Bacterial Photosynthesis Research Articles

EPR on Biological Samples beyond the Limits of Superconducting MagnetsThe Primary Donor Cation of Purple Bacterial Photosynthesis

The g-matrix of a free radical is an important observable that yields information on its electronic structure. It is usually measured by electron paramagnetic resonance (EPR) under “high field” conditions, where the spectral splitting of the principal g-factor components is larger than the line width due to unresolved hyperfine splitting. For large organic molecules such as the primary electron donor in photosynthetic reaction centers (RC) this usually requires fields above 11 T, or, for fields between 3 and 11 T, full deuteration and/or single-crystal work. When trying to obtain improved spectral resolution a major concern is the presence of g-strain which leads to extra line broadening. Here we show that g-strain is negligible for bacterial RCs up to a field of 24 T. We investigated the temperature dependence of the g-anisotropy for RCs from Rhodobacter (Rb.) sphaeroides using different detergents and find that within experimental errors there is no change in the principal g-matrix components up to 200 K. This is the first report of a successful EPR experiment on a biological sample above the limits of superconducting magnets.

Regulation of bacterial photosynthesis genes by oxygen and light.

Most bacteria have the capability to adapt to changes in their environment. Facultatively phototrophic bacteria like Rhodobacter can switch from aerobic respiration to anoxygenic photosynthesis in the absence of oxygen. The formation of the photosynthetic apparatus is primarily regulated by oxygen tension. The amount of photosynthetic complexes is influenced by the light intensity in anaerobic cultures. This review focuses on the molecular mechanisms involved in the regulation of Rhodobacter photosynthesis genes by oxygen and light.

Regulation of bacterial photosynthesis genes by oxygen and light

Most bacteria have the capability to adapt to changes in their environment. Facultatively phototrophic bacteria like Rhodobacter can switch from aerobic respiration to anoxygenic photosynthesis in the absence of oxygen. The formation of the photosynthetic apparatus is primarily regulated by oxygen tension. The amount of photosynthetic complexes is influenced by the light intensity in anaerobic cultures. This review focuses on the molecular mechanisms involved in the regulation of Rhodobacter photosynthesis genes by oxygen and light.

Hybrid density functional studies of a bacteriopheophytina model and its anion radical form: Geometry, spin densities, and hyperfine couplings

Hybrid density functional calculations are performed on models of the bacteriopheophytin a molecule, which acts as an electron acceptor in the initial electron transfer events of bacterial photosynthesis. The geometry of the neutral and one-electron reduced free radical form are calculated. Little variation in the main π framework geometry is predicted to occur on one-electron reduction. The electron density of the SOMO for the anion free radical form is also investigated, and the unpaired spin density distribution is also analyzed. Anisotropic and isotropic hyperfine couplings are calculated and discussed on the basis of the unpaired spin density distributions. The electron density of the SOMO is shown to extend on to the carbonyl group attached to ring A; the extent of this delocalization being dependent on the twist angle, this groupmakes with the main ring plane with the magnitude of the 1H hyperfine couplings of the 2methyl group strongly dependent on the value of this twist angle. Good agreement is observed between calculated and experimentally measured isotropic hyperfine couplings. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 1292–1298, 1999

Molecular Structure and Spectroscopy of a Bacteriopurpurin. A New Class of Bacteriochlorin Photosensitizers

Bacteriochlorins mediate energy and electron transfer in vivo and serve as biomimetic models of bacterial photosynthesis. Recently, the same physicochemical properties of the chromophores have made them attractive as photosensitizers for photodynamic therapy, a medical treatment that combines light, porphyrins, and oxygen to attack and destroy malignant tissue. Despite the intense biophysical, chemical, and medicinal interest in bacteriochlorins, very few high precision structures of the chromophores exist to provide the basis for theoretical calculations that seek to predict their extensively studied physicochemical properties. The authors report here the synthesis, spectral characterization, and structural determination of a metal-free bacteriopurpurin 1, a paradigm for a new class of photosensitizers comprised of porphyrins at the saturation level of bacteriochlorins flanked by two annelated, exocyclic cyclopentenyl rings. The molecule exhibits a very red-shifted Qy transition at 843 nm and is readily oxidized (at 0.39V vs SCE) to a {pi} cation radical with a distinctive EPR signal. The crystallographic results for 1 unambiguously identify the bacteriopurpurin, provide the first stereochemical parameters for this new class of expanded bacteriochlorins, extend the high resolution structural database for bacteriochlorins, and form the basis for theoretical calculations which correctly describe the optical features and electron density profile of 1more » and its cation radical.« less

Porphyrin analogs in bacterial photosynthesis

The electron transfer process in bacterial reaction centers is investigated. It is found that the side indole group Trp M252 in Rb sphaeroides (Trp M250 in Rps viridis) assists tunneling electron transfer by a distance of ∼14A from pheophytin to quinone.

The primary and secondary acceptors in bacterial photosynthesis III. Characterization of the quinone radicals Q A − ⋅ and Q B − ⋅ by EPR and ENDOR

Electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) techniques were used to investigate the electronic structure of the primary (Q A −⋅ ) and secondary (Q B −⋅ ) ubiquinone electron acceptors in reaction centers (RCs) of the photosynthetic bacteriumRhodobacter sphaeroides. To reduce the EPR linewidth, the high-spin Fe2+ present in native RCs was replaced by diamagnetic Zn2+. Experiments were performed both on frozen solutions and single crystals at microwave frequencies of 9, 35 and 94 GHz. Differences in the EPR/ENDOR spectra were observed for Q A −⋅ and Q B −⋅ , which are attributed to different environments of the quinones in the RC. The differences exhibited themselves in: (i) the g-tensors, (ii) the17O and13C hyperfine coupling (hfc) constants of the quinones labeled at the carbonyl group, (iii) the1H-hfcs of the quinone ring and (iv) the exchangeable protons hydrogen bonded to the carbonyl oxygens. From these results and from H/D exchange experiments, the following conclusions were drawn: both Q A −⋅ and Q B −⋅ have at least two hydrogen bonds of different strengths to the carbonyl oxygens. The hydrogen bonds for Q A −⋅ are stronger and more asymmetric than for Q B −⋅ . For Q A −⋅ the stronger bond (to O4) was assigned to His(M219) and the weaker (to O1) to Ala(M260). For Q B −⋅ the stronger bond (to O4) was assigned to His(L190), with several weaker bonds (to O1) to Ser(L223), Ile(224) and Gly(L225). From the temperature dependence of the hfcs of the exchangeable protons some dynamic properties of the RC were deduced. Hfcs with more distant nitrogens were observed by electron spin echo envelope modulation (ESEEM). For Q A −⋅ they were assigned to Nδ of His(M219) and to the peptide backbone nitrogen of Ala(M260) and for Q B −⋅ to Nδ of His(L190). These interactions indicate the extent of the electron wave function, which is important for the understanding of the electron transfer mechanism. Based on the magnetic resonance results, the function of the quinone acceptors in the reaction center is discussed.

The Electronic Structure of the Bacteriopheophytin a Anion Radical, in Vivo

Density functional calculations are used to calculate the electronic structure of the bacteriopheophytin a radical anion, φA, formed in the initial electron-transfer reactions of bacterial photosynthesis. Using the hybrid B3LYP functional together with the double ζ basis set EPR−II, 13C, 1H, 17O, and 14N isotropic and anisotropic hyperfine couplings are calculated and explained by reference to the electron density of the highest occupied molecular orbital and of the unpaired spin distribution around the radical. Good agreement is observed between calculated and experimental hyperfine couplings. Hydrogen bonding to the carbonyl group of ring E leads to minor changes in the unpaired spin density distribution and the resultant hyperfine couplings. The electronic structure of the anion radical form of the other bacteriopheophytin a molecule, φB, found along the inactive-electron-transfer (B) branch, is also studied, and the calculated electronic properties are compared with φA. It is shown that, whereas the e...

Supramolecular organization of the photosynthetic apparatus of Rhodobacter sphaeroides.

Native tubular membranes were purified from the purple non-sulfur bacterium Rhodobacter sphaeroides. These tubular structures contain all the membrane components of the photosynthetic apparatus, in the relative ratio of one cytochrome bc1 complex to two reaction centers, and approximately 24 bacteriochlorophyll molecules per reaction center. Electron micrographs of negative-stained membranes diffract up to 25 A and allow the calculation of a projection map at 20 A. The unit cell (a = 198 A, b = 120 A and gamma = 103 degrees) contains an elongated S-shaped supercomplex presenting a pseudo-2-fold symmetry. Comparison with density maps of isolated reaction center and light-harvesting complexes allowed interpretation of the projection map. Each supercomplex is composed of light-harvesting 1 complexes that take the form of two C-shaped structures of approximately 112 A in external diameter, facing each other on the open side and enclosing the two reaction centers. The remaining positive density is tentatively attributed to one cytochrome bc1 complex. These features shed new light on the association of the reaction center and the light-harvesting complexes. In particular, the organization of the light-harvesting complexes in C-shaped structures ensures an efficient exchange of ubihydroquinone/ubiquinone between the reaction center and the cytochrome bc1 complex.

The polarization model in bacterial photosynthesis.

The widely accepted model of reaction center /RC/ functioning is proved to come into contradiction with some recent data. In particular, it cannot explain why only a minor part of electronic excitations (approximately 10%) escapes from excited RC special pairs back to antenna BChls. Therefore we believe that the model must be substantially modernized. In 1981 we developed a new model/1,2/. We suggested a femtosecond state to precede primary e-transfer reaction due to reorientation of water molecule dipole in the electric field of excited RC dimer. This mechanism is responsible for energy trapping before the primary e-transport occurs. During last years his mechanism got support from various experimental works. Now this polarization model claims to fit all reliable experimental data at least in bacterial photosynthesis.

The primary and secondary acceptors in bacterial photosynthesis: II. The structure of the Fe2+-Q− complex

The primary processes of bacterial photosynthesis take place in a membrane-bound protein called the reaction center (RC). The process involves light-induced electron transfer from a primary electron donor to a sequence of electron acceptors. The terminal acceptors, operating sequentially, are two ubiquinones, QA and QB, that couple magnetically to a high-spin (S = 2) Fe2+ forming an Fe2+-Q− complex. We have used a variety of techniques to investigate the electronic as well as some features of the spatial structure of the Fe2+-Q− complex in RCs fromRb. sphaeroides. These include: (a) static magnetization measurements in the temperature range of 0.7 to 190 K and magnetic fields up to 8 kG, (b) EPR spectroscopy at helium temperatures at 1.2, 9, and 35 GHz, (c) Mossbauer spectroscopy and (d) extended X-ray fine structure (EXAFS) determinations. The results of these measurements showed that Fe2+ resides in an asymmetric ligand field environment forming 6 ligands with a combination of oxygens and nitrogens. From the EXAFS results the distances of the Fe2+ to the first and third coordination shells were determined. These spatial features were subsequently corroborated by the X-ray structure of the RC, which showed the environment of the Fe to be a distorted octahedron, the base plane of which is formed by three Nɛ’s of histidines and one carbonyl oxygen; the apex is formed by a fourth Nɛ and a second carbonyl oxygen. The distances from the Fe2+ to the first and third shell were in good agreement with the values obtained from EXAFS. The most detailed information of the electronic structure of the Fe2+-Q− complex was obtained from the EPR spectra using the spin Hamiltonian formalism. The following spin-Hamiltonian parameters were obtained: for the crystalline field parameters,D = 7.60 K,E/D = 0.25; for the electronicg-values, gFe, x = 2.16, gFe, y = 2.27, gFe, z = 2.04 and for the antiferromagnetic exchange interaction,J x = −0.13 K,J y = −0.58 K,J z = −0.58 K. The use of the spin Hamiltonian was validated by comparing the results obtained from it with those obtained from an exact numerical solution of the 25 lowest energy levels of the orbital Hamiltonian. Possible roles that Fe2+ may play in the function of the RC are discussed. They include a structural role in which Fe2+ liganded to four histidine nitrogens imparts a rigid structure to a four α-helix bundle and a role in the electron transfer kinetics, most notably from the intermediate acceptor I− (bacteriopheophytin) to QA. Replacement of Fe2+ by diamagnetic Zn2+ retained all observed native characteristics of the RC.

Theory on Rates of Excitation-Energy Transfer between Molecular Aggregates through Distributed Transition Dipoles with Application to the Antenna System in Bacterial Photosynthesis

It has often been observed in the light-harvesting antenna system of bacterial photosynthesis that excitation-energy transfer (EET) takes place very rapidly to or from a nearly (or completely) optically forbidden state. So far, the rate constant of EET has usually been calculated by Förster's formula, which regards EET as arising from the overlap integral between the luminescence spectrum of the excitation donor and the absorption spectrum of its acceptor. The observed EETs are much faster than expected from this formula, since the transition dipole as a whole nearly (or completely) vanishes in this state, giving a very small (or completely vanishing) Förster's overlap integral. We note in these EETs that at least one of the donor and acceptor is an aggregate of chromophores. Individual chromophores comprising the aggregate retain nonvanishing transition dipoles, even if their sum nearly (or completely) vanishes at an exciton state which is nearly (or completely) optically forbidden therein. EET to (or from) the exciton state can arise due to interactions to (or from) individual transition dipoles in the aggregate when the distance between the donor and the acceptor is not much larger than the physical size of the aggregate. A new formula is proposed for calculating the rate constant of such an EET. It fills well the gap between theory and experiment mentioned above.

Nonequilibrium Oscillatory Electron Transfer in Bacterial Photosynthesis

A theory to describe nonequilibrium electronic surface crossing during vibrational relaxation induced by ultrafast photoexcitation is developed and applied to the primary electron transfer (ET) in bacterial photosynthetic reaction centers. As a key concept, we define on a microscopic basis the angle between two reaction coordinates each representing the environmental nuclear displacements coupled to the initial photoexcitation (to the P* state) and to the subsequent ET processes, respectively. The “cross-spectral” density function, whose integral intensity gives the cosine of this angle, is also defined to give a consistent (nonphenomenological) description of the vibrational coherence and its dephasing. In the application to the primary ET in bacterial photosynthesis, we find (1) the time-dependent ET rate exhibits marked oscillation at low temperatures due to the nonequilibrium vibrational coherence in the P* state. However, it does not contribute very much to accelerate the primary ET rate with respect...

Mimicking bacterial photosynthesis

Abstract

The primary and secondary electron acceptors in bacterial photosynthesis: I. A chronological account of their identification by EPR

A chronological account is presented of the work that led to the identification of the primary and secondary acceptors in photosynthetic reaction centers ofRb. sphaeroides. Both acceptors when reduced give rise to broad EPR signals centered atg = 1.82. When the iron is removed from the reaction center, the EPR line, narrowed by approximately two orders of magnitude, is characteristic of a ubisemiquinone. The broad signal in native reaction centers (RCs) is, therefore, ascribed to an unpaired electron being localized on the ubisemiquinone interacting magnetically with the high spin Fe2+. These findings are consistent with the structure of the RC, as determined a decade later by X-ray diffraction.

Fluctuational tunnel effects and vibrational dispersion in primary electron transfer processes of bacterial photosynthesis

We have investigated forward electron transfer (ET) from reduced bacteriopheophytin (bph −) to the primary quinone (Q A), and the recombination ET from Q A − to the oxidized special pair (bch) 2 + in the photosynthetic reaction centre of Rh. sphaeroides. The same reactant, Q A, is here engaged in two ET reactions of widely different distances. The processes can also be followed over broad temperature ranges. Focus is on two elements not commonly considered in ET theory. One is continuous environmental dielectric dispersion which is both formally and physically important. The other one is environmental fluctuational modulation of the tunnel factor which is important when the donor and acceptor are separated by environmental matter. The analysis shows, first that a broad dielectric dispersion is in line with the ET data up to ≈180°K. Fluctuational modulation of the tunnel factor is, secondly, unimportant in shorter-range forward ET from bph − to Q A (13 Å centre-to-centre distance) but could be important in the much longer-range recombination ET from Q A − to (bch) 2 + (28 Å).

Robert Huber—Nobel Prize for Chemistry

Robert Huber, a German biochemist, shared the 1988 Nobel Prize for chemistry with two German scientists, Johann Deisenhofer (1943-) and Hartmut Michel (1948-), for their determination of the structure of a protein complex that is essential to photosynthesis in bacteria. Michel crystallized the membrane-bound protein photosynthetic reaction center of the Rhodopseudomonas viridis bacterium, and then Huber and Deisenhofer used xray crystallography to elucidate the positions of approximately 10,000 atoms in the protein complex. By 1985, the three scientists had succeeded in describing the complete structure of the protein that is essential to photosynthesis in certain bacteria. Although bacterial photosynthesis is somewhat simpler than that in plants, the work of Huber, Deisenhofer, and Michel substantially increased the understanding of the mechanisms of photosynthesis in general. Their work had other applications as well because membrane-bound proteins are important in the understanding of the membrane structure of cells in health and in disease. Huber was born on Feb. 20, 1937, in Munich, Germany. He graduated from the Technical University in Munich in 1960 and earned a Ph.D. degree there in 1963. After receiving his doctorate, he joined the Max Planck Institute for Biochemistry at Martinsried, where he worked with Deisenhofer and Michel. He also worked at the Technical University in Munich. In addition to the Nobel Prize, Huber was awarded many honors and medals for his scientific work, including honorary doctorates from the University of Louvain (Belgium) in 1987, the University of Ljubljana (Slovenia) in 1989, and the University of Rome (Italy) in 1991, the E. K. Frey Medal in 1972, the Otto Warburg Medal in 1977, and the Emil von Behring Medal in 1982. He was honored on a stamp issued by Sierra Leone in 1995.

A Density Functional Investigation of Model Molecules for Ubisemiquinone Radical Anions

We have applied density functional methods for the determination of structures, vibrational spectra, electron affinities, and isotropic hyperfine coupling constants of the radical anions of 2,3-dimethoxy-1,4-benzoquinone and 2,3-dimethoxy-5,6-dimethyl-1,4-benzoquinone. Our calculations predict three stable conformers with respect to the orientation of the two methoxy groups for both molecules. Different orientations of the methoxy groups are shown to affect force constants and vibrational spectra less dramatically than in the corresponding neutral quinone. The different mode decompositions predicted in symmetrical and nonsymmetrical conformers, respectively, suggest that site specific labeling of one carbonyl group might allow one to distinguish among these conformers. The presented calculations allow us to provide a mode assignment for the CC and CO modes which, in most cases, agrees well with previous assignments based solely on experimental data. Electron affinities are shown to vary by up to 40 kJ/mol for different orientations of the methoxy groups. Our calculations demonstrate that the two additional substituents at positions 5 and 6 have to be considered if the molecule is supposed to serve as a model compound for quinones participating in bacterial photosynthesis.

Artificial membrane mimics photosynthesis

Researchers at Arizona State University, Tempe, have created an artificial membrane that mimics the entire process of bacterial photosynthesis. Illumination of the membrane produces a proton gradient that powers the enzyme adenosine triphosphate (ATP) synthase to catalyze the phosphorylation of adenosine diphosphate (ADP) to ATP, the energy source of living cells. The work was carried out by postdoctoral associates Gali Steinberg-Yfrach and Edgardo N. Durantini and professors of chemistry and biochemistry Ana L. Moore, Devens Gust, and Thomas A. Moore at Arizona State, in collaboration with membrane biochemist Jean-Louis Rigaud of the Curie Institute, Paris [ Nature , 392 , 479 (1998)]. Last year, the Moores (a husband-and-wife team), Gust, and coworkers designed and assembled a proton pump that could translocate protons across a liposome membrane in response to light to produce a proton gradient [ Nature , 385 , 239 (1997)]. Now they've added ATP synthase to the membrane and have demonstrated that the pr...

Three decades of research in bacterial photosynthesis and the road leading to it: A personal account

In this minireview I present a very personal account of my life and research in bacterial photosynthesis. It is divided into two parts. The first part is autobiographical and narrates the events that led me to change fields from electrical engineering to physics and ultimately to biophysics. The second part describes the work on the primary processes in bacterial photosynthesis carried out with numerous collaborators in our laboratory over the past ∼ 30 years.