The distal heme center in Bacillus subtilis succinate:quinone reductase is crucial for electron transfer to menaquinone

Succinate:quinone reductases are membrane-bound enzymes that catalyze electron transfer from succinate to quinone. Some enzymes in vivo reduce ubiquinone (exergonic reaction) whereas others reduce menaquinone (endergonic reaction). The succinate:menaquinone reductases all contain two heme groups in the membrane anchor of the enzyme: a proximal heme (heme b(P)) located close to the negative side of the membrane and a distal heme (heme b(D)) located close to the positive side of the membrane. Heme b(D) is a distinctive feature of the succinate:menaquinone reductases, but the role of this heme in electron transfer to quinone has not previously been analyzed. His28 and His113 are the axial ligands to heme b(D) in Bacillus subtilis succinate:menaquinone reductase. We have individually replaced these His residues with Leu and Met, respectively, resulting in assembled membrane-bound enzymes. The H28L mutant enzyme lacks succinate:quinone reductase activity probably due to a defective quinone binding site. The H113M mutant enzyme contains heme b(D) with raised midpoint potential and is impaired in electron transfer to menaquinone. Our combined experimental data show that the heme b(D) center, into which we include a quinone binding site, is crucial for succinate:menaquinone reductase activity. The results support a model in which menaquinone is reduced on the positive side of the membrane and the transmembrane electrochemical potential provides driving force for electron transfer from succinate via heme b(P) and heme b(D) to menaquinone.     

Extracellular S100A4 stimulates the migration rate of astrocytic tumor cells by modifying the organization of their actin cytoskeleton

An overview of the present knowledge about succinate:quinone oxidoreductase in Paracoccus denitrificans and Bacillus subtilis is presented. P. denitrificans contains a monoheme succinate:ubiquinone oxidoreductase that is similar to that of mammalian mitochondria with respect to composition and sensitivity to carboxin. Results obtained with carboxin-resistant P. denitrificans mutants provide information about quinone-binding sites on the enzyme and the molecular basis for the resistance. B. subtilis contains a diheme succinate:menaquinone oxidoreductase whose activity is dependent on the electrochemical gradient across the cytoplasmic membrane. Data from studies of mutant variants of the B. subtilis enzyme combined with available crystal structures of a similar enzyme, Wolinella succinogenes fumarate reductase, substantiate a proposed explanation for the mechanism of coupling between quinone reductase activity and transmembrane potential.     

Experimental evidence for proton motive force-dependent catalysis by the diheme-containing succinate:menaquinone oxidoreductase from the Gram-positive bacterium Bacillus licheniformis

In Gram-positive bacteria and other prokaryotes containing succinate:menaquinone reductases, it has previously been shown that the succinate oxidase and succinate:menaquinone reductase activities are lost when the transmembrane electrochemical proton potential, Deltap, is abolished by the rupture of the bacteria or by the addition of a protonophore. It has been proposed that the endergonic reduction of menaquinone by succinate is driven by the electrochemical proton potential. Opposite sides of the cytoplasmic membrane were envisaged to be separately involved in the binding of protons upon the reduction of menaquinone and their release upon succinate oxidation, with the two reactions linked by the transfer of two electrons through the enzyme. However, it has previously been argued that the observed Deltap dependence is not associated specifically with the succinate:menaquinone reductase. Definitive insight into the mechanism of catalysis of this reaction requires a corresponding functional characterization of an isolated, membrane-bound succinate:menaquinone reductase from a Gram-positive bacterium. Here, we describe the purification, reconstitution into proteoliposomes, and functional characterization of the diheme-containing succinate:menaquinone reductase from the Gram-positive bacterium Bacillus licheniformis and, with the help of the design, synthesis, and characterization of quinones with finely tuned oxidation/reduction potentials, provide unequivocal evidence for Deltap-dependent catalysis of succinate oxidation by quinone as well as for Deltap generation upon catalysis of fumarate reduction by quinol.     

Characterization of the menaquinone reduction site in the diheme cytochrome b membrane anchor of Wolinella succinogenes NiFe-hydrogenase

The majority of bacterial membrane-bound NiFe-hydrogenases and formate dehydrogenases have homologous membrane-integral cytochrome b subunits. The prototypic NiFe-hydrogenase of Wolinella succinogenes (HydABC complex) catalyzes H2 oxidation by menaquinone during anaerobic respiration and contains a membrane-integral cytochrome b subunit (HydC) that carries the menaquinone reduction site. Using the crystal structure of the homologous FdnI subunit of Escherichia coli formate dehydrogenase-N as a model, the HydC protein was modified to examine residues thought to be involved in menaquinone binding. Variant HydABC complexes were produced in W. succinogenes, and several conserved HydC residues were identified that are essential for growth with H2 as electron donor and for quinone reduction by H2. Modification of HydC with a C-terminal Strep-tag II enabled one-step purification of the HydABC complex by Strep-Tactin affinity chromatography. The tagged HydC, separated from HydAB by isoelectric focusing, was shown to contain 1.9 mol of heme b/mol of HydC demonstrating that HydC ligates both heme b groups. The four histidine residues predicted as axial heme b ligands were individually replaced by alanine in Strep-tagged HydC. Replacement of either histidine ligand of the heme b group proximal to HydAB led to HydABC preparations that contained only one heme b group. This remaining heme b could be completely reduced by quinone supporting the view that the menaquinone reduction site is located near the distal heme b group. The results indicate that both heme b groups are involved in electron transport and that the architecture of the menaquinone reduction site near the cytoplasmic side of the membrane is similar to that proposed for E. coli FdnI.     

Isolation of a naphthaquinone with partly hydrogenated side chain from Corynebacterium diphtheriae

1. Corynebacterium diphtheriae contains relatively large amounts (6.6mumoles/g. dry wt.) of a naphthaquinone whose ultraviolet-absorption spectrum is that of a typical menaquinone (vitamin K(2)), the E(1%) (1 cm.) value corresponding with that of MK-8, but on reversed-phase paper chromatograms it runs with MK-9. 2. In the presence of Adams catalyst hydrogen uptake is 2 atoms/mol. less than that calculated for MK-8. 3. Hydrogenated samples of the Corynebacterium quinone and the hydrogenation product of authentic MK-8 ran together on reversed-phase chromatograms. 4. Infrared-absorption spectra indicated close relationship with the menaquinone series, and nuclear-magnetic-resonance measurements show that one, non-terminal, double bond of the side chain has been saturated. 5. The compound is thus designated MK-8(2H), indicating a menaquinone with eight isoprene units but only seven double bonds in the side chain.     

A C-methyltransferase involved in both ubiquinone and menaquinone biosynthesis: isolation and identification of the Escherichia coli ubiE gene.

Strains of Escherichia coli with mutations in the ubiE gene are not able to catalyze the carbon methylation reaction in the biosynthesis of ubiquinone (coenzyme Q) and menaquinone (vitamin K2), essential isoprenoid quinone components of the respiratory electron transport chain. This gene has been mapped to 86 min on the chromosome, a region where the nucleic acid sequence has recently been determined. To identify the ubiE gene, we evaluated the amino acid sequences encoded by open reading frames located in this region for the presence of sequence motifs common to a wide variety of S-adenosyl-L-methionine-dependent methyltransferases. One open reading frame in this region (o251) was found to encode these motifs, and several lines of evidence that confirm the identity of the o251 product as UbiE are presented. The transformation of a strain harboring the ubiE401 mutation with o251 on an expression plasmid restored both the growth of this strain on succinate and its ability to synthesize both ubiquinone and menaquinone. Disruption of o251 in a wild-type parental strain produced a mutant with defects in growth on succinate and in both ubiquinone and menaquinone synthesis. DNA sequence analysis of the ubiE401 allele identified a missense mutation resulting in the amino acid substitution of Asp for Gly142. E. coli strains containing either the disruption or the point mutation in ubiE accumulated 2-octaprenyl-6-methoxy-1,4-benzoquinone and demethylmenaquinone as predominant intermediates. A search of the gene databases identified ubiE homologs in Saccharomyces cerevisiae, Caenorhabditis elegans, Leishmania donovani, Lactococcus lactis, and Bacillus subtilis. In B. subtilis the ubiE homolog is likely to be required for menaquinone biosynthesis and is located within the gerC gene cluster, known to be involved in spore germination and normal vegetative growth. The data presented identify the E. coli UbiE polypeptide and provide evidence that it is required for the C methylation reactions in both ubiquinone and menaquinone biosynthesis.     

Taxonomy of alkaliphilic Bacillus strains

The DNA base compositions of 78 alkaliphilic Bacillus strains were determined. These strains were grouped as follows: DNA group A, guanine-plus-cytosine (G+C) content of 34.0 to 37.5 mol% (17 strains); DNA group B, G+C content of 38.2 to 40.8 mol% (33 strains); and DNA group C, G+C content of 42.1 to 43.9 mol% (28 strains). DNA group A includes the type strain of Bacillus alcalophilus Vedder 1934. DNA-DNA hybridization studies with DNA group A strains revealed that only one strain, strain DSM 2526, exhibited a high level of DNA homology with B. alcalophilus DSM 485T (T = type strain). Neither strain DSM 485T nor any other DNA group A strain is homologous to any of the Bacillus type strains with comparable base compositions. Six strains formed a distinct group containing three highly homologous strains and three strains exhibiting greater than 50% DNA homology.     

Comparative 16S rDNA sequence analysis of some alkaliphilic bacilli and the establishment of a sixth rRNA group within the genus Bacillus

Abstract Comparative sequence analysis of the 16S rDNA of 14 alkaliphilic or alkalitolerant, Gram-positive, aerobic, endo-spore forming bacterial strains was performed. Bacillus alcalophilus DSM 485^T and Bacillus cohnii DSM 6307^T were included to represent the two validly described alkaliphiles assigned to the genus Bacillus . The majority of isolates (8 strains) clustered with B. alcalophilus DSM 485^T forming a distinct phylogenetic group (rRNA group 6) within the radiation of the genus Bacillus and related taxa. Bacillus cohnii DSM 6307^T and two of the isolates, DSM 8719 and DSM 8723, grouped with B. fastidiosus and B. megaterium and are allocated to rRNA group 1. The remaining two strains DSM 8720 and DSM 8721 show an equidistant relationship to both groups.     

Generation of a proton potential by succinate dehydrogenase of Bacillus subtilis functioning as a fumarate reductase.

The membrane fraction of Bacillus subtilis catalyzes the reduction of fumarate to succinate by NADH. The activity is inhibited by low concentrations of 2-(heptyl)-4-hydroxyquinoline-N-oxide (HOQNO), an inhibitor of succinate: quinone reductase. In sdh or aro mutant strains, which lack succinate dehydrogenase or menaquinone, respectively, the activity of fumarate reduction by NADH was missing. In resting cells fumarate reduction required glycerol or glucose as the electron donor, which presumably supply NADH for fumarate reduction. Thus in the bacteria, fumarate reduction by NADH is catalyzed by an electron transport chain consisting of NADH dehydrogenase (NADH:menaquinone reductase), menaquinone, and succinate dehydrogenase operating in the reverse direction (menaquinol:fumarate reductase). Poor anaerobic growth of B. subtilis was observed when fumarate was present. The fumarate reduction catalyzed by the bacteria in the presence of glycerol or glucose was not inhibited by the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) or by membrane disruption, in contrast to succinate oxidation by O2. Fumarate reduction caused the uptake by the bacteria of the tetraphenyphosphonium cation (TPP+) which was released after fumarate had been consumed. TPP+ uptake was prevented by the presence of CCCP or HOQNO, but not by N,N'-dicyclohexylcarbodiimide, an inhibitor of ATP synthase. From the TPP+ uptake the electrochemical potential generated by fumarate reduction was calculated (Deltapsi = -132 mV) which was comparable to that generated by glucose oxidation with O2 (Deltapsi = -120 mV). The Deltapsi generated by fumarate reduction is suggested to stem from menaquinol:fumarate reductase functioning in a redox half-loop.     

Occurrence of menaquinone as the sole isoprenoid quinone in the photosynthetic bacterium Heliobacterium chlorum

Thin-layer chromatography, ultraviolet spectrophotometry, high-performance liquid chromatography, and mass spectroscopy revealed that Heliobacterium chlorum contained menaquinone as the sole quinone with an average amount of 0.35 mol per g dry weight of cells. A menaquinone homologue with nine isoprene units occurred as the major component.Non-standard abbreviations MK-n menaquinone with n isoprene units - TLC thin-layer chromatography - HPLC high-performance liquid chromatography     

Limited reversibility of transmembrane proton transfer assisting transmembrane electron transfer in a dihaem-containing succinate:quinone oxidoreductase

Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential (Δp), either by supporting transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by supporting transmembrane proton transfer. The first mechanism has been unequivocally demonstrated to be operational for Δp-dependent catalysis of succinate oxidation by quinone in the case of the dihaem-containing succinate:menaquinone reductase (SQR) from the Gram-positive bacterium Bacillus licheniformis. This is physiologically relevant in that it allows the transmembrane potential Δp to drive the endergonic oxidation of succinate by menaquinone by the dihaem-containing SQR of Gram-positive bacteria. In the case of a related but different respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of the ?-proteobacterium Wolinella succinogenes, evidence has been obtained that both mechanisms are combined, so as to facilitate transmembrane electron transfer by proton transfer via a both novel and essential compensatory transmembrane proton transfer pathway (“E-pathway”). Although the reduction of fumarate by menaquinol is exergonic, it is obviously not exergonic enough to support the generation of a Δp. This compensatory “E-pathway” appears to be required by all dihaem-containing QFR enzymes and results in the overall reaction being electroneutral. However, here we show that the reverse reaction, the oxidation of succinate by quinone, as catalysed by W. succinogenes QFR, is not electroneutral. The implications for transmembrane proton transfer via the E-pathway are discussed.     

Menaquinone-6 and thermoplasmaquinone-6 in Wolinella succinogenes

Abstract The respiratory quinone composition of Wolinella succinogenes was investigated. Unsaturated menaquinones with six isoprene units (2-methyl-3-hexaprenyl-1,4-naphthoquinone) was found to be the major isoprenoid quinone. Substantial levels of a methyl-substituted menaquinone was also present. Mass spectrometry and proton nuclear magnetic resonance spectrometry indicated the methyl-substituted quinone corresponded to 2-, [5 or 8]- dimethyl-3-hexaprenyl-1,4-naphthoquinone.     

Properties of the reaction center of the thermophilic purple photosynthetic bacterium Chromatium tepidum

Reaction centers were purified from the thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum. The reaction center consists of four polypeptides L, M, H and C, whose apparent molecular masses were determined to be 25, 30, 34 and 44 kDa, respectively, by polyacrylamide gel electrophoresis. The heaviest peptide corresponds to tightly bound cytochrome. The tightly bound cytochrome c contains two types of heme, high-potential c-556 and low-potential c-553. The low-potential heme is able to be photooxidized at 77 K. The reaction center exhibits laser-flash-induced absorption changes and circular dichroism spectra similar to those observed in other purple photosynthetic bacteria. Whole cells contain both ubiquinone and menaquinone. Reaction centers contain only a single active quinone; chemical analysis showed this to be menaquinone. Reaction center complexes without the tightly bound cytochrome were also prepared. The near-infrared pigment absorption bands are red-shifted in reaction centers with cytochrome compared to those without cytochrome.     

Purification and characterization of succinate:menaquinone oxidoreductase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Succinate:menaquinone oxidoreductase from Corynebacterium glutamicum, a high-G+C, Gram-positive bacterium, was purified to homogeneity. The enzyme contained two heme B molecules and three polypeptides with apparent molecular masses of 67, 29 and 23 kDa, which corresponded to SdhA (flavoprotein), SdhB (iron–sulfur protein), and SdhC (membrane anchor protein), respectively. In non-denaturating polyacrylamide gel electrophoresis, the enzyme migrated as a single band with an apparent molecular mass of 410 kDa, suggesting that it existed as a trimer. The succinate dehydrogenase activity assayed using 2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone and 2,6-dichloroindophenol as the electron acceptor was inhibited by 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), and the Dixon plots were biphasic. In contrast, the succinate dehydrogenase activity assayed using phenazine methosulfate and 2,6-dichloroindophenol was inhibited by p-benzoquinone and not by HQNO. These findings suggested that the C. glutamicum succinate:menaquinone oxidoreductase had two quinone binding sites. In the phylogenetic tree of SdhA, Corynebacterium species do not belong to the high-G+C group, which includes Mycobacterium tuberculosis and Streptomyces coelicolor, but are rather close to the group of low-G+C, Gram-positive bacteria such as Bacillus subtilis. This situation may have arisen due to the horizontal gene transfer.     

Fumarate reduction systems in members of the family Rhodospirillaceae with different quinone types

Nineteen established and one undesignated species of the Rhodospirillaceae were examined for fumarate reduction in connection with their quinone systems. The fumarate reductase activity with reduced methyl viologen (MVH) or FMNH₂ as electron donor was found in membrane (chromatophore) preparations from phototrophically grown cells of all species containing menaquinone (MK) and/or rhodoquinone. The species having ubiquinone as the sole quinone contained no fumarate reductase activity, except some Rhodobacter species showing the FMNH₂-dependent activity. The MVH-fumarate reductase activity of the MK-type species was not inhibited by Triton X-100 or acetone treatment, suggesting the presence of a fumarate reductase reacting directly with MVH, while such an enzyme was absent in the MK-lacking strains, with few exceptions. The FMNH₂-fumarate reduction system was abolished by a detergent or acetone extraction in all bacteria but differed much among species with different quinone types as to the response to respiratory inhibitors. These differences in fumarate-reducing properties and quinone systems among the phototrophic bacteria are discussed from evolutionary and taxonomic viewpoints.Non-standard abbreviations RQ rhodoquinone - MK menaquinone - MVH reduced methyl viologen - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - TTFA 2-thenoyltrifluoroacetone     

Properties of the reaction center of the thermophilic purple photosynthetic bacterium Chromatium tepidum

Reaction centers were purified from the thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum. The reaction center consists of four polypeptides L, M, H and C, whose apparent molecular masses were determined to be 25, 30, 34 and 44 kDa, respectively, by polyacrylamide gel electrophoresis. The heaviest peptide corresponds to tightly bound cytochrome. The tightly bound cytochrome c contains two types of heme, high-potential c-556 and low-potential c-553. The low-potential heme is able to be photooxidized at 77 K. The reaction center exhibits laser-flash-induced absorption changes and circular dichroism spectra similar to those observed in other purple photosynthetic bacteria. Whole cells contain both ubiquinone and menaquinone. Reaction centers contain only a single active quinone; chemical analysis showed this to be menaquinone. Reaction center complexes without the tightly bound cytochrome were also prepared. The near-infrared pigment absorption bands are red-shifted in reaction centers with cytochrome compared to those without cytochrome.     

Polyamine metabolism in an obligately alkalophilic Bacillus alcalophilus that grows at pH 11.0

Bacillus alcalophilus, an obligately alkalophilic bacterium that grows at pH 11.0, has an intracellular pH of 9.5 or less. Unlike all other living organisms, polyamines (putrescine, spermidine and spermine) in B. alcalophilus, if present, will be largely unprotonated. HPLC analysis indicated that spermidine is the major polyamine in B. alcalophilus, accounting for more than 90% of total polyamines, and the level of spermidine varies during growth. Ornithine decarboxylase activity was not detectable in B. alcalophilus under all conditions examined. When [3H]arginine was added to the culture medium, the radioactivity can be recovered from polyamine pool; the distribution is 3% for putrescine, 94% for spermidine, and 3% for spermine, suggesting the presence of arginine pathway for polyamine biosynthesis. The polyamine transport system in B. alcalphilus appears to be Na+-dependent and is highly sensitive to the inhibition of gramicidin S and valinomycin.     

Sequence analysis and specificity of distinct types of menaquinone methyltransferases indicate the widespread potential of methylmenaquinone production in bacteria and archaea

Menaquinone (MK) serves as an essential membranous redox mediator in various electron transport chains of aerobic and anaerobic respiration. In addition, the composition of the quinone/quinol pool has been widely used as a biomarker in microbial taxonomy. The HemN-like class C radical SAM methyltransferases (RSMTs) MqnK, MenK and MenK2 have recently been shown to facilitate specific menaquinone methylation reactions at position C-8 (MqnK/MenK) or C-7 (MenK2) to synthesize 8-methylmenaquinone, 7-methylmenaquinone and 7,8-dimethylmenaquinone. However, the vast majority of protein sequences from the MqnK/MenK/MenK2 family belong to organisms, whose capacity to produce methylated menaquinones has not been investigated biochemically. Here, representative putative menK and menK2 genes from Collinsella tanakaei and Ferrimonas marina were individually expressed in Escherichia coli (wild-type or ubiE deletion mutant) and the corresponding cells were found to produce methylated derivatives of the endogenous MK and 2-demethylmenaquinone. Cluster and phylogenetic analyses of 828 (methyl)menaquinone methyltransferase sequences revealed signature motifs that allowed to discriminate enzymes of the MqnK/MenK/MenK2 family from other radical SAM enzymes and to identify C-7-specific menaquinone methyltransferases of the MenK2 subfamily. This study will help to predict the methylation status of the quinone/quinol pool of a microbial species (or even a microbial community) from its (meta)genome and contribute to the future design of microbial quinone/quinol pools in a Synthetic Biology approach.     

High-performance liquid chromatographic analysis of demethylmenaquinone and menaquinone mixtures from bacteria

Demethylmenaquinone and menaquinone mixtures from some species of enterobacteria were analysed by reverse-phase partition high-performance liquid chromatography. This method allowed clear separation and quantitative determination of these quinone components.     

High-performance liquid chromatographic analysis of demethylmenaquinone and menaquinone mixtures from bacteria

Demethylmenaquinone and menaquinone mixtures from some species of enterobacteria were analysed by reverse-phase partition high-performance liquid chromatography. This method allowed clear separation and quantitative determination of these quinone components.