura et al. (1992) J. Biol. Chem. 267, 4489-4496) that the D. desulfuricans protein contains two [6Fe-6S] clusters.

The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate.

A flavodoxin from Azotobacter vinelandii is chosen as a model system to study the folding of alpha/beta doubly wound proteins. The guanidinium hydrochloride induced unfolding of apoflavodoxin is demonstrated to be reversible. Apoflavodoxin thus can fold in the absence of the FMN cofactor. The unfolding curves obtained for wild-type, C69A and C69S apoflavodoxin as monitored by circular dichroism and fluorescence spectroscopy do not coincide. Apoflavodoxin unfolding occurs therefore not via a simple two-state mechanism. The experimental data can be described by a three-state mechanism of apoflavodoxin equilibrium unfolding in which a relatively stable intermediate is involved. The intermediate species lacks the characteristic tertiary structure of native apoflavodoxin as deduced from fluorescence spectroscopy, but has significant secondary structure as inferred from circular dichroism spectroscopy. Both spectroscopic techniques show that thermally-induced unfolding of apoflavodoxin also proceeds through formation of a similar molten globule-like species. Thermal unfolding of apoflavodoxin is accompanied by anomalous circular dichroism characteristics: the negative ellipticity at 222 nM increases in the transition zone of unfolding. This effect is most likely attributable to changes in tertiary interactions of aromatic side chains upon protein unfolding. From the presented results and hydrogen/deuterium exchange data, a model for the equilibrium unfolding of apoflavodoxin is presented.

Apparent local stability of the secondary structure of Azotobacter vinelandii holoflavodoxin II as probed by hydrogen exchange: implications for redox potential regulation and flavodoxin folding.

As a first step to determine the folding pathway of a protein with an alpha/beta doubly wound topology, the 1H, 13C, and 15N backbone chemical shifts of Azotobacter vinelandii holoflavodoxin II (179 residues) have been determined using multidimensional NMR spectroscopy. Its secondary structure is shown to contain a five-stranded parallel beta-sheet (beta2-beta1-beta3-beta4-beta5) and five alpha-helices. Exchange rates for the individual amide protons of holoflavodoxin were determined using the hydrogen exchange method. The amide protons of 65 residues distributed throughout the structure of holoflavodoxin exchange slowly at pH* 6.2 [kex < 10(-5) s(-1)] and can be used as probes in future folding studies. Measured exchange rates relate to apparent local free energies for transient opening. We propose that the amide protons in the core of holoflavodoxin only exchange by global unfolding of the apo state of the protein. The results obtained are discussed with respect to their implications for flavodoxin folding and for modulation of the flavin redox potential by the apoprotein. We do not find any evidence that A. vinelandii holoflavodoxin II is divided into two subdomains based on its amide proton exchange rates, as opposed to what is found for the structurally but not sequentially homologous alpha/beta doubly wound protein Che Y.

The operon for the Fe-hydrogenase in Desulfovibrio vulgaris (Hildenborough): mapping of the transcript and regulation of expression.

The genes for the subunits of the Fe-only hydrogenase from Desulfovibrio vulgaris are transcribed as a 1.9 kb mRNA; the operon contains no other genes besides those encoding the two subunits. The transcriptional start site of the operon was mapped. Determination of hydrogenase activity and hydrogenase mRNA levels indicates a growth-phase dependent regulation of hydrogenase expression at transcriptional level. However, it has not yet been possible to localize the sequences required for regulation and expression of the genes.

Molecular cloning of a gene coding for a Vibrio cholerae haemagglutinin.

Recombinant plasmids encoding a Vibrio cholerae haemagglutinin were isolated from the highly virulent V. cholerae strain C5 by cosmid cloning. Both Escherichia coli HB101 containing the recombinant plasmids and V. cholerae C5 were able to agglutinate a variety of erythrocytes from human and animal origin; this haemagglutination was not inhibited by D-mannose or L-fucose. Subcloning of the recombinant cosmid DNA revealed that a 1.3 kb DNA fragment was sufficient for haemagglutinin production in E. coli HB101. Under direction of this 1.3 kb Vibrio DNA fragment, two proteins were made in E. coli minicells, of 27 and 10 kDa. Haemagglutinin-encoding sequences were not detected in every V. cholerae strain.

Reduction of the amount of periplasmic hydrogenase in Desulfovibrio vulgaris (Hildenborough) with antisense RNA: direct evidence for an important role of this hydrogenase in lactate metabolism.

To establish the function of the periplasmic Fe-only hydrogenase in the anaerobic sulfate reducer Desulfovibrio vulgaris (Hildenborough), derivatives with a reduced content of this enzyme were constructed by introduction of a plasmid that directs the synthesis of antisense RNA complementary to hydrogenase mRNA. It was demonstrated that the antisense RNA technique allowed specific suppression of the synthesis of this hydrogenase in D. vulgaris by decreasing the amount of hydrogenase mRNA but did not result in the complete elimination of the enzyme, as is usual with most conventional mutagenesis techniques. The hydrogenase content in these antisense RNA-producing D. vulgaris clones was two- to threefold lower than in the parental strain when the strains were grown in batch cultures with lactate as a substrate and sulfate as a terminal electron acceptor. Under these conditions, several differences in growth parameters were measured between the hydrogenase-suppressed clones and wild-type D. vulgaris: growth rates of the clones decreased two- to threefold, and at excess lactate, growth yields were reduced by 20%. Furthermore, the amount of hydrogen measured in the culture headspaces was reduced three- to fivefold for the clones. These observations indicate that this hydrogenase has an important function during growth on lactate and is involved in hydrogen production from protons and electrons originating from at least one of the two oxidation reactions in the conversion of lactate to acetate. The implications for the energy metabolism of D. vulgaris are discussed.

The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and [2Fe-2S].

Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe-2S-2O] cluster, which can attain four redox states. Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe-2S] cluster instead of a [4Fe-4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe-2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 degrees C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (Em NADH/NAD+ couple: -320 mV) are transferred along the [2Fe-2S] cluster of the NADH oxidoreductase (Em = -220 mV) and the [2Fe-2S] cluster of the hybrid-cluster protein (Em = -35 mV) to the hybrid cluster (Em = -50, +85 and +365 mV for the three redox transitions). The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration.

Characterization of a ferredoxin from Desulfovibrio vulgaris (Hildenborough) that interacts with RNA.

The purification and characterization of a ferredoxin from Desulfovibrio vulgaris (Hildenborough) is described. The protein can be isolated in two forms; the major form is strongly complexed to RNA, while a minor form is free from nucleic acid. Bound RNA cannot be removed by digestion with nucleases, or by heating to 70 degrees C, and it can only be partially removed by rechromatography. The ultraviolet/visible spectrum shows typical absorption maxima at 280 nm and 400 nm for the RNA-free ferredoxin. The RNA-bound protein exhibits an additional strong peak at 260 nm. The RNA can be extracted from the protein with phenol. The ferredoxin is a dimer of subunits, each of 7.5 kDa; its pI is 3.9. The protein contains a [4Fe-4S](2+;1+) cluster with an EPR spectrum (g = 1.90, 1.93 and 2.05) in the reduced state. A reduction potential of -360 mV was determined for the RNA-free ferredoxin with reversible voltammetry at glassy carbon. From the temperature dependence of the reduction potential, the unusually high standard reaction entropy was calculated as delta S degree = -230 J.K-1.mol-1. No electrochemical response was obtained with the RNA-bound ferredoxin. Binding of RNA appears to require the presence of an intact cluster, since the absence of absorption at 400 nm runs in parallel with the absence of absorption at 260 nm. The possibility is discussed that the binding to the RNA has a regulatory function and is controlled by the state of the cluster.

The accumulation of the maternal pool of histone H1A during oogenesis in Xenopus laevis.

The accumulation of the maternal pool of histone H1A in Xenopus laevis oocytes was measured by the use of a semi-quantitative immunoradiographic method. This method implies the size-fractionation of total basic protein extracts from oocytes on a polyacrylamide gel, blotting of the proteins to diazotized paper and detection of histone proteins on the blots with the aid of antibodies to histone H1A and of [125I]protein A. It is shown that the maternal H1A pool that is present in fully-grown oocytes is accumulated very early in oogenesis, mainly in early vitellogenic oocytes. Stage III oocytes (Dumont, 1972) already contain approximately 80% of the final amount of H1A that is found in mature oocytes. The minimum amount of histone H1A estimated to be present in mature oocytes is 8-10 ng.

Histone gene expression in early development of Xenopus laevis. Analysis of histone mRNA in oocytes and embryos by blot-hybridization and cell-free translation.

This study comprises the hybridization analysis of electrophoretically separated histone mRNAs from oocytes and embryos of Xenopus laevis, and analysis of in vitro translation products of these mRNAs on polyacrylamide gels containing sodium dodecyl sulfate (SDS) or Triton X-100. In oocytes and embryos up to the tailbud stage, four types of mRNAs complementary to histone H2B DNA and two complementary to histone H4 DNA can be discriminated by their different electrophoretic mobilities on polyacrylamide gels. Electrophoretic heterogeneity was not detected for messengers for histones H2A and H3. Histone mRNA, purified by hybridization under stringent conditions with a cloned histone gene cluster, was used to direct histone protein synthesis in a wheat-germ cell free system. The proteins synthesized comigrate with purified marker histones when electrophoresed on SDS-gels or acid-urea gels containing Triton X-100. When hybrid-selected histone mRNAs from oocytes and embryos in different developmental stages are translated, the proteins made by the mRNA from one stage can not be discriminated from those made by the mRNA from another stage after electrophoresis on SDS-gels or acid urea Triton X-100 gels.

Molecular cloning and sequence determination of the lpd gene encoding lipoamide dehydrogenase from Pseudomonas fluorescens.

The lpd gene encoding lipoamide dehydrogenase (dihydrolipoamide dehydrogenase; EC 1.8.1.4) was isolated from a library of Pseudomonas fluorescens DNA cloned in Escherichia coli TG2 by use of serum raised against lipoamide dehydrogenase from Azotobacter vinelandii. Large amounts (up to 15% of total cellular protein) of the P. fluorescens lipoamide dehydrogenase were produced by the E. coli clone harbouring plasmid pCJB94 with the lipoamide dehydrogenase gene. The enzyme was purified to homogeneity by a three-step procedure. The gene was subcloned from plasmid pCJB94 and the complete nucleotide sequence of the subcloned fragment (3610 bp) was determined. The derived amino acid sequence of P. fluorescens lipoamide dehydrogenase showed 84% and 42% homology when compared to the amino acid sequences of lipoamide dehydrogenase from A. vinelandii and E. coli, respectively. The lpd gene of P. fluorescens is clustered in the genome with genes for the other components of the 2-oxoglutarate dehydrogenase complex.

Overproduction of prismane protein in Desulfovibrio vulgaris (Hildenborough): evidence for a second S = 1/2-spin system in the one-electron reduced state.

The gene encoding the prismane protein from Desulfovibrio vulgaris (Hildenborough) was inserted into broad-host-range vector pSUP104. The recombinant plasmid, pJSP104, was transferred to D. vulgaris by conjugal plasmid transfer. In the transconjugant D. vulgaris cells the prismane protein was 25-fold overproduced. The overproduced prismane protein was characterized by molecular mass, isoelectric point, iron content and spectroscopical properties. Both the iron content and the ultraviolet/visible spectrum are identical to the wild-type protein indicating that iron incorporation in the overproduced protein is complete. EPR spectra of the dithionite-reduced form of the overproduced protein indicated that the Fe-S cluster might occur in a similar structure as found in inorganic model compounds containing a [6Fe-6S] prismane core. The as-isolated overproduced protein showed the presence of a second S = 1/2 spin system that was also detected in the corresponding prismane protein from D. desulfuricans (ATCC 27774), but not in the protein from wild-type D. vulgaris. This additional signal was irreversibly transformed to the 'wild-type' high-spin and low-spin systems upon two reduction/re-oxidation cycles. It is shown that the EPR spectroscopy of the overproduced prismane protein is very similar to that of the D. desulfuricans enzyme and, with the exception of the second S = 1/2 spin system, to that of the prismane protein from wild-type D. vulgaris. Contrary to claims for the D. desulfuricans protein, it is shown here that all data can be fully explained assuming a single [6Fe-6S] cluster, that might be titrated into four different redox states and occurs in up to three different spin systems in the one-electron reduced state.

The primary structure of a protein containing a putative [6Fe-6S] prismane cluster from Desulfovibrio vulgaris (Hildenborough).

The gene encoding a protein containing a putative [6Fe-6S] prismane cluster has been cloned from Desulfovibrio vulgaris (Hildenborough) and sequenced. The gene encodes a polypeptide composed of 553 amino acids (60,161 Da). The DNA-derived amino acid sequence was partly confirmed by N-terminal sequencing of the purified protein and of fragments of the protein generated by CNBr cleavage. Furthermore, the C-terminal sequence was verified by digestion with carboxypeptidases A and B. The polypeptide contains nine Cys residues. Four of these residues are gathered in a Cys-Xaa2-Cys-Xaa7-Cys-Xaa5-Cys motif located towards the N-terminus of the protein. No relevant sequence similarity was found with other proteins, including those with high-spin Fe-S clusters (nitrogenase, hydrogenase), with one significant exception: the stretch containing the first four Cys residues spans two submotifs, Cys-Xaa2-Cys and Lys-Gly-Xaa-Cys-Gly, separated by 11 residues, that are also present in high-spin Fe-S cluster containing CO dehydrogenase. Western-blot analysis demonstrates cross-reactivity of antibodies raised against the purified protein both in Desulfovibrio strains and other sulfate-reducing bacteria. Hybridization of the cloned gene with genomic DNA of several other Desulfovibrio species indicates that homologous sequences are generally present in the genus Desulfovibrio.

Effect of hydrogen-bond networks in controlling reduction potentials in Desulfovibrio vulgaris (Hildenborough) cytochrome C3 probed by site-specific mutagenesis.

Cytochromes C3 isolated from Desulfovibrio spp. are periplasmic proteins that play a central role in energy transduction by coupling the transfer of electrons and protons from hydrogenase. Comparison between the oxidized and reduced structures of cytochrome C3 isolated from Desulfovibrio vulgaris (Hildenborough) show that the residue threonine 24, located in the vicinity of heme III, reorients between these two states [Messias, A. C., Kastrau, D. H. W., Costa, H. S., LeGall, J., Turner, D. L., Santos, H., and Xavier, A. V. (1998) J. Mol. Biol. 281, 719-739]. Threonine 24 was replaced with valine by site-directed mutagenesis to elucidate its effect on the redox properties of the protein. The NMR spectra of the mutated protein are very similar to those of the wild type, showing that the general folding and heme core architecture are not affected by the mutation. However, thermodynamic analysis of the mutated cytochrome reveals a large alteration in the microscopic reduction potential of heme III (75 and 106 mV for the protonated forms of the fully reduced and oxidized states, respectively). The redox interactions involving this heme are also modified, while the remaining heme-heme interactions and the redox-Bohr interactions are less strongly affected. Hence, the order of oxidation of the hemes in the mutated cytochrome is different from that in the wild type, and it has a higher overall affinity for electrons. This is consistent with the replacement of threonine 24 by valine preventing the formation of a network of hydrogen bonds, which stabilizes the oxidized state. The mutated protein is unable to perform a concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild-type protein. Thus, replacing a single residue unbalances the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer and may affect the functionality of the protein.

The influence of the peptide chain on the kinetics and stability of microperoxidases.

Microperoxidases with increasing lengths of the peptide attached to the heme moiety have been isolated after proteolytic digestion of horse-heart cytochrome c (microperoxidases 6, 8, and 11) and of cytochrome c550 from Thiobacillus versutus (microperoxidase 17). The different microperoxidases catalyze the H2O2-dependent para-hydroxylation of aniline relatively efficiently but are rapidly inactivated under turnover conditions. The horse-heart cytochrome-c-derived microperoxidases have identical values for Vmax but show a decrease of the K(m) for aniline and a higher stability when the attached peptide is longer. The kinetic constants obtained for microperoxidase 17, differ markedly from the microperoxidases derived from horse-heart cytochrome c. Possible factors underlying the observed differences are discussed.

Replacement of lysine 45 by uncharged residues modulates the redox-Bohr effect in tetraheme cytochrome c3 of Desulfovibrio vulgaris (Hildenborough).

The structural basis for the pH dependence of the redox potential in the tetrahemic Desulfovibrio vulgaris (Hildenborough) cytochrome c3 was investigated by site-directed mutagenesis of charged residues in the vicinity of heme I. Mutation of lysine 45, located in the neighborhood of the propionates of heme I, by uncharged residues, namely threonine, glutamine and leucine, was performed. The replacement of a conserved charged residue, aspartate 7, present in the N-terminal region and near heme I was also attempted. The analysis of the redox interactions as well as the redox-Bohr behavior of the mutated cytochromes c3 allowed the conclusion that residue 45 has a functional role in the control of the pKa of the propionate groups of heme I and confirms the involvement of this residue in the redox-Bohr effect.