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W Junge

Publications and source records attributed to W Junge.

At least 19 recordsLinked to original sources
PubMed2000

F-ATPase: specific observation of the rotating c subunit oligomer of EF(o)EF(1).

The rotary motion in response to ATP hydrolysis of the ring of c subunits of the membrane portion, F(o), of ATP synthase, F(o)F(1), is still under contention. It was studied with EF(o)EF(1) (Escherichia coli) using microvideography with a fluorescent actin filament. To overcome the limited specificity of actin attachment through a Cys-maleimide couple which might have hampered the interpretation of previous work, we engineered a 'strep-tag' sequence into the C-terminal end of subunit c. It served (a) to purify the holoenzyme and (b) to monospecifically attach a fluorescent actin filament to subunit c. EF(o)EF(1) was immobilized on a Ni-NTA-coated glass slide by the engineered His-tag at the N-terminus of subunit beta. In the presence of MgATP we observed up to five counterclockwise rotating actin filaments per picture frame of 2000 microm(2) size, in some cases yielding a proportion of 5% rotating over total filaments. The rotation was unequivocally attributable to the ring of subunit c. The new, doubly engineered construct serves as a firmer basis for ongoing studies on torque and angular elastic distortions between F(1) and F(o).

Actins↗
PubMed1999

Proton transfer from the bulk to the bound ubiquinone Q(B) of the reaction center in chromatophores of Rhodobacter sphaeroides: retarded conveyance by neutral water.

The mechanism of proton transfer from the bulk into the membrane protein interior was studied. The light-induced reduction of a bound ubiquinone molecule Q(B) by the photosynthetic reaction center is accompanied by proton trapping. We used kinetic spectroscopy to measure (i) the electron transfer to Q(B) (at 450 nm), (ii) the electrogenic proton delivery from the surface to the Q(B) site (by electrochromic carotenoid response at 524 nm), and (iii) the disappearance of protons from the bulk solution (by pH indicators). The electron transfer to Q(B)(-) and the proton-related electrogenesis proceeded with the same time constant of approximately 100 microseconds (at pH 6.2), whereas the alkalinization in the bulk was distinctly delayed (tau approximately 400 microseconds). We investigated the latter reaction as a function of the pH indicator concentration, the added pH buffers, and the temperature. The results led us to the following conclusions: (i) proton transfer from the surface-located acidic groups into the Q(B) site followed the reduction of Q(B) without measurable delay; (ii) the reprotonation of these surface groups by pH indicators and hydronium ions was impeded, supposedly, because of their slow diffusion in the surface water layer; and (iii) as a result, the protons were slowly donated by neutral water to refill the proton vacancies at the surface. It is conceivable that the same mechanism accounts for the delayed relaxation of the surface pH changes into the bulk observed previously with bacteriorhodopsin membranes and thylakoids. Concerning the coupling between proton pumps in bioenergetic membranes, our results imply a tendency for the transient confinement of protons at the membrane surface.

Ion Transport↗
PubMed1999

On the stator of rotary ATP synthase: the binding strength of subunit delta to (alpha beta)3 as determined by fluorescence correlation spectroscopy.

ATP synthase is conceived as a rotary enzyme. Proton flow drives the rotor (namely, subunits c12 epsilon gamma) relative to the stator (namely, subunits ab2 delta(alpha beta)3) and extrudes spontaneously formed ATP from three symmetrically arranged binding sites on (alpha beta)3 into the solution. We asked whether the binding of subunit delta to (alpha beta)3 is of sufficient strength to hold against the elastic strain, which is generated during the operation of this enzyme. According to current estimates, the elastically stored energy is about 50 kJ/mol. Subunit delta was specifically labeled without impairing its function. Its association with solubilized (alpha beta)3 gamma in detergent-free buffer was studied by fluorescence correlation spectroscopy (FCS). A very strong tendency of delta to dimerize in detergent-free buffer was apparent (K(d) </= 0.2 nM). Taking the upper limit of this figure into account, the dissociation constant between monomeric delta and (alpha beta)3 gamma was 0.8 nM if not smaller. It is equivalent to a free energy of binding of at least 52 kJ/mol and therewith is sufficient for the assumed hold-function of delta in the stator. Our data were compatible with a single binding site for delta on the hexagon of (alpha beta)3.

Detergents↗
PubMed1999

Rotation of Escherichia coli F(1)-ATPase.

By applying the same method used for F(1)-ATPase (TF(1)) from thermophilic Bacillus PS3 (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299-302), we observed ATP-driven rotation of a fluorescent actin filament attached to the gamma subunit in Escherichia coli F(1)-ATPase. The torque value and the direction of the rotation were the same as those observed for TF(1). F(1)-ATPases seem to share common properties of rotation irrespective of the sources.

Actin Cytoskeleton↗
PubMed1999

Evidence for impaired hydrogen-bonding of tyrosine YZ in calcium-depleted photosystem II

Photosystem II (PS II) evolves oxygen from two bound water molecules in a four-stepped reaction that is driven by four quanta of light, each oxidizing the chlorophyll moiety P680 to yield P+680. When starting from its dark equilibrium (mainly state S1), the catalytic center can be clocked through its redox states (S0ellipsisS4) by a series of short flashes of light. The center involves at least a Mn4-cluster and a special tyrosine residue, named YZ, as redox cofactors plus two essential ionic cofactors, Cl- and Ca2+. Centers which have lost Ca2+ do not evolve oxygen. We investigated the stepped progression in dark-adapted PS II core particles after the removal of Ca2+. YZ was oxidized from the first flash on. The difference spectrum of YZ-->YoxZ differed from the one in competent centers, where it has been ascribed to a hydrogen-bonded tyrosinate. The rate of the electron transfer from YZ to P+680 was slowed down by three orders of magnitude and its kinetic isotope effect rose up from 1.1 to 2.5. Proton release into the bulk was now a prerequisite for the electron transfer from YZ to P+680. On the basis of these results and similar effects in Mn-(plus Ca2+-)depleted PS II (M. Haumann et al., Biochemistry, 38 (1999) 1258-1267) we conclude that the presence of Ca2+ in the catalytic center is required to tune the apparent pK of a base cluster, B, to which YZ is linked by hydrogen bonds. The deposition of a proton on B within close proximity of YZ (not its release into the bulk!) is a necessary condition for the reduction in nanoseconds of P+680 and for the functioning of water oxidation. The removal of Ca2+ rises the pK of B, thereby disturbing the hydrogen bonded structure of YZB.

Journal Article↗
PubMed1999

Transient accumulation of elastic energy in proton translocating ATP synthase.

ATP synthase is conceived as a rotatory engine with two reversible drives, the proton-transporting membrane portion, F0, and the catalytic peripheral portion, F1. They are mounted on a central shaft (subunit gamma) and held together by an eccentric bearing. It is established that the hydrolysis of three molecules of ATP in F1 drives the shaft over a full circle in three steps of 120 degrees each. Proton flow through F0 probably generates a 12-stepped rotation of the shaft so that four proton-translocating steps of 30 degrees each drive the synthesis of one molecule of ATP. We addressed the elasticity of the transmission between F0 and F1 in a model where the four smaller steps in F0 load a torsional spring which is only released under liberation of ATP from F1. The kinetic model of an elastic ATP synthase described a wealth of published data on the synthesis/hydrolysis of ATP by F0F1 and on proton conduction by F0 as function of the pH and the protonmotive force. The pK values of the proton-carrying group interacting with the acidic and basic sides of the membrane were estimated as 5.3-6.4 and 8.0-8.3, respectively.

Energy Metabolism↗
PubMed1999

ATP-synthase of Rhodobacter capsulatus: coupling of proton flow through F0 to reactions in F1 under the ATP synthesis and slip conditions.

A stepwise increasing membrane potential was generated in chromatophores of the phototrophic bacterium Rhodobacter capsulatus by illumination with short flashes of light. Proton transfer through ATP-synthase (measured by electrochromic carotenoid bandshift and by pH-indicators) and ATP release (measured by luminescence of luciferin-luciferase) were monitored. The ratio between the amount of protons translocated by F0F1 and the ATP yield decreased with the flash number from an apparent value of 13 after the first flash to about 5 when averaged over three flashes. In the absence of ADP, protons slipped through F0F1. The proton transfer through F0F1 after the first flash contained two kinetic components, of about 6 ms and 20 ms both under the ATP synthesis conditions and under slip. The slower component of proton transfer was substantially suppressed in the absence of ADP. We attribute our observations to the mechanism of energy storage in the ATP-synthase needed to couple the transfer of four protons with the synthesis of one molecule of ATP. Most probably, the transfer of initial protons of each tetrad creates a strain in the enzyme that slows the translocation of the following protons.

Adenosine Diphosphate↗
PubMed1999

Tyrosine-Z in oxygen-evolving photosystem II: a hydrogen-bonded tyrosinate.

In oxygen-evolving photosystem II (PSII), a tyrosine residue, D1Tyr161 (YZ), serves as the intermediate electron carrier between the catalytic Mn cluster and the photochemically active chlorophyll moiety P680. A more direct catalytic role of YZ, as a hydrogen abstractor from bound water, has been postulated. That YZox appears as a neutral (i.e. deprotonated) radical, YZ*, in EPR studies is compatible with this notion. Data based on electrochromic absorption transients, however, are conflicting because they indicate that the phenolic proton remains on or near to YZox. In Mn-depleted PSII the electron transfer between YZ and P680+ can be almost as fast as in oxygen-evolving material, however, only at alkaline pH. With an apparent pK of about 7 the fast reaction is suppressed and converted into an about 100-fold slower one which dominates at acid pH. In the present work we investigated the optical difference spectra attributable to the transition YZ --> YZox as function of the pH. We scanned the UV and VIS range and used Mn-depleted PSII core particles and also oxygen-evolving ones. Comparing these spectra with published in vitro and in vivo spectra of phenolic compounds, we arrived at the following conclusions: In oxygen-evolving PSII YZ resembles a hydrogen-bonded tyrosinate, YZ(-).H(+).B. The phenolic proton is shifted toward a base B already in the reduced state and even more so in the oxidized state. The retention of the phenolic proton in a hydrogen-bonded network gives rise to a positive net charge in the immediate vicinity of the neutral radical YZ*. It may be favorable both for the very rapid reduction by YZ of P680+ and for electron (not hydrogen) abstraction by YZ* from the Mn-water cluster.

Hydrogen Bonding↗
PubMed1999

Monitoring daily milk yields with a recursive test day repeatability model (Kalman filter).

Mixed model methodology and recursive estimation techniques (Kalman filter) were combined to detect significant changes in the performance level of both individual cows and an entire herd. Yields were predicted for the next time of recording using all data available up to that time. Predicted yields were compared with actual measurements. If the error of prediction, or innovation, exceeded +/- 2 times its standard deviation, the observation was considered to be significantly different from the former yield level. The data comprised 30,199 records for 135 cows and 366 d. Effects fitted in the model were test day, breed, and lactation class as fixed effects and cow within lactation number as a random effect. A lactation curve was fitted within lactation class. Of the observed milk yields, 9.2% deviated significantly from the expected value in a negative direction. None of the innovation of the fixed-day effect exceeded the threshold of two standard deviations. Compared with the results of rolling average, which were calculated as the average of a 10-d period, over 20% of the observations of the daily milk yield were classified differently by the two methods. The mixed-model method for recursive estimation takes better account of environmental and lactational effects influencing daily milk yields as the rolling average. The mixed-model recursive estimation method was applicable for the detection of suspicious (i.e., outside a specified prediction interval) observations of individual cows at the time of the actual recording.

Animals↗
PubMed1998

Oxygenic photosystem II: the mutation D1-D61N in Synechocystis sp. PCC 6803 retards S-state transitions without affecting electron transfer from YZ to P680+.

Photosynthetic oxygen evolution is powered by photosystem II (PSII), in particular by the oxidized chl a-aggregate P680+, and catalyzed by the oxygen-evolving complex (Mn4X-entity) as well as a tyrosine residue (YZ). The role of particular amino acids as cofactors of electron and proton transfer or as modulators of the activity is still ill-defined. The effects of single-site mutations at the donor side of PSII on the partial reactions of water oxidation have been primarily studied in whole cells. Because of better signal-to-noise in oxygen-evolving core preparations more detailed information on the electronic, protonic, and electrostatic events is expected from studies with such material. We investigated cells and oxygen-evolving core preparations from the wildtype of Synechocystis sp. PCC 6803 and point-mutants of D1-D61. In cells, oxygen-release was slowed drastically in D61A (8-fold) and D61N (10-fold) compared to WT, whereas it remained unchanged in D61E within the time resolution of the measurements. In core preparations, the S1 --> S2 and S2 --> S3 transitions were slowed approximately 2-fold in D61N compared to WT. However, the nanosecond components of electron transfer from YZ to P680+ were unchanged in the same mutant. We conclude that substitution of a neutral residue for D1-D61 selectively affects electron-transfer events on the donor side of YZ.

Asparagine↗
PubMed1998

The cytochrome bc1 complex of Rhodobacter capsulatus: ubiquinol oxidation in a dimeric Q-cycle?

We studied the cytochrome bc1 complex (hereafter bc) by flash excitation of Rhodobacter capsulatis chromatophores. The reduction of the high-potential heme b(h), of cytochrome b (at 561 nm) and of cytochromes c (at 552 nm) and the electrochromic absorption transients (at 524 nm) were monitored after the first and second flashes of light, respectively. We kept the ubiquinone pool oxidized in the dark and concerned for the ubiquinol formation in the photosynthetic reaction center only after the second flash. Surprisingly, the first flash caused the oxidation of about one ubiquinol per bc dimer. Based on these and other data we propose a dimeric Q-cycle where the energetically unfavorable oxidation of the first ubiquinol molecule by one of the bc monomers is driven by the energetically favorable oxidation of the second ubiquinol by the other bc monomer resulting in a pairwise oxidation of ubiquinol molecules by the dimeric bc in the dark. The residual unpaired ubiquinol supposedly remains on the enzyme and is then oxidized after the first flash.

Electron Transport↗
PubMed1998

Three-stepped rotation of subunits gamma and epsilon in single molecules of F-ATPase as revealed by polarized, confocal fluorometry.

The proton translocating ATP synthase is conceived as a rotatory molecular engine. ATP hydrolysis by its headpiece, CF1, drives the rotation of subunit gamma relative to the hexagonally arranged large subunits, (alphabeta)3. We investigated transition states of the rotatory drive by polarized confocal fluorometry (POCOF) as applied to single molecules of engineered, immobilized and load-free spinach-CF1. We found that the hydrolysis of ATP caused the stepped and sequential progression of subunit gamma through three discrete angular positions, with the transition states of gamma being too shortlived for detection. We also observed the stepped motion of epsilon, whereas delta was immobile as (alphabeta)3.

Fluorometry↗
PubMed1998

Function of tyrosine Z in water oxidation by photosystem II: electrostatical promotor instead of hydrogen abstractor.

Photosynthetic water oxidation by photosystem II is mediated by a Mn4 cluster, a cofactor X still chemically ill-defined, and a tyrosine, YZ (D1-Tyr161). Before the final reaction with water proceeds to yield O2 (transition S4-->S0), two oxidizing equivalents are stored on Mn4 (S0-->S1-->S2), a third on X (S2-->S3), and a forth on YZ(S3-->S4). It has been proposed that YZ functions as a pure electron transmitter between Mn4X and P680, or, more recently, that it acts as an abstractor of hydrogen from bound water. We scrutinized the coupling of electron and proton transfer during the oxidation of YZ in PSII core particles with intact or impaired oxygen-evolving capacity. The rates of electron transfer to P680+, of electrochromism, and of pH transients were determined as a function of the pH, the temperature, and the H/D ratio. In oxygen-evolving material, we found only evidence for electrostatically induced proton release from peripheral amino acid residues but not from YZox itself. The positive charge stayed near YZox, and the rate of electron transfer was nearly independent of the pH. In core particles with an impaired Mn4 cluster, on the other hand, the rate of the electron transfer became strictly dependent on the protonation state of a single base (pK approximately 7). At pH < 7, the rate of electron transfer revealed the same slow rate (t1/2 approximately 35 microseconds) as that of proton release into the bulk. The deposition of a positive charge around YZox was no longer detected. A large H/D isotope effect (approximately 2.5) on these rates was also indicative of a steering of electron abstraction by proton transfer. That YZox was deprotonated into the bulk in inactive but not in oxygen-evolving material argues against the proposed role of YZox as an acceptor of hydrogen from water. Instead, the positive charge in its vicinity may shift the equilibrium from bound water to bound peroxide upon S3-->S4 as a prerequisite for the formation of oxygen upon S4-->S0.

Chlorophyll↗
PubMed1997

Cross-linking of chloroplast F0F1-ATPase subunit epsilon to gamma without effect on activity. Epsilon and gamma are parts of the rotor.

Cys residues were directed into positions 17, 28, 41 and 85 of a Cys6-->Ser mutant of subunit epsilon of spinach chloroplast F0F1 ATP synthase. Wild-type and engineered epsilon were expressed in Escherichia coli, purified in the presence of urea, refolded and reassembled with spinach chloroplast F1 lacking the epsilon subunit [F1(-epsilon)]. Cys-containing epsilon variants were modified with a sulfhydryl-reactive photolabile cross-linker. Photocross-linking of epsilon to F1(-epsilon) yielded the same SDS gel pattern of cross-link products independent of the presence or absence of Mg2+ x ADP, phosphate and Mg2+ x ATP. Epsilon (wild type) [Ser6,Cys28]epsilon and [Ser6,Cys41]epsilon were cross-linked with subunit gamma. With chloroplast F0F1 the same cross-link pattern was obtained, except for one extra cross-link, probably between [Ser6,Cys28]epsilon and F0 subunit III. [Ser6,Cys17]epsilon and [Ser6,Cys85]epsilon did not produce cross-links. Cross-linking of epsilon, [Ser6,Cys28]epsilon, [Ser6,Cys41]epsilon to gamma in soluble chloroplast F1 impaired the ability of epsilon to inhibit Ca2+-ATPase activity. The Mg2+-ATPase activity of soluble F1 (measured in the presence of 30% MeOH) was not affected by cross-linking epsilon with gamma. Functional reconstitution of photophosphorylation in F1-depleted thylakoids was observed with F1 in which gamma was cross-linked to [Ser6,Cys28]epsilon or [Ser6,Cys41]epsilon but not with wild-type epsilon. In view of the intersubunit rotation of gamma relative to (alphabeta)3, which is driven by ATP hydrolysis, gamma and epsilon would seem to act concertedly as parts of the 'rotor' relative to the 'stator' (alphabeta)3.

Amino Acid Sequence↗
PubMed1997

ATP synthase: a tentative structural model.

Adenosine triphosphate (ATP) synthase produces ATP from ADP and inorganic phosphate at the expense of proton- or sodium-motive force across the respective coupling membrane in Archaea, Bacteria and Eucarya. Cation flow through the intrinsic membrane portion of this enzyme (Fo, subunits ab2c9-12) and substrate turnover in the headpiece (F1, subunits alpha3beta3 gammadeltaepsilon) are mechanically coupled by the rotation of subunit gamma in the center of the catalytic hexagon of subunits (alphabeta)3 in F1. ATP synthase is the smallest rotatory engine in nature. With respect to the headpiece alone, it probably operates with three steps. Partial structures of six out of its at least eight different subunits have been published and a 3-dimensional structure is available for the assembly (alphabeta)3gamma. In this article, we review the available structural data and build a tentative topological model of the holoenzyme. The rotor portion is proposed to consist of a wheel of at least nine copies of subunits c, epsilon and a portion of gamma as a spoke, and another portion of gamma as a crankshaft. The stator is made up from a, the transmembrane portion of b2, delta and the catalytic hexagon of (alphabeta)3. As an educated guess, the model may be of heuristic value for ongoing studies on this fascinating electrochemical-to-mechanical-to-chemical transducer.

ATP Synthetase Complexes↗
PubMed1997

Electrogenicity of electron and proton transfer at the oxidizing side of photosystem II.

The electrogenicity of electron and proton transfer at the oxidizing side of PSII was monitored by transmembrane electrochromism of carotenoids in thylakoids and, independently, by electrometry in oxygen-evolving photosystem II core particles. It yielded dielectrically weighted distances between cofactors. They were related to the one between YZox and QA- (=100%). The electron transfer from YZ to P680+ ranged over a relative distance of 15%, while the one from Mn4 to YZox ranged over less than 3.5%. The latter result placed Mn4 and YZ at about the same weighted depth in the membrane. The oxidation of cofactor X by YZox during S2 --> S3 ranged over 10%. We tentatively attributed 7% to proton transfer into the lumen and 3% to electron transfer, in line with our notion that one proton is liberated from Xox itself. This placed X at the same depth in the membrane as Mn. Proton release upon the final oxidation of water during the oxygen-evolving step S4 --> S0 revealed relative electrogenic components of 5.5% in core particles and between 10.5% (pH 7.4) and 2% (pH 6.2) in thylakoids. The former likely reflected proton transfer from bound water into the lumen and the latter to intraprotein bases that were created in the foregoing transitions. A tentative scheme for the arrangement of cofactors at the oxidizing side of photosystem II is presented.

Carotenoids↗