Biomedical subjects

Marianne Schiffer

Publications and source records attributed to Marianne Schiffer.

13 recordsLinked to original sources

PubMed2006

Thermodynamic characterization of triheme cytochrome PpcA from Geobacter sulfurreducens: evidence for a role played in e-/H+ energy transduction.

The facultative aerobic bacterium Geobacter sulfurreducens produces a small periplasmic c-type triheme cytochrome with 71 residues (PpcA) under anaerobic growth conditions, which is involved in the iron respiration. The thermodynamic properties of the PpcA redox centers and of a protonatable center were determined using NMR and visible spectroscopy techniques. The redox centers have negative and different reduction potentials (-162, -143, and -133 mV for heme I, III, and IV, respectively, for the fully reduced and protonated protein), which are modulated by redox interactions among the hemes (covering a range from 10 to 36 mV) and by redox-Bohr interactions (up to -62 mV) between the hemes and a protonatable center located in the proximity of heme IV. All the interactions between the four centers are dominated by electrostatic effects. The microscopic reduction potential of heme III is the one most affected by the oxidation of the other hemes, whereas heme IV is the most affected by the protonation state of the molecule. The thermodynamic properties of PpcA showed that pH strongly modulates the redox behavior of the individual heme groups. A preferred electron transfer pathway at physiologic pH is defined, showing that PpcA has the necessary thermodynamic properties to perform e-/H+ energy transduction, contributing to a H+ electrochemical potential gradient across the periplasmic membrane that drives ATP synthesis. PpcA is 46% identical in sequence to and shares a high degree of structural similarity with a periplasmic triheme cytochrome c7 isolated from Desulfuromonas acetoxidans, a bacterium closely related to the Geobacteracea family. However, the results obtained for PpcA are quite different from those published for D. acetoxidans c7, and the physiological consequences of these differences are discussed.

Cytochromes↗

PubMed2006

Primary structure-based function characterization of BRCT domain replicates in BRCA1.

BRCA1 is a large protein that exhibits a multiplicity of functions in its apparent role in DNA repair. Certain mutations of BRCA1 are known to have exceptionally high penetrance with respect to familial breast and ovarian cancers. The structures of the N-terminus and C-terminus of the protein have been determined. The C-terminus unit consists of two alpha-beta-alpha domains designated BRCT. We predicated two homologous BRCT regions in the BRCA1 internal region, and subsequently produced and purified these protein domains. Both recombinant domains show significant self-association capabilities as well as a preferential tendency to interact with each other. These results suggest a possible regulatory mechanism for BRCA1 function. We have demonstrated p53-binding activity by an additional region, and confirmed previous results showing that two regions of BRCA1 protein bind p53 in vitro. Based on sequence analysis, we predict five p53-binding sites. Our comparison of binding by wild-type and mutant domains indicates the sequence specificity of BRCA1-p53 interaction.

Amino Acid Sequence↗

PubMed2006

Characterization of a c-type heme-containing PAS sensor domain from Geobacter sulfurreducens representing a novel family of periplasmic sensors in Geobacteraceae and other bacteria.

Geobacter sulfurreducens encodes one of the largest numbers of proteins annotated as parts of the two-component signal transduction and/or chemotaxis pathways. Ten of these signal transducers have homologous periplasmic sensor domains that contain the sequence signature for c-type hemes. One such sensor domain encoded by gene GSU0303 was isolated and characterized. The protein was expressed in Escherichia coli and was isolated as two colored species (green and red). The green species is a monomer of the sensor domain with a five-coordinated high-spin heme and the red species is probably a noncovalent dimer of the sensor domain which might have an uncharacterized ligand bound to the dimer. The UV-VIS spectrum of the green species indicates that it has a c'-type heme, but its structure is predicted to be homologous to CitA, a periplasmic PAS domain that does not contain heme. The GSU0303 sensor domain represents a previously unreported family of PAS-type periplasmic sensor domains that contain c-type hemes; these proteins could be part of an important mechanism for sensing redox potential or small ligands in the periplasm. Homologs to the sensor domains we identified in G. sulfurreducens are observed in various bacteria although they occur in larger numbers in the Geobacteraceae.

Amino Acid Sequence↗

PubMed2005

Heterologous expression of dodecaheme "nanowire" cytochromes c from Geobacter sulfurreducens.

Multiheme cytochromes c are difficult to produce in heterologous systems. The genome of delta-proteobacterium Geobacter sulfurreducens contains more than a hundred genes coding for c-type cytochromes. Among those are two dodecaheme cytochromes c representing a new class of multiheme cytochromes, whose putative structure is a one-dimensional array of small highly homologous domains that contain three hemes and are covalently bound by short linkers. They are likely to form "nanowires" that are part of the electron transfer chain. We cloned the genes coding for the two cytochromes into a vector we developed for ligation-independent cloning of proteins targeted to the Escherichia coli periplasmic space. We expressed the proteins in E. coli co-transformed with a plasmid harboring the cytochrome c maturation genes. Expression levels were optimized by varying IPTG concentrations used for induction. Although both proteins appeared insoluble or strongly associated with cell membranes, they were solubilized using 0.5 M sodium chloride which was more selective than conventional solubilizing agents, such as HEGA-10 or beta-octylglucoside. The solubilized proteins were dialyzed and purified by cation exchange chromatography followed by gel filtration. Mass-spectrometry analysis confirmed that both purified proteins contained the complete set of covalently attached hemes, 12 per molecule. Their visible spectra were typical of c-type cytochromes. Both proteins were successfully crystallized.

Amino Acid Sequence↗

PubMed2005

A novel Geobacteraceae-specific outer membrane protein J (OmpJ) is essential for electron transport to Fe(III) and Mn(IV) oxides in Geobacter sulfurreducens.

BACKGROUND: Metal reduction is thought to take place at or near the bacterial outer membrane and, thus, outer membrane proteins in the model dissimilatory metal-reducing organism Geobacter sulfurreducens are of interest to understand the mechanisms of Fe(III) reduction in the Geobacter species that are the predominant Fe(III) reducers in many environments. Previous studies have implicated periplasmic and outer membrane cytochromes in electron transfer to metals. Here we show that the most abundant outer membrane protein of G. sulfurreducens, OmpJ, is not a cytochrome yet it is required for metal respiration. RESULTS: When outer membrane proteins of G. sulfurreducens were separated via SDS-PAGE, one protein, designated OmpJ (outer membrane protein J), was particularly abundant. The encoding gene, which was identified from mass spectrometry analysis of peptide fragments, is present in other Geobacteraceae, but not in organisms outside this family. The predicted localization and structure of the OmpJ protein suggested that it was a porin. Deletion of the ompJ gene in G. sulfurreducens produced a strain that grew as well as the wild-type strain with fumarate as the electron acceptor but could not grow with metals, such as soluble or insoluble Fe(III) and insoluble Mn(IV) oxide, as the electron acceptor. The heme c content in the mutant strain was ca. 50% of the wild-type and there was a widespread loss of multiple cytochromes from soluble and membrane fractions. Transmission electron microscopy analyses of mutant cells revealed an unusually enlarged periplasm, which is likely to trigger extracytoplasmic stress response mechanisms leading to the degradation of periplasmic and/or outer membrane proteins, such as cytochromes, required for metal reduction. Thus, the loss of the capacity for extracellular electron transport in the mutant could be due to the missing c-type cytochromes, or some more direct, but as yet unknown, role of OmpJ in metal reduction. CONCLUSION: OmpJ is a putative porin found in the outer membrane of the model metal reducer G. sulfurreducens that is required for respiration of extracellular electron acceptors such as soluble and insoluble metals. The effect of OmpJ in extracellular electron transfer is indirect, as OmpJ is required to keep the integrity of the periplasmic space necessary for proper folding and functioning of periplasmic and outer membrane electron transport components. The exclusive presence of ompJ in members of the Geobacteraceae family as well as its role in metal reduction suggest that the ompJ sequence may be useful in tracking the growth or activity of Geobacteraceae in sedimentary environments.

Amino Acid Sequence↗

PubMed2005

Heterologous expression of hexaheme fragments of a multidomain cytochrome from Geobacter sulfurreducens representing a novel class of cytochromes c.

Multiheme cytochromes c are of great interest for researchers for a variety of reasons but difficult to obtain in quantities sufficient for the majority of studies. The genome of delta-proteobacterium Geobacter sulfurreducens contains more than a hundred genes coding for c-type cytochromes. Three of them represent a new class of multiheme cytochromes characterized by a mixed type of heme coordination and multidomain organization. We cloned and expressed in Escherichia coli three hexaheme fragments corresponding to two-domain fragments of one such protein containing 12 heme binding motifs and believed to consist of four triheme domains. Despite high sequence similarity among the fragments, expression levels varied significantly. Expression was optimized either by host strain variation or by reducing the rate of apoprotein synthesis. All three fragments were purified by cation exchange followed by gel filtration and were shown to contain six covalently attached hemes as confirmed by mass spectrometry. Their visible spectra are typical of c-type cytochromes. One of the fragments was crystallized and its preliminary X-ray structure shows two separate domains.

Amino Acid Sequence↗

PubMed2004

Redox characterization of Geobacter sulfurreducens cytochrome c7: physiological relevance of the conserved residue F15 probed by site-specific mutagenesis.

The complete genome sequence of the delta-proteobacterium Geobacter sulfurreducens reveals a large abundance of multiheme cytochromes. Cytochrome c(7), isolated from this metal ion-reducing bacterium, is a triheme periplasmic electron-transfer protein with M(r) 9.6 kDa. This protein is involved in metal ion-reducing pathways and shares 56% sequence identity with a triheme cytochrome isolated from the closely related delta-proteobacterium Desulfuromonas acetoxidans (Dac(7)). In this work, two-dimensional NMR was used to monitor the heme core and the general folding in solution of the G. sulfurreducens triheme cytochrome c(7) (PpcA). NMR signals obtained for the three hemes of PpcA at different stages of oxidation were cross-assigned to the crystal structure [Pokkuluri, P. R., Londer, Y. Y., Duke, N. E. C., Long, W. C., and Schiffer, M. (2004) Biochemistry 43, 849-859] using the complete network of chemical exchange connectivities, and the order in which each heme becomes oxidized was determined at pH 6.0 and 8.2. Redox titrations followed by visible spectroscopy were also performed in order to monitor the macroscopic redox behavior of PpcA. The results obtained showed that PpcA and Dac(7) have different redox properties: (i) the order in which each heme becomes oxidized is different; (ii) the reduction potentials of the heme groups and the global redox behavior of PpcA are pH dependent (redox-Bohr effect) in the physiological pH range, which is not observed with Dac(7). The differences observed in the redox behavior of PpcA and Dac(7) may account for the different functions of these proteins and constitute an excellent example of how homologous proteins can perform different physiological functions. The redox titrations followed by visible spectroscopy of PpcA and two mutants of the conserved residue F15 (PpcAF15Y and PpcAF15W) lead to the conclusion that F15 modulates the redox behavior of PpcA, thus having an important physiological role.

Amino Acid Substitution↗

PubMed2004

Temperature and cryoprotectant influence secondary quinone binding position in bacterial reaction centers.

We have determined the first de novo position of the secondary quinone QB in the Rhodobacter sphaeroides reaction center (RC) using phases derived by the single wavelength anomalous dispersion method from crystals with selenomethionine substitution. We found that in frozen RC crystals, QB occupies primarily the proximal binding site. In contrast, our room temperature structure showed that QB is largely in the distal position. Both data sets were collected in dark-adapted conditions. We estimate that the occupancy of the QB site is 80% with a proximal: distal ratio of 4:1 in frozen RC crystals. We could not separate the effect of freezing from the effect of the cryoprotectants ethylene glycol or glycerol. These results could have far-reaching implications in structure/function studies of electron transfer in the acceptor quinone complex because the above are the most commonly used cryoprotectants in spectroscopic experiments.

Benzoquinones↗

PubMed2004

Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer.

The structure of a novel c(7)-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 A resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c(7)-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c(7) molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.

Amino Acid Sequence↗

PubMed2004

Family of cytochrome c7-type proteins from Geobacter sulfurreducens: structure of one cytochrome c7 at 1.45 A resolution.

The structure of a cytochrome c(7) (PpcA) from Geobacter sulfurreducens was determined by X-ray diffraction at 1.45 A resolution; the R factor is 18.2%. The protein contains a three-heme core that is surrounded by 71 amino acid residues. An unusual feature of this cytochrome is that it has 17 lysine residues, but only nine hydrophobic residues that are larger than alanine. The details of the structure are described and compared with those of cytochrome c(7) from Desulfuromonas acetoxidans and with cytochromes c(3). The two cytochrome c(7) molecules have sequences that are 46% identical, but the arrangements of the hemes in the two structures differ; the rms deviation of all alpha-carbons is 2.5 A. These cytochromes can reduce various metal ions. The reduction site of the chromate ion in D. acetoxidans is occupied by a sulfate ion in the crystal structure of PpcA. We identified four additional homologues of cytochrome c(7) in the G. sulfurreducens genome and three polymers of c(7)-type domains. Of the polymers, two have four repeats and one has nine repeats. On the basis of sequence alignments, one of the hemes in each of the cytochrome c(7)-type domains does not have the bis-histidine coordination. The packing of the molecules in the crystal structure of PpcA suggests that the polymers have an elongated conformation and might form a "nanowire".

Amino Acid Sequence↗

PubMed2003

Inter- and intraspecific variation in excited-state triplet energy transfer rates in reaction centers of photosynthetic bacteria.

In protein-cofactor reaction center (RC) complexes of purple photosynthetic bacteria, the major role of the bound carotenoid (C) is to quench the triplet state formed on the primary electron donor (P) before its sensitization of the excited singlet state of molecular oxygen from its ground triplet state. This triplet energy is transferred from P to C via the bacteriochlorophyll monomer B(B). Using time-resolved electron paramagnetic resonance (TREPR), we have examined the temperature dependence of the rates of this triplet energy transfer reaction in the RC of three wild-type species of purple nonsulfur bacteria. Species-specific differences in the rate of transfer were observed. Wild-type Rhodobacter capsulatus RCs were less efficient at the triplet transfer reaction than Rhodobacter sphaeroides RCs, but were more efficient than Rhodospirillum rubrum RCs. In addition, RCs from three mutant strains of R. capsulatus carrying substitutions of amino acids near P and B(B) were examined. Two of the mutant RCs showed decreased triplet transfer rates compared with wild-type RCs, whereas one of the mutant RCs demonstrated a slight increase in triplet transfer rate at low temperatures. The results show that site-specific changes within the RC of R. capsulatus can mimic interspecies differences in the rates of triplet energy transfer. This application of TREPR was instrumental in defining critical energetic and coupling factors that dictate the efficiency of this photoprotective process.

Electron Spin Resonance Spectroscopy↗

PubMed2002

Production and preliminary characterization of a recombinant triheme cytochrome c(7) from Geobacter sulfurreducens in Escherichia coli.

Multiheme cytochromes c have been found in a number of sulfate- and metal ion-reducing bacteria. Geobacter sulfurreducens is one of a family of microorganisms that oxidize organic compounds, with Fe(III) oxide as the terminal electron acceptor. A triheme 9.6 kDa cytochrome c(7) from G. sulfurreducens is a part of the metal ion reduction pathway. We cloned the gene for cytochrome c(7) and expressed it in Escherichia coli together with the cytochrome c maturation gene cluster, ccmABCDEFGH, on a separate plasmid. We designed two constructs, with and without an N-terminal His-tag. The untagged version provided a good yield (up to 6 mg/l of aerobic culture) of the fully matured protein, with all three hemes attached, while the N-terminal His-tag appeared to be detrimental for proper heme incorporation. The recombinant protein (untagged) is properly folded, it has the same molecular weight and displays the same absorption spectra, both in reduced and in oxidized forms, as the protein isolated from G. sulfurreducens and it is capable of reducing metal ions in vitro. The shape parameters for the recombinant cytochrome c(7) determined by small angle X-ray scattering are in good agreement with the ones calculated from a homologous cytochrome c(7) of known structure.

Cytochrome c Group↗

PubMed2002

Proton uptake of rhodobacter capsulatus reaction center mutants modified in the primary quinone environment.

Flash-induced absorbance spectroscopy was used to analyze the proton uptake and electron transfer properties of photosynthetic reaction centers (RC) of Rhodobacter capsulatus that have been genetically modified near the primary quinone electron acceptor (Q(A)). M246Ala and M247Ala, which are symmetry-related to the positions of two acidic groups, L212Glu and L213Asp, in the secondary quinone electron acceptor (QB) protein environment, have been mutated to Glu and Asp, respectively. The pH dependence of the stoichiometry of proton uptake upon formation of the P+Q(A)- (H+/P+Q(A)-) and PQ(A) (H+/Q(A)-) (P is the primary electron donor, a noncovalently linked bacteriochlorophyll dimer) states have been measured in the M246Ala --> Glu and the M247Ala --> Asp mutant RC, in the M246Ala-M247Ala --> Glu-Asp double mutant and in the wild type (WT). Our results show that the introduction of an acidic group (Glu or Asp) in the QA protein region induces notable additional proton uptake over a large pH region (approximately 6-9), which reflects a delocalized response of the protein to the formation of Q(A)-. This may indicate the existence of a widely spread proton reservoir in the cytoplasmic region of the protein. Interestingly, the pH titration curves of the proton release caused by the formation of P+ (H+/P+: difference between H+/P+Q(A)- and H+/PQ(A)- curves) are nearly superimposable in the WT and the M246Ala --> Glu mutant RC, but substantial additional proton release is detected between pH 7 and 9 in the M247Ala --> Asp mutant RC. This effect can be accounted for by an increased proton release by the P+ environment in the M247Ala --> Asp mutant. The M247Ala --> Asp mutation reveals the existence of an energetic and conformational coupling between donor and acceptor sides of the RC at a distance of nearly 30A.

Amino Acid Substitution↗