Photosynthetic oxygen production.
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Catalytic Domain↗
Biomedical subjects
Wolfgang Junge
Publications and source records attributed to Wolfgang Junge.
Evidence that bicarbonate is not the substrate in photosynthetic oxygen evolution.
It is widely accepted that the oxygen produced by photosystem II of cyanobacteria, algae, and plants is derived from water. Earlier proposals that bicarbonate may serve as substrate or catalytic intermediate are almost forgotten, though not rigorously disproved. These latter proposals imply that CO2 is an intermediate product of oxygen production in addition to O2. In this work, we investigated this possible role of exchangeable HCO3- in oxygen evolution in two independent ways. (1) We studied a possible product inhibition of the electron transfer into the catalytic Mn4Ca complex during the oxygen-evolving reaction by greatly increasing the pressure of CO2. This was monitored by absorption transients in the near UV. We found that a 3,000-fold increase of the CO2 pressure over ambient conditions did not affect the UV transient, whereas the S(3) --> S(4) --> S(0) transition was half-inhibited by raising the O2 pressure only 10-fold over ambient, as previously established. (2) The flash-induced O2 and CO2 production by photosystem II was followed simultaneously with membrane inlet mass spectrometry under approximately 15% H2(18)O enrichment. Light flashes that revealed the known oscillatory O2 release failed to produce any oscillatory CO2 signal. Both types of results exclude that exchangeable bicarbonate is the substrate for (and CO2 an intermediate product of) oxygen evolution by photosynthesis. The possibility that a tightly bound carbonate or bicarbonate is a cofactor of photosynthetic water oxidation has remained.
Absorption↗
Photosynthetic water oxidation at high O2 backpressure monitored by delayed chlorophyll fluorescence.
The atmospheric dioxygen is produced by photosynthetic organisms. This light-driven process culminates in what appears as one step: a four-electron abstraction from two water molecules bound to the Mn4Ca complex of photosystem II. Recently, an intermediate of the O2-producing reaction sequence was stabilized by elevated oxygen backpressure and detected by UV flash photometry [Clausen, J., and Junge, W. (2004) Nature 430, 480]. We scrutinized its properties by delayed chlorophyll fluorescence measurements. Half-suppression of oxygen evolution was observed at a similar O2 pressure of 2.3 bar, as previously, now with photosystem II membrane particles from spinach, without artificial electron acceptors, and at a high signal-to-noise ratio. The data are tentatively interpreted as the stabilization of a 2-fold oxidized state of the catalytic center (S2*) with bound peroxide and its slow conversion into the normal S2 state by the release of peroxide.
Chlorophyll↗
Regulation of the F0F1-ATP synthase: the conformation of subunit epsilon might be determined by directionality of subunit gamma rotation.
F(0)F(1)-ATP synthase couples ATP synthesis/hydrolysis with transmembrane proton transport. The catalytic mechanism involves rotation of the gamma epsilon c(approximately 10)-subunits complex relative to the rest of the enzyme. In the absence of protonmotive force the enzyme is inactivated by the tight binding of MgADP. Subunit epsilon also modulates the activity: its conformation can change from a contracted to extended form with C-terminus stretched towards F(1). The latter form inhibits ATP hydrolysis (but not synthesis). We propose that the directionality of the coiled-coil subunit gamma rotation determines whether subunit epsilon is in contracted or extended form. Block of rotation by MgADP presumably induces the extended conformation of subunit epsilon. This conformation might serve as a safety lock, stabilizing the ADP-inhibited state upon de-energization and preventing spontaneous re-activation and wasteful ATP hydrolysis. The hypothesis merges the known regulatory effects of ADP, protonmotive force and conformational changes of subunit epsilon into a consistent picture.
Adenosine Diphosphate↗
Structural biology. Nature's rotary electromotors.
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Adenosine Diphosphate↗
Proton slip in the ATP synthase of Rhodobacter capsulatus: induction, proton conduction, and nucleotide dependence.
FOF1-ATP synthase converts two energetic "currencies" of the cell (ATP and protonmotive force, pmf) by coupling two rotary motors/generators. Their coupling efficiency is usually very high. Uncoupled proton leakage (slip) has only been observed in chloroplast enzyme at unphysiologically low nucleotide concentration. We investigated the properties of proton slip in chromatophores (sub-bacterial vesicles) from Rhodobacter capsulatus in the single-enzyme-per-vesicle mode. The membrane was energized by excitation with flashing light and the relaxation of the transmembrane voltage and pH difference was photometrically detected. We found that: (1) Proton slip occurred only at low nucleotide concentration (<1 microM) and after pre-illumination over several seconds. (2) Slip induction by pmf was accompanied by the release of approximately 0.25 mol ADP per mole of enzyme. There was no detectable detachment of F1 from FO. (3) The transmembrane voltage and the pH difference were both efficient in slip induction. Once induced, slip persisted for hours, and was only partially reverted by the addition of ADP or ATP (>1 microM). (4) There was no pmf threshold for the proton transfer through the slipping enzyme; slip could be driven both by voltage and pH difference. (5) The conduction was ohmic and weakly pH-dependent in the range from 5.5 to 9.5. The rate constant of proton transfer under slip conditions was 185 s(-1) at pH 8. Proton slip probably presents the free-wheeling of the central rotary shaft, subunit gamma, in an open structure of the (alphabeta)3 hexagon with no nucleotides in the catalytic sites.
Bacterial Chromatophores↗
Search for intermediates of photosynthetic water oxidation.
Photosystem II of cyanobacteria and plants incorporates the catalytic centre of water oxidation. Powered and clocked by quanta of light the centre accumulates four oxidising equivalents before oxygen is released. The first three oxidising equivalents are stored on the Mn(4)Ca-cluster, raising its formal oxidation state from S0 to S3 and the third on YZ, producing S3 YZ ox. From there on water oxidation proceeds in what appears as a single reaction step (S3 YZ ox(H2O)2 <==>O2 + 4H+ + S0. Intermediate oxidation products of bound water had not been detected, until our recent report on the stabilisation of such an intermediate by high oxygen pressure (NATURE 430, 2004, 480-483). Based on the oxygen titration (half-point 2.3 bar) the standard free-energy profile of a reaction sequence with a single intermediate was calculated. It revealed a rather small difference (-3 kJ mol-1) between the starting state [S3YZ OX(H2O2) and the product state S0YZ + O2 + 4H+ . Here we describe the tests for side effects of exposing core particles to high oxygen pressure. We found the reduction of P680 +* in ns and the reduction/dismutation of quinones at the acceptor side of PSII both unaffected, and the inhibition of the oxygen evolving reaction by exposure to high O2-pressure was fully reversible by decompression to atmospheric conditions.
Absorption↗
Detection of an intermediate of photosynthetic water oxidation.
The oxygen that we breathe is produced by photosystem II of cyanobacteria and plants. The catalytic centre, a Mn4Ca cluster, accumulates four oxidizing equivalents before oxygen is formed, seemingly in a single reaction step 2H2O<==>O2 + 4H+ + 4e-. The energy and cycling of this reaction derives solely from light. No intermediate oxidation product of water has been detected so far. Here, we shifted the equilibrium of the terminal reaction backward by increasing the oxygen pressure and monitoring (by absorption transients in the near-ultraviolet spectrum) the electron transfer from bound water into the catalytic centre. A tenfold increase of ambient oxygen pressure (2.3 bar) half-suppressed the full progression to oxygen. The remaining electron transfer at saturating pressure (30 bar) was compatible with the formation of a stabilized intermediate. The abstraction of four electrons from water was probably split into at least two electron transfers: mildly endergonic from the centre's highest oxidation state to an intermediate, and exergonic from the intermediate to oxygen. There is little leeway for photosynthetic organisms to push the atmospheric oxygen concentration much above the present level.
Absorption↗
Time-resolved oxygen production by PSII: chasing chemical intermediates.
Photosystem II (PSII) produces dioxygen from water in a four-stepped process, which is driven by four quanta of light and catalysed by a Mn-cluster and tyrosine Z. Oxygen is liberated during one step, coined S(3)=>S(0). Chemical intermediates on the way from reversibly bound water to dioxygen have not yet been tracked, however, a break in the Arrhenius plot of the oxygen-evolving step has been taken as evidence for its existence. We scrutinised the temperature dependence of (i) UV-absorption transients attributable to the reduction of the Mn-cluster and tyrosine Z by water, and (ii) polarographic transients attributable to the release of dioxygen. Using a centrifugatable and kinetically competent Pt-electrode, we observed no deviation from a linear Arrhenius plot of oxygen release in the temperature range from -2 to 32 degrees C, and hence no evidence, by this approach, for a sufficiently long-lived chemical intermediate. The half-rise times of oxygen release differed between Synechocystis WT* (at 20 degrees C: 1.35 ms) and a point mutant (D1-D61N: 13.1 ms), and the activation energies differed between species (Spinacia oleracea, 30 kJ/mol versus Synechocystis, 41 kJ/mol) and preparations (PSII membranes, 41 kJ/mol versus core complexes, 33 kJ/mol, Synechocystis). Correction for polarographic artefacts revealed, for the first time, a temperature-dependent lag-phase of the polarographic transient (duration at 20 degrees C: 0.45 ms, activation energy: 31 kJ/mol), which was indicative of a short-lived intermediate. It was, however, not apparent in the UV-transients. Thus the "intermediate" was probably newly formed and transiently bound oxygen.
Cyanobacteria↗
Determination of reference intervals for serum creatinine, creatinine excretion and creatinine clearance with an enzymatic and a modified Jaffé method.
OBJECTIVES: The aim of this study was to determine the reference intervals for serum creatinine, the renal creatinine output and the creatinine clearance (CrCl) with two new methods for accurate creatinine determination. METHODS: The reference population consisted of 252 healthy subjects (127 males and 125 females) at the age between 18 and 74 years, median 27. Urine was collected for exactly 24 h. Creatinine in serum and urine was measured with an enzymatic assay ("Creatinine Plus") and a modification of the kinetic Jaffé reaction, named "Jaffé compensated". RESULTS: Reference values for serum creatinine were almost identical to previously published ones obtained with the same methods: 0.73-1.18 and 0.55-1.02 mg/dl for males and females, respectively, with the enzymatic, 0.72-1.16 and 0.55-0.96 mg/dl with the compensated Jaffé method (Jcomp). CrCl values were normally distributed and showed no gender difference in contrast to some previous studies. The reference interval for the entire group was found to be 66-143 ml/min with the enzymatic assay and 71-151 ml/min with the chemical one.
Adolescent↗
Proton transfer dynamics at the membrane/water interface: dependence on the fixed and mobile pH buffers, on the size and form of membrane particles, and on the interfacial potential barrier.
Crossing the membrane/water interface is an indispensable step in the transmembrane proton transfer. Elsewhere we have shown that the low dielectric permittivity of the surface water gives rise to a potential barrier for ions, so that the surface pH can deviate from that in the bulk water at steady operation of proton pumps. Here we addressed the retardation in the pulsed proton transfer across the interface as observed when light-triggered membrane proton pumps ejected or captured protons. By solving the system of diffusion equations we analyzed how the proton relaxation depends on the concentration of mobile pH buffers, on the surface buffer capacity, on the form and size of membrane particles, and on the height of the potential barrier. The fit of experimental data on proton relaxation in chromatophore vesicles from phototropic bacteria and in bacteriorhodopsin-containing membranes yielded estimates for the interfacial potential barrier for H(+)/OH(-) ions of approximately 120 meV. We analyzed published data on the acceleration of proton equilibration by anionic pH buffers and found that the height of the interfacial barrier correlated with their electric charge ranging from 90 to 120 meV for the singly charged species to >360 meV for the tetra-charged pyranine.
Bacterial Chromatophores↗
Protons, proteins and ATP.
The machinery of life has been disclosed in the second half of the 20th century to a degree not in the least envisioned previously by even the most daring players in this field. It has been extremely rewarding to start out from the fogs and to enjoy the brightness at the end of one's active career. Perhaps the most astounding lesson to learn is how conservative and modular is the construction of key devices. Oxidative and photophosphorylation are carried out by ATP synthase, which is unique in converting electrochemical, mechanical and chemical forms of energy within one nano-machine. This complex protein consists of more than 20 polypeptides of at least eight different kinds. Still, its activity survives in engineered chimerical constructs joining parts from organisms that underwent billions of years of separate evolution. The path of discovery of its structure and function is sketched here from a personal viewpoint. It has been a long way from before-structure-bioenergetics to the post-structural one (which now dominates the biology textbooks), but there is still a long way to go for a rigorous physical understanding. The author has been privileged to enjoy the friendship, cooperation and competition of excellent scientists from widely different backgrounds and expertise.
Journal Article↗
Rotary F1-ATPase. Is the C-terminus of subunit gamma fixed or mobile?
F-ATP synthase synthesizes ATP at the expense of ion motive force by a rotary coupling mechanism. A central shaft, subunit gamma, functionally connects the ion-driven rotary motor, F(O), with the rotary chemical reactor, F(1). Using polarized spectrophotometry we have demonstrated previously the functional rotation of the C-terminal alpha-helical portion of gamma in the supposed 'hydrophobic bearing' formed by the (alpha beta)(3) hexagon. In apparent contradiction with these spectroscopic results, an engineered disulfide bridge between the alpha-helix of gamma and subunit alpha did not impair enzyme activity. Molecular dynamics simulations revealed the possibility of a 'functional unwinding' of the alpha-helix to form a swivel joint. Furthermore, they suggested a firm clamping of that part of gamma even without the engineered cross-link, i.e. in the wild-type enzyme. Here, we rechecked the rotational mobility of the C-terminal portion of gamma relative to (alpha beta)(3). Non-fluorescent, engineered F(1) (alpha P280C/gamma A285C) was oxidized to form a (nonfluorescent) alpha gamma heterodimer. In a second mutant, containing just the point mutation within alpha, all subunits were labelled with a fluorescent dye. Following disassembly and reassembly of the combined preparations and cystine reduction, the enzyme was exposed to ATP or 5'-adenylyl-imidodiphosphate (AMP-PNP). After reoxidation, we found fluorescent alpha gamma dimers in all cases in accordance with rotary motion of the entire gamma subunit under these conditions. Molecular dynamics simulations covering a time range of nanoseconds therefore do not necessarily account for motional freedom in microseconds. The rotation of gamma within hours is compatible with the spectroscopically detected blockade of rotation in the AMP-PNP-inhibited enzyme in the time-range of seconds.
Adenosine Triphosphate↗
Determination of lipase catalytic activity in two reference materials: BCR 693 and BCR 694 by titrimetry at constant pH.
Because routine assays for pancreatic lipase catalytic activity are not yet standardized, between-method comparability is very poor. This is mainly due to the lack of reference materials (RMs). The aim of this study was to assign values of catalytic concentration to two human pancreatic lipase RMs, one prepared from human pancreatic juice (BCR 693), the other obtained by recombinant technology (BCR 694). Lipase catalytic activity was assayed in five experienced laboratories, using aliquots from the same lot of triolein emulsion and a standardized titrimetric procedure, optimized with regard to substrate, cofactors and pH. The accepted sets of data (n = 4) gave a mean +/- the corresponding uncertainty expressed as the 0.95 confidence interval of 1732 +/- 72 U/l and 1043 +/- 60 U/l for BCR 693 and 694, respectively. Transferability of the whole operating procedure proved to be quite satisfactory. The authors conclude that both RMs can be used to verify the correct implementation of the standardized measurement procedure and to assign values to secondary lipase materials (commercial calibrators, control products) which, in turn, ensures traceability to the standardized procedure in this study, and contributes to the harmonization of laboratory results according to the Directive for in vitro Diagnostic Medical Devices.
Animals↗
Creation of the necessary analytical quality for generating and using reference intervals.
This is a brief compilation of criteria on analytical quality that are prerequisites for the establishment and use of reference intervals in laboratory medicine. In this context, analytical quality is characterized by the precision, accuracy and long-term stability of laboratory methods performed with diagnostic kits, including their application on various instrument platforms. Systematically, analytical quality can be differentiated into quality of the method and its performance, and quality of long-term use. Factors influencing these types of quality in the development of diagnostic kits are discussed. The responsibility for maintaining analytical quality is shared by the two parties involved in the provision and use of reference intervals: The manufacturers of diagnostic kits are committed to providing complete description and traceability of their methods, whereas users must consider the effects of physiological, biometrical, preanalytical and analytical influences on the reference interval.
Calibration↗
The proton-driven rotor of ATP synthase: ohmic conductance (10 fS), and absence of voltage gating.
The membrane portion of F(0)F(1)-ATP synthase, F(0), translocates protons by a rotary mechanism. Proton conduction by F(0) was studied in chromatophores of the photosynthetic bacterium Rhodobacter capsulatus. The discharge of a light-induced voltage jump was monitored by electrochromic absorption transients to yield the unitary conductance of F(0). The current-voltage relationship of F(0) was linear from 7 to 70 mV. The current was extremely proton-specific (>10(7)) and varied only slightly ( approximately threefold) from pH 6 to 10. The maximum conductance was approximately 10 fS at pH 8, equivalent to 6240 H(+) s(-1) at 100-mV driving force, which is an order-of-magnitude greater than of coupled F(0)F(1). There was no voltage-gating of F(0) even at low voltage, and proton translocation could be driven by deltapH alone, without voltage. The reported voltage gating in F(0)F(1) is thus attributable to the interaction of F(0) with F(1) but not to F(0) proper. We simulated proton conduction by a minimal rotary model including the rotating c-ring and two relay groups mediating proton exchange between the ring and the respective membrane surface. The data fit attributed pK values of approximately 6 and approximately 10 to these relays, and placed them close to the membrane/electrolyte interface.
ATP Synthetase Complexes↗
Low dielectric permittivity of water at the membrane interface: effect on the energy coupling mechanism in biological membranes.
Protonmotive force (the transmembrane difference in electrochemical potential of protons, ) drives ATP synthesis in bacteria, mitochondria, and chloroplasts. It has remained unsettled whether the entropic (chemical) component of relates to the difference in the proton activity between two bulk water phases (deltapH(B)) or between two membrane surfaces (deltapH(S)). To scrutinize whether deltapH(S) can deviate from deltapH(B), we modeled the behavior of protons at the membrane/water interface. We made use of the surprisingly low dielectric permittivity of interfacial water as determined by O. Teschke, G. Ceotto, and E. F. de Souza (O. Teschke, G. Ceotto, and E. F. de Sousa, 2001, PHYS: Rev. E. 64:011605). Electrostatic calculations revealed a potential barrier in the water phase some 0.5-1 nm away from the membrane surface. The barrier was higher for monovalent anions moving toward the surface (0.2-0.3 eV) than for monovalent cations (0.1-0.15 eV). By solving the Smoluchowski equation for protons spreading away from proton "pumps" at the surface, we found that the barrier could cause an elevation of the proton concentration at the interface. Taking typical values for the density of proton pumps and for their turnover rate, we calculated that a potential barrier of 0.12 eV yielded a steady-state pH(S) of approximately 6.0; the value of pH(S) was independent of pH in the bulk water phase under neutral and alkaline conditions. These results provide a rationale to solve the long-lasting problem of the seemingly insufficient protonmotive force in mesophilic and alkaliphilic bacteria.
Cell Membrane↗
Electrostatics and proton transfer in photosynthetic water oxidation.
Photosystem II (PSII) oxidizes two water molecules to yield dioxygen plus four protons. Dioxygen is released during the last out of four sequential oxidation steps of the catalytic centre (S(0) --> S(1), S(1) --> S(2), S(2) --> S(3), S(3) --> S(4) --> S(0)). The release of the chemically produced protons is blurred by transient, highly variable and electrostatically triggered proton transfer at the periphery (Bohr effect). The extent of the latter transiently amounts to more than one H(+)/e(-) under certain conditions and this is understood in terms of electrostatics. By kinetic analyses of electron-proton transfer and electrochromism, we discriminated between Bohr-effect and chemically produced protons and arrived at a distribution of the latter over the oxidation steps of 1 : 0 : 1 : 2. During the oxidation of tyr-161 on subunit D1 (Y(Z)), its phenolic proton is not normally released into the bulk. Instead, it is shared with and confined in a hydrogen-bonded cluster. This notion is difficult to reconcile with proposed mechanisms where Y(Z) acts as a hydrogen acceptor for bound water. Only in manganese (Mn) depleted PSII is the proton released into the bulk and this changes the rate of electron transfer between Y(Z) and the primary donor of PSII P(+)(680) from electron to proton controlled. D1-His190, the proposed centre of the hydrogen-bonded cluster around Y(Z), is probably further remote from Y(Z) than previously thought, because substitution of D1-Glu189, its direct neighbour, by Gln, Arg or Lys is without effect on the electron transfer from Y(Z) to P(+)(680) (in nanoseconds) and from the Mn cluster to Y(ox)(Z).
Binding Sites↗