Alternative sites for proton entry from the cytoplasm to the quinone binding site in Escherichia coli succinate dehydrogenase

Escherichia coli succinate dehydrogenase (Sdh) belongs to the highly conserved complex II family of enzymes that reduce ubiquinone. These enzymes do not generate a protonmotive force during catalysis and are electroneutral. Because of its electroneutrality, the quinone reduction reaction must consume cytoplasmic protons which are released stoichiometrically during succinate oxidation. The X-ray crystal structure of E. coli Sdh shows that residues SdhB (G227), SdhC (D95), and SdhC (E101) are located at or near the entrance of a water channel that has been proposed to function as a proton wire connecting the cytoplasm to the quinone binding site. However, the pig and chicken Sdh enzymes show an alternative entrance to the water channel via the conserved SdhD (Q78) residue. In this study, site-directed mutants of these four residues were created and characterized by in vivo growth assays, in vitro activity assays, and electron paramagnetic resonance spectroscopy. We show that the observed water channel in the E. coli Sdh structure is the functional proton wire in vivo, while in vitro results indicate an alternative entrance for protons. In silico examination of the E. coli Sdh reveals a possible H-bonding network leading from the cytoplasm to the quinone binding site that involves SdhD (D15). On the basis of these results we propose an alternative proton pathway in E. coli Sdh that might be functional only in vitro.     

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.     

Out of plane distortions of the heme b of Escherichia coli succinate dehydrogenase

The role of the heme b in Escherichia coli succinate dehydrogenase is highly ambiguous and its role in catalysis is questionable. To examine whether heme reduction is an essential step of the catalytic mechanism, we generated a series of site-directed mutations around the heme binding pocket, creating a library of variants with a stepwise decrease in the midpoint potential of the heme from the wild-type value of +20 mV down to -80 mV. This difference in midpoint potential is enough to alter the reactivity of the heme towards succinate and thus its redox state under turnover conditions. Our results show both the steady state succinate oxidase and fumarate reductase catalytic activity of the enzyme are not a function of the redox potential of the heme. As well, lower heme potential did not cause an increase in the rate of superoxide production both in vitro and in vivo. The electron paramagnetic resonance (EPR) spectrum of the heme in the wild-type enzyme is a combination of two distinct signals. We link EPR spectra to structure, showing that one of the signals likely arises from an out-of-plane distortion of the heme, a saddled conformation, while the second signal originates from a more planar orientation of the porphyrin ring.     

The iron-sulfur clusters in Escherichia coli succinate dehydrogenase direct electron flow

Succinate dehydrogenase is an indispensable enzyme involved in the Krebs cycle as well as energy coupling in the mitochondria and certain prokaryotes. During catalysis, succinate oxidation is coupled to ubiquinone reduction by an electron transfer relay comprising a flavin adenine dinucleotide cofactor, three iron-sulfur clusters, and possibly a heme b556. At the heart of the electron transport chain is a [4Fe-4S] cluster with a low midpoint potential that acts as an energy barrier against electron transfer. Hydrophobic residues around the [4Fe-4S] cluster were mutated to determine their effects on the midpoint potential of the cluster as well as electron transfer rates. SdhB-I150E and SdhB-I150H mutants lowered the midpoint potential of this cluster; surprisingly, the His variant had a lower midpoint potential than the Glu mutant. Mutation of SdhB-Leu-220 to Ser did not alter the redox behavior of the cluster but instead lowered the midpoint potential of the [3Fe-4S] cluster. To correlate the midpoint potential changes in these mutants to enzyme function, we monitored aerobic growth in succinate minimal medium, anaerobic growth in glycerol-fumarate minimal medium, non-physiological and physiological enzyme activities, and heme reduction. It was discovered that a decrease in midpoint potential of either the [4Fe-4S] cluster or the [3Fe-4S] cluster is accompanied by a decrease in the rate of enzyme turnover. We hypothesize that this occurs because the midpoint potentials of the [Fe-S] clusters in the native enzyme are poised such that direction of electron transfer from succinate to ubiquinone is favored.     

The binding site for oxaloacetate on succinate dehydrogenase

Oxaloacetate, a competitive inhibitor of succinate dehydrogenase, bound with a sulfhydryl group of the enzyme to abolish the enzymic activity. Subsequently a thiosemiacetal was apparently formed to render the inhibition practically irreversible. The dehydrogenase, after taking up 25 silver equivalents per flavin, bound little oxaloacetate.     

The Escherichia coli cytochrome b556 gene, cybA, is assignable as sdhC in the succinate dehydrogenase gene cluster

Abstract The cytochrome b556 -deficient mutant Escherichia coli K12 strain TK3D11 [7] could not grow with succinate as the sole carbon source, but could grow well on dl -lactate. This finding suggested that cytochrome b556 is primarily responsible for oxidative metabolism and utilization of succinate. 24 Amino acid residues at the amino-terminal of purified cytochrome b556 were determined. This sequence coincided completely with amino acid residues 4 to 27, predicted from the DNA sequence of the sdhC gene, one of the unassigned open reading frames of the sdh gene cluster recently reported by Wood et al. [16]. Based on these and other results, we concluded that cybA , the gene for cytochrome b556 , is assignable as sdhC .     

The low-spin heme site of cytochrome o from Escherichia coli is promiscuous with respect to heme type.

Cytochrome o of Escherichia coli is able to incorporate two different structures of heme, either heme B (protoheme) or heme O, in its low-spin heme site. In contrast, the heme of the binuclear O2 reduction site is invariably heme O. Heme O is a newly discovered heme that is related to heme A, but with the formyl group of the latter replaced by methyl. Enzyme isolated from wild type E. coli has predominantly heme B in the low-spin site, whereas enzyme isolated from various overexpressing strains contains both types of enzyme in different proportions. In some strains, 70% of the enzyme has heme O in the low-spin site. Despite this variation in the structure of one of the prosthetic groups, the enzymatic activity and polypeptide composition of the enzyme remain virtually constant. EPR and activity data both indicate that heme B and heme O occupy the same low-spin heme site in the enzyme. With heme O in this site, the alpha-absorption band is narrower and further to the blue, and the Em,7 is lower, than when there is heme B in the site. In contrast to previous proposals, we show here that the enzyme does not exhibit significant spectral interactions between the hemes. The structural heterogeneity of the low-spin heme accounts for the variation in the optical spectra and redox properties of the enzyme as isolated from different strains of E. coli.     

Menaquinone as well as ubiquinone as a bound quinone crucial for catalytic activity and intramolecular electron transfer in Escherichia coli membrane-bound glucose dehydrogenase

Escherichia coli membrane-bound glucose dehydrogenase (mGDH), which is one of quinoproteins containing pyrroloquinoline quinone (PQQ) as a coenzyme, is a good model for elucidating the function of bound quinone inside primary dehydrogenases in respiratory chains. Enzymatic analysis of purified mGDH from cells defective in synthesis of ubiquinone (UQ) and/or menaquinone (MQ) revealed that Q-free mGDH has very low levels of activity of glucose dehydrogenase and UQ2 reductase compared with those of UQ-bearing mGDH, and both activities were significantly increased by reconstitution with UQ1. On the other hand, MQ-bearing mGDH retains both catalytic abilities at the same levels as those of UQ-bearing mGDH. A radiolytically generated hydrated electron reacted with the bound MQ to form a semiquinone anion radical with an absorption maximum at 400 nm. Subsequently, decay of the absorbance at 400 nm was accompanied by an increase in the absorbance at 380 nm with a first order rate constant of 5.7 x 10(3) s(-1). This indicated that an intramolecular electron transfer from the bound MQ to the PQQ occurred. EPR analysis revealed that characteristics of the semiquinone radical of bound MQ are similar to those of the semiquinone radical of bound UQ and indicated an electron flow from PQQ to MQ as in the case of UQ. Taken together, the results suggest that MQ is incorporated into the same pocket as that for UQ to perform a function almost equivalent to that of UQ and that bound quinone is involved at least partially in the catalytic reaction and primarily in the intramolecular electron transfer of mGDH.     

The DNA binding activity of MutL is required for methyl-directed mismatch repair in Escherichia coli

The DNA binding properties of the mismatch repair protein MutL and their importance in the repair process have been controversial for nearly two decades. We have addressed this issue using a point mutant of MutL (MutL-R266E). The biochemical and genetic data suggest that DNA binding by MutL is required for dam methylation-directed mismatch repair. We demonstrate that purified MutL-R266E retains wild-type biochemical properties that do not depend on DNA binding, such as basal ATP hydrolysis in the absence of DNA and the ability to interact with other mismatch repair proteins. However, purified MutL-R266E binds DNA poorly in vitro as compared with MutL, and consistent with this observation, its DNA-dependent biochemical activities, like DNA-stimulated ATP hydrolysis and helicase II stimulation, are severely compromised. In addition, there is a modest effect on stimulation of MutH-catalyzed nicking. Finally, genetic assays show that MutL-R266E has a strong mutator phenotype, demonstrating that the mutant is unable to function in dam methylation-directed mismatch repair in vivo.     

An Escherichia coli mutant defective in the NAD-dependent succinate semialdehyde dehydrogenase

Escherichia coli mutants, unable to grown on 4-hydroxyphenylacetate, have been isolated and found to be defective in the NAD-dependent succinate semialdehyde dehydrogenase. When the mutants are grown with 4-aminobutyrate as sole nitrogen source an NAD-dependent succinate semialdehyde dehydrogenase seen in the parental strain is absent but, as in the parental strain, an NADP-dependent enzyme is induced. Growth of the mutants is inhibited by 4-hydroxyphenylacetate due to the accumulation of succinate semialdehyde. The mutants are more sensitive to inhibition by exogenous succinate semialdehyde than is the parental strain. Secondary mutants able to grow in the presence of 4-hydroxyphenylacetate but still unable to use it as sole carbon source were defective in early steps of 4-hydroxyphenylacetate catabolism and so did not form succinate semialdehyde from 4-hydroxyphenylacetate. The gene encoding the NAD-dependent succinate semialdehyde dehydrogenase of Escherichia coli K-12 was located at min 34.1 on the genetic map.     

Effect of heme binding on the structure and stability of Escherichia coli apocytochrome b562

The structure and stability of apocytochrome b562 were explored using absorption and circular dichroism spectroscopic methods. The polypeptide chain retains a well-defined structure when the prosthetic heme group is removed from cytochrome b562. Circular dichroism measurements estimate 60% helicity for apocytochrome b562, compared with 80% helicity found in holocytochrome b562. At low pH, apocytochrome b562 displays a midpoint pH of 2.9, while ferricytochrome b562 displays a midpoint pH of 2.3. The unfolding of the apoprotein by urea and heat can be well approximated by the two-state transition model. The stability of apocytochrome b562 is significantly reduced from that of the holoprotein. The free energy of stabilization (delta G degrees) and the midpoint transition temperature (Tm) for apocytochrome b562 are found to be 3.2 +/- 0.5 kcal/mol and 52.3 +/- 0.9 degrees C, respectively, compared with 6.6 +/- 0.5 kcal/mol and 67.2 +/- 0.5 degrees C for ferricytochrome b562. The smaller heat capacity change upon unfolding of apocytochrome b562 than that of ferricytochrome b562, estimated from the thermodynamic parameters, indicates that apocytochrome b562 possesses a smaller hydrophobic core than holocytochrome b562. Size-exclusion chromatography studies indicate that the apoprotein is slightly more extended in molecular dimension than ferricytochrome b562. The data suggest that apocytochrome b562 resembles a "molten globule" or a "collapsed form" of the holoprotein, in which secondary structure formation is largely complete while the global folding is either only partially complete or dynamically expanded.     

The epsilon subunit of Escherichia coli coupling factor 1 is required for its binding to the cytoplasmic membrane.

The coupling factor, F1-ATPase of Escherichia coli (ECF1) contains five different subunits, alpha, beta, gamma, delta, and epsilon. Properties of delta-deficient ECF1 have previously been described. F1-ATPase containing only the alpha, beta, and gamma subunits was prepared from E. coli by passage of delta-deficient ECF1 through an affinity column containing immobilized antibodies to the epsilon subunit. The delta, epsilon-deficient enzyme has normal ATPase activity but cannot bind to ECF1-depleted membrane vesicles. Both the delta and epsilon subunits are required for the binding of delta, epsilon-deficient ECF1 to membranes and the restoration of oxidative phosphorylation. Either delta or epsilon will bind to the deficient enzyme to form a four-subunit complex. Neither four-subunit enzyme binds to depleted membranes. The epsilon subunit, does, however, slightly improve the binding affinity between delta and delta-deficient enzyme suggesting a possible interaction between the two subunits. Neither subunit binds to trypsin-treated ECF1, which contains only the alpha and beta subunits. A role for gamma in the binding of epsilon to F1 is suggested. epsilon does not bind to ECF1-depleted membranes. Therefore, the in vitro reconstitution of depleted membranes requires an initial complex formation between epsilon and the rest of ECF1 prior to membrane attachment. Reconstitution experiments indicate that only one epsilon is required per functional ECF1 molecule.     

SdhE is a conserved protein required for flavinylation of succinate dehydrogenase in bacteria

Conserved uncharacterized genes account for ~30% of genes in both eukaryotic and bacterial genomes and are predicted to encode what are often termed "conserved hypothetical proteins." Many of these proteins have a wide phylogenetic distribution and might play important roles in conserved cellular pathways. Using the bacterium Serratia as a model system, we have investigated two conserved uncharacterized proteins, YgfY (a DUF339 protein, renamed SdhE; succinate dehydrogenase protein E) and YgfX (a DUF1434 protein). SdhE was required for growth on succinate as a sole carbon source and for the function, but not stability, of succinate dehydrogenase, an important component of the electron transport chain and the tricarboxylic acid cycle. SdhE interacted with the flavoprotein SdhA, directly bound the flavin adenine dinucleotide co-factor, and was required for the flavinylation of SdhA. This is the first demonstration of a protein required for FAD incorporation in bacteria. Furthermore, the loss of SdhE was highly pleiotropic, suggesting that SdhE might flavinylate other flavoproteins. Our findings are of wide importance to central metabolism because SdhE homologues are present in α-, β-, and γ-proteobacteria and multiple eukaryotes, including humans and yeast.     

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.     

Glyceraldehyde-3-phosphate dehydrogenase is required for efficient repair of cytotoxic DNA lesions in Escherichia coli

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with diverse biological functions in human cells. In bacteria, moonlighting GAPDH functions have only been described for the secreted protein in pathogens or probiotics. At the intracellular level, we previously reported the interaction of Escherichia coli GAPDH with phosphoglycolate phosphatase, a protein involved in the metabolism of the DNA repair product 2-phosphoglycolate, thus suggesting a putative role of GAPDH in DNA repair processes. Here, we provide evidence that GAPDH is required for the efficient repair of DNA lesions in E. coli. We show that GAPDH-deficient cells are more sensitive to bleomycin or methyl methanesulfonate. In cells challenged with these genotoxic agents, GAPDH deficiency results in reduced cell viability and filamentous growth. In addition, the gapA knockout mutant accumulates a higher number of spontaneous abasic sites and displays higher spontaneous mutation frequencies than the parental strain. Pull-down experiments in different genetic backgrounds show interaction between GAPDH and enzymes of the base excision repair pathway, namely the AP-endonuclease Endo IV and uracil DNA glycosylase. This finding suggests that GAPDH is a component of a protein complex dedicated to the maintenance of genomic DNA integrity. Our results also show interaction of GAPDH with the single-stranded DNA binding protein. This interaction may recruit GAPDH to the repair sites and implicates GAPDH in DNA repair pathways activated by profuse DNA damage, such as homologous recombination or the SOS response.