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B Ludwig

Publications and source records attributed to B Ludwig.

At least 37 records · Page 2Linked to original sources
PubMed2000

Single-electron reduction of the oxidized state is coupled to proton uptake via the K pathway in Paracoccus denitrificans cytochrome c oxidase.

The reductive part of the catalytic cycle of cytochrome c oxidase from Paracoccus denitrificans was examined by using time-resolved potential measurements on black lipid membranes. Proteoliposomes were adsorbed to the black lipid membranes and Ru(II)(2, 2'-bipyridyl)(3)(2+) was used as photoreductant to measure flash-induced membrane potential generation. Single-electron reduction of the oxidized wild-type cytochrome c oxidase reveals two phases of membrane potential generation (tau(1) approximately 20 micros and tau(2) approximately 175 micros) at pH 7.4. The fast phase is not sensitive to cyanide and is assigned to electron transfer from Cu(A) to heme a. The slower phase is inhibited completely by cyanide and shows a kinetic deuterium isotope effect by a factor of 2-3. Although two enzyme variants mutated in the so-called D pathway of proton transfer (D124N and E278Q) show the same time constants and relative amplitudes as the wild-type enzyme, in the K pathway variant K354M, tau(2) is increased to 900 micros. This result suggests uptake of a proton through the K pathway during the transition from the oxidized to the one-electron reduced state. After the second laser flash under anaerobic conditions, a third electrogenic phase with a time constant of approximately 1 ms appears. The amplitude of this phase grows with increasing flash number. We explain this growth by injection of a second electron into the single-electron reduced enzyme. On multiple flashes, both D pathway mutants behave differently compared with the wild type and two additional slow phases of tau(3) approximately 2 ms and tau(4) approximately 15 ms are observed. These results suggest that the D pathway is involved in proton transfer coupled to the uptake of the second electron.

Amino Acid Substitution↗
PubMed2000

Structure of the soluble domain of cytochrome c(552) from Paracoccus denitrificans in the oxidized and reduced states.

The crystal structure of the soluble domain of the membrane bound cytochrome c(552) (cytochrome c(552)') from Paracoccus denitrificans was determined using the multiwavelength anomalous diffraction technique and refined at 1.5 A resolution for the oxidized and at 1. 4 A for the reduced state. This is the first high-resolution crystal structure of a cytochrome c at low ionic strength in both redox states. The atomic model allowed for a detailed assessment of the structural properties including the secondary structure, the heme geometry and interactions, and the redox-coupled structural changes. In general, the structure has the same features as that of known eukaryotic cytochromes c. However, the surface properties are very different. Cytochrome c(552)' has a large strongly negatively charged surface part and a smaller positively charged area around the solvent-exposed heme atoms. One of the internal water molecules conserved in all structures of eukaryotic cytochromes c is also present in this bacterial cytochrome c. It contributes to the interactions between the side-chain of Arg36 and the heme propionate connected to pyrrole ring A. Reduction of the oxidized crystals does not influence the conformation of cytochrome c(552)' in contrast to eukaryotic cytochromes c. The oxidized cytochrome c(552)', especially the region of amino acid residues 40 to 56, appears to be more flexible than the reduced one.

Crystallography, X-Ray↗
PubMed2000

Solution structure of the functional domain of Paracoccus denitrificans cytochrome c552 in the reduced state.

In order to determine the solution structure of Paracoccus denitrificans cytochrome c552 by NMR, we cloned and isotopically labeled a 10.5-kDa soluble fragment (100 residues) containing the functional domain of the 18.2-kDa membrane-bound protein. Using uniformly 15N-enriched samples of cytochrome c552 in the reduced state, a variety of two-dimensional and three-dimensional heteronuclear double-resonance NMR experiments was employed to achieve complete 1H and 15N assignments. A total of 1893 distance restraints was derived from homonuclear 2D-NOESY and heteronuclear 3D-NOESY spectra; 1486 meaningful restraints were used in the structure calculations. After restrained energy minimization a family of 20 structures was obtained with rmsd values of 0.56 +/- 0. 10 A and 1.09 +/- 0.09 A for the backbone and heavy atoms, respectively. The overall topology is similar to that seen in previously reported models of this class of proteins. The global fold consists of two long helices at the N-terminus and C-terminus and three shorter helices surrounding the heme moiety; the helices are connected by well-defined loops. Comparison with the X-ray structure shows some minor differences in the positions of the Trp57 and Phe65 side-chain rings as well as the heme propionate groups.

Amino Acid Sequence↗
PubMed2000

13C and 15N distributions in three spodic dystric cambisols under beech and spruce.

The study of natural isotopic abundance signatures is useful to gain further insights in the processes resulting in depthwise changes in the composition of soil organic matter (SOM). Objectives were to describe the delta 13C and delta 15N abundances of SOM with depth in soils from a 153-year old beech (B1), a 119-year old spruce (F1) and a 61-year old spruce (F2) stand at Solling, north-west Germany, and to study, how podzolisation affects the isotopic abundances of 13C and 15N in the SOM. The degree of podzolisation decreased in the order F1 > B1 > F2. At the surface of the humus layer of all three sites, delta 13C values are approximately 1 to 4/1000 higher than in the leaves and needles, probably mainly due to the discrimination of 13C by microbial decomposition. 13C abundances in the organic layers of B1 and F2 increased only slightly from -27.6/1000 PDB (B1, L) to -27.2/1000 PDB (B1, Oh) and from -26.3/1000 PDB (F2, L) to -25.9/1000 PDB (F2, Oh), suggesting that biotic activity resulted in mixing of organic matter. At F1, however, 13C abundance increased from -27.5/1000 PDB (L) to -26.0/1000 PDB (Oh) which reflects the lack of mixing by animals. In the upper 2-4 cm of the mineral soil, i.e., in the eluvial horizons Aeh, 13C values showed a minimum at the spruce sites which was presumably related to a translocation of 13C enriched fulvic acids. Depthwise changes in delta 15N values were not related to podzolisation processes. At all three sites, a 13N enrichment with depth occurred in the mineral soil which is the result of the discrimination of 15N by microbial decomposition.

Carbon Isotopes↗
PubMed2000

Use of 13C and 15N mass spectrometry to study the decomposition of Calamagrostis epigeios in soil column experiments with and without ash additions.

The dynamics of C and N in terrestrial ecosystems are not completely understood and the use of stable isotopes may be useful to gain further insight in the pathways of CO2 emissions and leaching of dissolved organic carbon (DOC) and nitrogen (DON) during decomposition of litter. Objectives were (i) to study the decomposition dynamics of Calamagrostis epigeios, a common grass species in forests, using 13C-depleted and 15N-enriched plants and (ii) to quantify the effect wood ash addition on the decomposition and leaching of DOC and DON. Decomposition was studied for 128 days under aerobic conditions at 8 degrees C and moisture close to field capacity in a spodic dystric Cambisol with mor-moder layer. Variants included control plots and additions of (i) Calamagrostis litter and (ii) Calamagrostis litter plus 4 kg ash m-2. (i) Decomposition of Calamagrostis resulted in a CO2 production of 76.2 g CO2-C m-2 (10% of added C) after 128 days and cumulative DOC production was 14.0 g C m-2 out of which 0.9 g C m-2 was Calamagrostis-derived (0.1% of added C). The specific CO2 formation and specific DOC production from Calamagrostis were 6 times higher (CO2) and 4 times smaller (DOC) than those from the organic layer. The amount of Calamagrostis-derived total N (NH4+, NO3-, DON) leached was 0.7 g N m-2 (4.8% of added N). Cumulative DON production was 0.8 g N m-2 which was slightly higher than for the control. During soil passage, much of the DOC and DON was removed due to sorption or decomposition. DOC and DON releases from the mineral soil (17 cm depth) were 6.3 g C m-2 and 0.5 g N m-2. (ii) Addition of ash resulted in a complete fixing of CO2 for 40 days due to carbonatisation. Afterwards, the CO2 production rates were similar to the variant without ash addition. Production of DOC (98.6 g C m-2) and DON (2.5 g N m-2) was marked, mainly owing to humus decay. However, Calamagrostis-derived DOC and Calamagrostis-derived total N were only 3.9 g C m-2 (0.5% of added C) and 0.5 g N m-2 (3.4% of added N). The specific DOC production rate from the organic layer was 6 times higher than that from Calamagrostis. The results suggest that with increasing humification from fresh plant residues to more decomposed material (OF and OH layers) the production ratio of DOC/CO2-C increases. Addition of alkaline substances to the forest floor can lead to a manifold increase in DOC production.

Carbon Isotopes↗
PubMed1999

Mutation of Arg-54 strongly influences heme composition and rate and directionality of electron transfer in Paracoccus denitrificans cytochrome c oxidase.

The effect of a single site mutation of Arg-54 to methionine in Paracoccus denitrificans cytochrome c oxidase was studied using a combination of optical spectroscopy, electrochemical and rapid kinetics techniques, and time-resolved measurements of electrical membrane potential. The mutation resulted in a blue-shift of the heme a alpha-band by 15 nm and partial occupation of the low-spin heme site by heme O. Additionally, there was a marked decrease in the midpoint potential of the low-spin heme, resulting in slow reduction of this heme species. A stopped-flow investigation of the reaction with ferrocytochrome c yielded a kinetic difference spectrum resembling that of heme a(3). This observation, and the absence of transient absorbance changes at the corresponding wavelength of the low-spin heme, suggests that, in the mutant enzyme, electron transfer from Cu(A) to the binuclear center may not occur via heme a but that instead direct electron transfer to the high-spin heme is the dominating process. This was supported by charge translocation measurements where Deltapsi generation was completely inhibited in the presence of KCN. Our results thus provide an example for how the interplay between protein and cofactors can modulate the functional properties of the enzyme complex.

Arginine↗
PubMed1999

Mutations in the Ca2+ binding site of the Paracoccus denitrificans cytochrome c oxidase.

Recent structure determinations suggested a new binding site for a non-redox active metal ion in subunit I of cytochrome c oxidase both of mitochondrial and of bacterial origin. We analyzed the relevant metal composition of the bovine and the Paracoccus denitrificans enzyme and of bacterial site-directed mutants in several residues presumably liganding this ion. Unlike the mitochondrial enzyme where a low, substoichiometric content of Ca2+ was found, the bacterial wild-type (WT) oxidase showed a stoichiometry of one Ca per enzyme monomer. Mutants in Asp-477 (in immediate vicinity of this site) were clearly diminished in their Ca content and the isolated mutant enzyme revealed a spectral shift in the heme a visible absorption upon Ca addition, which was reversed by Na ions. This spectral behavior, largely comparable to that of the mitochondrial enzyme, was not observed for the bacterial WT oxidase. Further structure refinement revealed a tightly bound water molecule as an additional Ca2+ ligand.

Animals↗
PubMed1999

Time-resolved FT-IR studies on the CO adduct of Paracoccus denitrificans cytochrome c oxidase: comparison of the fully reduced and the mixed valence form.

The rebinding of CO to cytochrome c oxidase from Paracoccus denitrificans in the fully reduced and in the half-reduced (mixed valence) form as a function of temperature was investigated using time-resolved rapid-scan FT-IR spectroscopy in the mid-IR (1200-2100 cm-1). For the fully reduced enzyme, rebinding was complete in approximately 2 s at 268 K and showed a biphasic reaction. At 84 K, nonreversible transfer of CO from heme a3 to CuB was observed. Both photolysis at 84 K and photolysis at 268 K result in FT-IR difference spectra which show similarities in the amide I, amide II, and heme modes. Both processes, however, differ in spectral features characteristic for amino acid side chain modes and may thus be indicative for the motional constraint of CO at low temperature. Rebinding of photodissociated CO for the mixed-valence enzyme at 268 K is also biphasic, but much slower as compared to the fully reduced enzyme. FT-IR difference spectra show band features similar to those for the fully reduced enzyme. Additional strong bands in the amide I and amide II range indicate local conformational changes induced by electron and coupled proton transfer. These signals disappear when the temperature is lowered to 84 K. At 268 K, a difference signal at 1746 cm-1 is observed which is shifted by 6 cm-1 to 1740 cm-1 in 2H2O. The absence of this signal for the mutant Glu 278 Gln allows assignment to the COOH stretching mode of Glu 278, and indicates changes of the conformation, proton position, or protonation of this residue upon electron transfer.

Carbon Monoxide↗
PubMed1999

Heterologous expression of soluble fragments of cytochrome c552 acting as electron donor to the Paracoccus denitrificans cytochrome c oxidase.

A membrane-bound c-type cytochrome, c552, acts as the electron mediator between the cytochrome bc1 complex and cytochrome c oxidase in the branched respiratory chain of the bacterium Paracoccus denitrificans. Unlike in mitochondria where a soluble cytochrome c interacts with both complexes, the bacterial c552, the product of the cycM gene, shows a tripartite structure, with an N-terminal membrane anchor separated from a typical class I cytochrome domain by a highly charged region. Two derivative fragments, lacking either only the membrane spanning region or both N-terminal domains, were constructed on the genetic level, and expressed in Escherichia coli cotransformed with the ccm gene cluster encoding host-specific cytochrome c maturation factors. High levels of cytochromes c were expressed and located in the periplasm as holo-proteins; both these purified c552 fragments are functional in electron transport to oxidase, as ascertained by kinetic measurements, and will prove useful for future structural studies of complex formation by NMR and X-ray diffraction.

Cloning, Molecular↗
PubMed1999

Ion selectivity reversal and induction of voltage-gating by site-directed mutations in the Paracoccus denitrificans porin.

The porin from Paracoccus denitrificans, a slightly anion specific outer membrane pore protein, was expressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of urea and detergents. The purified recombinant protein was reconstituted into black lipid bilayer membranes and showed no difference in its functional properties in comparison to the native porin isolated from P.denitrificans membranes. To investigate the molecular basis of its ion selectivity and voltage-gating, a series of site-directed mutants was constructed, comprising acidic residues located on the third extracellular loop (L3), which forms the constriction zone of the channel, and basic residues along the opposing barrel wall. Measurements using zero-current membrane potentials indicated that the selectivity changed drastically from a slight anion to a distinct cation selectivity with the exchange of residues R29 and R31 by glutamate, whereas replacements on the L3 loop went largely unaffected. However, when assaying the voltage-dependent closure of channels, only mutations located on the L3 loop showed an effect, in contrast to the voltage-independent recombinant and native Paracoccus porin.

Electric Conductivity↗
PubMed1999

Electrochemical and ultraviolet/visible/infrared spectroscopic analysis of heme a and a3 redox reactions in the cytochrome c oxidase from Paracoccus denitrificans: separation of heme a and a3 contributions and assignment of vibrational modes.

Cytochrome c oxidase from Paracoccus denitrificans was studied with a combined electrochemical and ultraviolet/visible/infrared (UV/vis/IR) spectroscopic approach. Global fit analysis of oxidative electrochemical redox titrations was used to separate the spectral contributions coupled to heme a and a3 redox transitions, respectively. Simultaneous adjustment of the midpoint potentials and of the amplitudes for a user-defined number of redox components (here heme a and a3) at all wavelengths in the UV/vis (900-400 nm) and at all wavenumbers in the infrared (1800-1250 cm-1) yielded difference spectra for the number of redox potentials selected. With an assumption of two redox components, two spectra for the redox potential at -0.03 +/- 0.01 V and 0.22 +/- 0.04 V (quoted vs Ag/AgCl) were obtained. The method used here allows the separation of the heme signals from the electrochemically induced visible difference spectra of native cytochrome c oxidase without the addition of any inhibitors. The separated heme a and a3 UV/vis difference spectra essentially correspond to spectra obtained for high/low-spin and 5/6-coordinated heme a/a3 model compounds presented by Babcock [(1988) in Biological Applications of Resonance Raman Spectroscopy (Spiro, T., Ed.) Wiley and Sons, New York]. Single-component Fourier transform infrared (FTIR) difference spectra were calculated for both hemes on the basis of these fits, thus revealing contributions from the reorganization of the polypeptide backbone, from the hemes, and from single amino acids upon electron transfer of the cofactors (heme a/a3, CuA, and CuB), as well from coupled processes such as proton transfer. A tentative assignment of heme vibrational modes is presented and the assignment of the signals to the reorganization of the polypeptide backbone and to perturbations of single amino acids, in particular Asp, Glu, Arg, or Tyr, is discussed.

Arginine↗
PubMed1998

Electrochemically induced FT-IR difference spectra of the two- and four-subunit cytochrome c oxidase from P. denitrificans reveal identical conformational changes upon redox transitions.

In order to study the role of subunits III and IV of the cytochrome c oxidase from P. denitrificans for electron and proton transfer, electrochemically induced FT-IR difference spectra of the two- and of the four-subunit enzyme have been compared. These spectra reflect the alterations in the protein upon electron and proton transfer. Since the spectra are essentially identical, they clearly indicate that the additional subunits III and IV do not contribute to the FT-IR difference spectra of the four-subunit oxidase. Subunits III and IV are thus not involved in the reorganization of the polypeptide backbone and of single amino acids upon electron transfer and coupled proton transfer observed in the difference spectra in addition to heme contributions. The subtle differences between the FT-IR difference spectra that are attributed to the influence of protein-protein interactions between the subunits are discussed.

Binding Sites↗
PubMed1998

Electron entry in a CuA mutant of cytochrome c oxidase from Paracoccus denitrificans. Conclusive evidence on the initial electron entry metal center.

A cytochrome c oxidase subunit II C216S mutant from Paracoccus denitrificans in which the CuA site was changed by site-directed mutagenesis to a mononuclear copper site [Zickermann, V., Wittershagen, A., Kolbesen, B.O. and Ludwig, B. Biochemistry 36 (1997) 3232-3236] was investigated by stopped-flow spectroscopy. Contrary to the behavior of the wild type enzyme, in this mutant cytochrome a cannot be reduced by excess cytochrome c in the millisecond time scale in which cytochrome c oxidation is observed. The results conclusively identify and establish CuA as the initial electron entry site in cytochrome c oxidase. Partial rapid reduction (ca. 20%) of the modified CuA site suggests that the mononuclear copper ion has a redox potential ca. 100 mV lower than the wild type, and that internal electron transfer to cytochrome a is > or = 10(3)-fold slower than with the wild type enzyme.

Copper↗
PubMed1998

Expression of the Escherichia coli cyo operon in Paracoccus denitrificans results in a fully active quinol oxidase of unexpected heme composition.

The cyo operon coding for the membrane-bound bo3-type quinol oxidase of Escherichia coli has been expressed in a Paracoccus denitrificans strain deleted in its endogenous ba3 quinol oxidase. Using the P. denitrificans qox promoter, the His tagged protein complex is synthesized to a level comparable to that in E. coli and the enzyme purified in a single step on a metal-chelating column. Whereas the activity of the isolated complex matches that of the oxidase purified directly from E. coli, the heterologously expressed oxidase does not show the characteristic heme composition but now carries heme a in its binuclear site.

Cytochrome b Group↗
PubMed1998

Paracoccus denitrificans cytochrome c oxidase: a kinetic study on the two- and four-subunit complexes.

Cytochrome c oxidase from Paracoccus denitrificans has been purified in two different forms differing in polypeptide composition. An enzyme containing polypeptides I-IV is obtained when the purification procedure is performed in beta-d-dodecylmaltoside. If, however, Triton X-100 is used to purify the enzyme under otherwise identical conditions, an enzyme is obtained containing only subunits I-II. The two enzymes are undistinguishable by optical spectroscopy but show significant differences in the transient and steady-state time regimes, as studied by stopped-flow spectroscopy. The observed differences, however, are not due to removal of subunits III and IV, but rather to a specific effect of Triton X-100 which appears to affect cytochrome c binding. From these results it is not expected that subunits III and IV play any significant role in cytochrome c binding and, possibly, in the subsequent electron transfer processes. The results also suggest that both electrostatic and hydrophobic interactions may be important in the initial electron transfer process from cytochrome c.

Journal Article↗
PubMed1998

Mutational analysis of residues forming hydrogen bonds in the Rieske [2Fe-2S] cluster of the cytochrome bc1 complex in Paracoccus denitrificans.

Two mutations (S157A and Y159F) in the Rieske iron-sulfur subunit of the ubihydroquinone-cytochrome c oxidoreductase from Paracoccus denitrificans have been characterized with respect to the protein and [2Fe-2S] cluster stability, the enzyme activity and the redox potential of the [2Fe-2S] cluster. In the structure of the water-soluble fragment of the Rieske iron-sulfur protein of the bovine heart mitochondrial bc1 complex, both residues (S163 and Y165 in the bovine sequence) form the following hydrogen bonds to sulfur atoms in the [2Fe-2S] cluster: Ser163 O gamma-S-1 and Tyr165 O eta-Cys139 S gamma [Iwata, S., Saynovits, M., Link, T. A. & Michel, H. (1996) Structure 4, 567-579]. They are conserved in all known Rieske iron-sulfur proteins from bc1 complexes including that from P. denitrificans. Both amino acid exchanges, introduced as separate point mutations on plasmids containing the fbc operon, lead to a fully assembled three-subunit complex in P. denitrificans, with a metal and heme content as well as subunit composition indistinguishable from the parental strain. The purified complexes show decreased turnover numbers and redox potentials of the Rieske cluster. Whereas the turnover number of the bc1 complex isolated from the parental strain was 145 s(-1), the turnover numbers for the mutants S157A and Y159F were 10 s(-1) and 52 s(-1), respectively, corresponding to 7% and 36% activity, respectively. The midpoint potential Em of the Rieske cluster at pH 6 and 5 degrees C was +360 mV for the bc1 complex from the parental strain; the values in the mutant complexes were +316 mV (Y159F) and +265 mV (S157A). Shifts of the g values in the electron paramagnetic resonance spectra indicate an altered electron distribution in the mutants compared to in the wild-type protein.

Amino Acid Sequence↗