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A J Hoff

Publications and source records attributed to A J Hoff.

At least 37 records · Page 2Linked to original sources
PubMed1994

QA binding in reaction centers of the photosynthetic purple bacterium Rhodobacter sphaeroides R26 investigated with electron spin polarization spectroscopy.

The relation between quinone (QA) binding and electron transport in reaction centers (RCs) of photosynthetic purple bacteria is investigated, using electron spin polarization (ESP) X-band (9 GHz) EPR as a tool to probe for structural changes resulting from charge separation and stabilization and from replacing the native QA molecule with other quinones. We present a study of possible changes in QA-binding that might be responsible for the remarkably prolonged lifetime of the charge-separated state at cryogenic temperatures for RCs of Rhodobacter sphaeroides R26 cooled under illumination [Kleinfeld, D., et al. (1984) Biochemistry 23, 5780-5786]. It is shown that this effect is not caused by a major reorientation of the chromophores. Furthermore, we studied the effects of structurally different quinones functioning as primary electron acceptor in different purple bacteria. With simulations of ESP X-band spectra of the spin-polarized secondary radical pair P.+QA.+- in menaquinone-reconstituted, Zn(2+)-substituted RCs of Rb. sphaeroides R26, we show that quinone reconstitution is highly selective for site and orientation. Furthermore, we find that a very small exchange interaction between P.+ and QA.+- (magnitude of JPQ approximately 1 microT) is needed to account accurately for the observed relative line intensities at X-band, without affecting the accuracy of the simulations of reported ESP K-band spectra [Füchsle, G., et al. (1993) Biochim. Biophys. Acta 1142, 23-35; Van der Est, A., et al. (1993) Chem. Phys. Lett. 212, 561-568]. This pronounced influence of small values for JPQ on the X-band ESP line shape results from cancellation effects of absorptive and emissive contributions to the spectrum, such that small shifts can be observed. The exchange interaction has opposite sign for the native, ubiquinone-containing RC [viz. JP.UQ = (-0.8 +/- 0.2) microT] and the menaquinone-substituted RC [JP.MK = (+0.3 +/- 0.2) microT]. The implications of these observations for electron-transport theory are discussed.

Electrochemistry↗
PubMed1994

Asymmetric binding of the primary acceptor quinone in reaction centers of the photosynthetic bacterium Rhodobacter sphaeroides R26, probed with Q-band (35 GHz) EPR spectroscopy.

The reaction center (RC)-bound primary acceptor quinone QA of the photosynthetic bacterium Rhodobacter sphaeroides R26 functions as a one-electron gate. The radical anion QA.- is proposed to have an asymmetric electron distribution, induced by the protein environment. We replace the native ubiquinone-10 (UQ10) with specifically 13C-labelled UQ10, and use Q-band (35 GHz) EPR spectroscopy to investigate this phenomenon in closer detail. The direct observation of the 13C-hyperfine splitting of the gz-component of UQ10A.- in the RC and in frozen isopropanol shows that the electron spin distribution is symmetric in the isopropanol glass, and asymmetric in the RC. Our results allow qualitative assessment of the spin and charge distribution for QA.- in the RC. The carbonyl oxygen of the semiquinone anion nearest to the S = 2 Fe(2+)-ion and QB is shown to acquire the highest (negative) charge density.

1-Propanol↗
PubMed1994

Determination of the number of detergent molecules associated with the reaction center protein isolated from the photosynthetic bacterium Rhodopseudomonas viridis. Effects of the amphiphilic molecule 1,2,3-heptanetriol.

Detergent-free reaction center (RC) proteins from the photosynthetic bacterium Rhodopseudomonas viridis were obtained using Bio-Beads SM-2. With these RCs, the amount of detergent molecules associated with the protein was measured by determining the detergent concentration at which re-solubilization occurred as a function of the RC concentration. For N,N-dimethyl dodecylamine-N-oxide (LDAO), Triton X-100 and beta-octylglucoside 260 +/- 30,105 +/- 10 and 360 +/- 100 detergent molecules were necessary to dissolve the protein, respectively. With this technique we have studied the effect of the amphiphilic molecule 1,2,3-heptanetriol, which is essential in the crystallization process of these RCs. Addition of 5% 1,2,3-heptanetriol reduces the value for LDAO to 120 +/- 20 LDAO/RC, supporting the notion that crystallization of the RCs is promoted by increasing the number of protein-protein contacts.

Crystallization↗
PubMed1992

13C magic angle spinning NMR evidence for a 15,15'-cis configuration of the spheroidene in the Rhodobacter sphaeroides photosynthetic reaction center.

The photosynthetic reaction center of Rhodobacter sphaeroides 2.4.1 contains one carotenoid that protects the protein complex against photodestruction. The structure around the central (15,15') double bond of the bound spheroidene carotenoid was investigated with low-temperature magic angle spinning 13C NMR, which allows an in situ characterization of the configuration of the central double bond in the carotenoid. Carotenoidless reaction centers of R. sphaeroides R26 were reconstituted with spheroidene specifically labeled at the C-14' or C-15' position, and the signals from the labels were separated from the natural abundance background using 13C MAS NMR difference spectroscopy. The resonances shift 5.2 and 3.8 ppm upfield upon incorporation in the protein complex, similar to the 5.6 and 4.4 ppm upfield shift occurring in the model compound beta-carotene upon trans to 15,15'-cis isomerization. Hence the MAS NMR favors a cis configuration, as opposed to the trans configuration deduced from X-ray data.

Carbon Isotopes↗
PubMed1992

13C magic angle spinning NMR study of the light-induced and temperature-dependent changes in Rhodobacter sphaeroides R26 reaction centers enriched in [4'-13C]tyrosine.

Solid-state 13C magic angle spinning (MAS) NMR has been used to investigate detergent-solubilized photosynthetic reaction centers of Rhodobacter sphaeroides R26, selectively enriched in [4-13C]-tyrosine. The reaction centers were frozen, in the dark and while subject to intense illumination, and studied at temperatures between approximately 215 and approximately 260 K. The signal consists of at least seven narrow lines superimposed on a broad doublet. The chemical shift anisotropy is similar to that for crystalline tyrosine. The two narrowest resonances, corresponding to signals from individual tyrosines, are 28 +/- 5 Hz wide, comparable to what is observed for quaternary carbons in linearly elastic organic solids. The line width as well as the chemical shift of these signals is essentially independent of temperature. This provides strong evidence for an unusually ordered, well-shielded, and structurally, electrostatically, and thermodynamically stable interior of the protein complex without structural heterogeneities. As the temperature is lowered, additional signal from the labels develops and the natural abundance resonances from the detergent broaden, providing evidence for considerable flexibility at the exterior of the protein complex and in the detergent belt at the higher temperatures. In addition, the NMR provides evidence for an electrostatically uniform and neutral complex, since the total dispersion in isotropic shifts for the labels is < 5 ppm and corresponds to electron density variations of less than 0.03 electronic equivalents with respect to tyrosine in the solid state or in solution. When the sample is frozen while subject to intense illumination, a substantial part of the protein is brought into the charge-separated state P.+QA.-. At least three sharp resonances, including the narrowest lines, are substantially reduced in intensity. It is argued that this effect is caused by the electronic spin density associated with the oxidized primary donor P.+. These results strongly suggest that the environment of the special pair is extremely rigid and question the role of protein conformational distortions during the primary photoprocess.

Carbon Isotopes↗
PubMed1991

ESE relaxation measurements in photosystem II. The influence of the reaction center non-heme iron on the spin-lattice relaxation of Tyr D.

The spin-lattice relaxation of the tyrosine radical D. in Photosystem II particles was studied at 4.2 K in samples in which flash-induced oscillations of the oxidation state of the Mn-cluster of the oxygen evolving system were abolished by addition of ANT2P, leaving Fe2+/Fe3+ oscillations intact. Samples subjected to 0, 1 or 2 light-flashes all showed the same relaxation kinetics. No period-2 oscillation in the spin-lattice relaxation corresponding to the Fe2+/Fe(3+)-oscillation was observed. Thus the T1-oscillations of D. as a function of flash number in untreated samples are solely caused by the charge-oscillations of the Mn-cluster (1989, Biochim, Biophys. Acta 973, 428-442).

Electron Spin Resonance Spectroscopy↗
PubMed1991

Influence of the H-subunit and Fe2+ on electron transport from I- to QA in Fe(2+)-free and/or H-free reaction centers from Rhodobacter sphaeroides R-26.

From reaction centres (RC) of Rhodobacter sphaeroides R-26 two LM preparations with 0.90 Fe2+/RC (LM) and 0.10 Fe2+/RC (LM/dFe) were prepared. Reconstitution of LM/dFe with the H-subunit and subsequently with Zn2+ yielded LMH/dFe and LMH/dFe and LMH/dFe + Zn preparations, respectively. In these four samples the decay of the primary radical pair P+I- was studied by means of transient absorption spectroscopy and compared with that in native RC. In LMH/dFe the reduction of QA by Bpheo a occurred in 5 ns, with concomitant increase in the yield of PT, the triplet state of the primary donor. In the LM/dFe, LM and LMH/dFe + Zn preparations the decay of I- had the same rate (200 ps)-1 as in native RC. Thus, neither the H-subunit in the RC nor a divalent metal as Fe2+ or Zn2+ are necessary per se for fast reduction of QA. Only demetallation in the presence of the H-subunit slows down the reduction of QA.

Cations, Divalent↗
PubMed1990

Mass spectrometric determination of isotopically labeled tyrosines and tryptophans in photosynthetic reaction centers of Rhodobacter sphaeroides R-26.

A synthetic medium for growing Rhodobacter sphaeroides R-26 is developed. This medium opened the way to the preparation of photosynthetic reaction centers incorporated with L-[4'-13C]tyrosine or L-[1'-15N]tryptophan. Gas chromatography combined with mass spectroscopy was used to estimate the metabolic incorporation of the labeled amino acid into the protein. Conditions were found for near-quantitative incorporation of labeled aromatic amino acids into the reaction center.

Chromatography, Gas↗
PubMed1989

Fluorescence spectra of hematoporphyrin and hematoporphyrin-diacetate aggregates in buffer solution.

Different fluorescence spectra are observed on preferential excitation of monomer and aggregate species of hematoporphyrin and hematoporphyrin-diacetate, respectively. From these measurements we obtain the fluorescence spectrum of aggregate species in buffer solution, which shows two maxima, at 628 nm and around 700 nm. The fluorescence intensity in the region between these maxima is very sensitive to environmental changes. It is suggested that the changes in the fluorescence of hematoporphyrin in vivo as compared with that in buffer solution is associated with both hematoporphyrin-substrate interactions and an enhanced contribution from aggregate species.

Buffers↗
PubMed1987

Exciton interactions in reaction centers of the photosynthetic bacterium Rhodopseudomonas viridis probed by optical triplet-minus-singlet polarization spectroscopy at 1.2 K monitored through absorbance-detected magnetic resonance.

Linear dichroic triplet-minus-singlet [LD-(T - S)] spectra of isolated reaction centers of the photosynthetic bacterium Rhodopseudomonas viridis have been measured at 1.2 K with the linear dichroic absorbance-detected magnetic resonance (LD-ADMR) technique for two mutually perpendicular directions of the preferred axis. The LD-(T - S) spectra have been calibrated with respect to the corresponding (T - S) spectra as a function of applied microwave power and quantitatively interpreted using the formalism of photoselection. The transition moment of the optical transition at 1007 nm makes angles of 72 degrees +/- 5 degrees and 15 degrees +/- 5 degrees with the triplet x and y spin axes, respectively. The experimental spectra have been simulated employing exciton theory and using the atomic coordinates of the resolved crystal structure of the reaction center. The spectral interpretation yields the angles between the transition moments of the various absorption bands of the (T - S) spectra and the triplet axes, and between the moments themselves, with the triplet state of the primary donor (3)P localized on the P-bacteriochlorophyll b in the "active" (L) chain.

Journal Article↗
PubMed1986

Role of charge-transfer states in bacterial photosynthesis.

Photon echo, photon-echo excitation, and "hole-burning" data recorded in the 800-990 nm region of Rhodobacter sphaeroides R26 and Rhodopseudomonas viridis reaction centers are reported. The primary process in these reaction centers, following excitation, was found to occur in approximately 25 fsec; the long-wavelength band of the primary electron donor (P) was largely homogeneously broadened. In accordance with our previous explanation of hole-burning and photon-echo measurements on Rb. sphaeroides [Meech, S. R., Hoff, A. J. & Wiersma, D. A. (1985) Chem. Phys. Lett. 121, 287-292], we interpret this as resulting from a dephasing of the excitation in P into a background of strongly coupled charge-transfer states. The previously reported picosecond lifetime of the excited P state is assigned to decay of these strongly mixed states. Further, a coupling between P and an adjacent bacteriochlorophyll was observed. The extent of this coupling and the role of charge-transfer states in the functioning of reaction centers is discussed.

Journal Article↗
PubMed1986

Femtosecond spectroscopy of electron transfer in the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26: Direct electron transfer from the dimeric bacteriochlorophyll primary donor to the bacteriopheophytin acceptor with a time constant of 2.8 +/- 0.2 psec.

The primary light-induced charge separation in reaction centers from Rhodopseudomonas sphaeroides R-26 has been investigated after excitation with laser pulses of 150 fsec duration within the longwave absorption band of the primary donor at 850 nm. An excited state of the primary donor, characterized by a broad absorption spectrum extending over the whole spectral range investigated (545-1240 nm), appeared within 100 fsec and gave rise to stimulated emission in the 870- to 1000-nm region with a 2.8-psec lifetime. The photooxidation of the primary donor, as measured at 1240 nm, and the photoreduction of the bacteriopheophytin acceptor, monitored at 545 nm and 675 nm, have been found to proceed simultaneously with a time constant of 2.8 +/- 0.2 psec. Kinetics of absorbance changes at other probe wavelengths gave no indication that an accessory bacteriochlorophyll is involved as a transient electron acceptor.

Journal Article↗
PubMed1981

The balance between primary forward and back reactions in bacterial photosynthesis.

The temperature dependence of the bacteriochlorophyll fluorescence and reaction center triplet yield in while cells of Rhodopseudomonas sphaeroides strain 2.4.1 and of the magnetic field-induced fluorescence increase are calculated, taking into account rate constants of losses in the antenna system and of charge separation and recombination in the reaction center. Triplet and singlet yield after recombination in the reaction center are described by the radical pair mechanism. Good fits of the theoretically calculated temperature dependence with published experimental results could be obtained, assuming that ks, the rate constant for recombination of the charges on the primary donor P+ and the reduced intermediate acceptor I- to the lowest excited singlet state P*I of the reaction center bacteriochlorophyll, is temperature-dependent via the Boltzmann factor Kso exp(-delta E/kT), where delta E is the energy difference between P*I and P+I- and kso is the frequency factor. kg and/or kt, the rate constants for recombination to the singlet ground and triplet states, respectively, were assumed to be temperature-independent, or temperature-dependent via their exothermicity factors ki = CiT-1/2 exp(-Ei/kT) with i = g, t. Depending on the particular choice for the temperature dependence of kg and kt, best fits were obtained for delta E = 45-75 meV and recombination rate constants at 300 K of ks = 0.4-0.8 ns-1, kg = 0.08-0.12 ns-1, and kt = 0.3-0.5 ns-1. The model predicts a lifetime of the radical pair P+I- that is somewhat larger than that of delayed fluorescence; a magnetic field increases both.

Kinetics↗
PubMed1980

Carotenoid triplet yields in normal and deuterated Rhodospirillum rubrum.

Quantum yields of carotenoid triplet formation in Rhodospirillum rubrum wild type and fully deuterated cells and chromatophores were determined in weak laser flashes for excitation wavelength lambda i = 530 nm (mainly absorbed by the carotenoid spirilloxanthin) and for lambda i = 608 nm (mainly absorbed by bacteriochlorophyll) in the presence and absence of magnetic fields. All experiments were performed at room temperature and in the absence of oxygen. The quantum yield of reaction center bacteriochlorophyll oxidation in wild type preparations, in which all reaction centers are in state PIX, at lambda i = 608 nm is close to unity, whereas the quantum yield of antenna carotenoid triplet formation is low (about 5%); P is the primary electron donor, a bacteriochlorophyll dimer, I the primary acceptor, a bacteriopheophytin, and X the secondary acceptor, an iron-ubiquinone complex. In cells in which the reaction centers are in the state P+IX(-), the antenna carotenoid triplet yield is about 0.2. In contrast, at lambda i = 530 nm, the quantum yield of P+ formation is relatively low (0.3) and the yield of the antenna carotenoid triplet state in state PIX unusually high (0.3). At increasing light intensities of 530 nm only about 3 carotenoids per reaction center of the 15 carotenoids present are efficiently photoconverted into the triplet state, which indicates that there are two different pools of carotenoids, one with a low efficiency for transfer of electronic excitation to bacteriochlorophyll and a high yield for triplet formation, the other with a high transfer efficiency and a low triplet yield. The absorption difference spectrum of the antenna carotenoid triplet, excited by 608 or 530 nm light in the state P+IX(-) does not show the peak at 430 nm, that is present in the difference spectrum of the reaction center carotenoid triplet, mainly observed at lambda i = 608 nm with weak flashes. The yield of the reaction center carotenoid triplet, generated in chromatophores in the state PIX(-), is decreased by about 10% by a magnetic field of 0.6 T. In a magnetic field of 0.6 T the yield of the carotenoid triplet, formed by 530 nm excitation in chromatophores at ambient redox potential, is decreased by about 45%. The high quantum yield of formation and the pronounced magnetic field effect for the carotenoid triplet generated by direct excitation at 530 nm can be explained by assuming that this triplet is not formed by intersystem crossing, but by fission of the singlet excitation into two triplet excitations and subsequent annihilation (triplet pair mechanism), or by charge separation and subsequent recombination (radical pair mechanism). Fully deuterated bacteria give essentially the same triplet yields, both in the reaction center and in the antenna carotenoids and show the same magnetic field effects as non-deuterated samples. This indicates that hyperfine interactions do not play a major role in the dephasing of the spins in the radical pair P+I- nor in the formation of the antenna carotenoid triplet.

Carotenoids↗
PubMed1980

Electron spin echo studies on chloroplasts. Spectral characteristics of electron transport components and light-induced transients.

Electron-spin resonance echoes are used to study the complex overlapping ESR spectra of whole chloroplasts. By varying the repetition rate of the microwave pulse sequence, delay time, and pulse width, signals with different longitudinal and transverse relaxation times were extracted. We have identified the echo signals due to plastocyanin and ferredoxins. In addition, we have found a strong signal at g = 4.3, that possibly arises from distorted cytochrome, and weak signals in the region g = 6-9. The strong echo signal at g = 2.0047 (Signal II), is made up of at least three "dark" components that differ in their relaxation times. Upon illumination at 1.2 K several of the echo signals including Signal II show reversible light-induced components. The kinetics of these transients depend on the addition of 3(3,4-dichlorophenyl)-1,1-dimethyl urea. Part of the transients are believed to arise from cyclic electron flow around Photosystem I.

Chloroplasts↗
PubMed1979

Transfer of light-induced electron-spin polarization from the intermediary acceptor to the prereduced primary acceptor in the reaction center of photosynthetic bacteria.

In reaction centers and chromatophores of photosynthetic bacteria strong light-induced emissive ESR signals have been found, not only after a flash but also under continuous illumination. The signal, with g = 2.0048 and delta Hpp = 7.6 G, is only present under reducing conditions in material in which the primary acceptor, ubiquinone, U and its associated high-spin ferrous ion are magnetically uncoupled. its amplitude under continuous illumination is strongly dependent on light intensity and on microwave power. The emissive signal is attributed to the prereduced primary acceptor, U-, which becomes polarized through transfer of spin polarization by a magnetic exchange interaction with the photoreduced, spin polarized intermediary acceptor, I-. A kinetic model is presented which explains the observed dependence of emissivity on light intensity and microwave power. Applying this analysis to the light saturation data, a value of the exchange rate between I- and U- of 4.10(8) s-1 is derived, corresponding to an exchange interaction of 3--5 G.

Bacterial Chromatophores↗