ubiquinone and bacteriopheophytin

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography

Topics: Bacteriochlorophylls; Binding Sites; Chlorophyll; Crystallography; Cytoplasm; Electron Transport; Electrons; Hyphomicrobiaceae; Lasers; Models, Molecular; Oxidation-Reduction; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Protons; Ubiquinone; Vitamin K 2

Using high-throughput methods for mutagenesis, protein isolation and charge-separation functionality, we have assayed 40 Rhodobacter capsulatus reaction center (RC) mutants for their P(+)QB(-) yield (P is a dimer of bacteriochlorophylls and Q is a ubiquinone) as produced using the normally inactive B-side cofactors BB and HB (where B is a bacteriochlorophyll and H is a bacteriopheophytin). Two sets of mutants explore all possible residues at M131 (M polypeptide, native residue Val near HB) in tandem with either a fixed His or a fixed Asn at L181 (L polypeptide, native residue Phe near BB). A third set of mutants explores all possible residues at L181 with a fixed Glu at M131 that can form a hydrogen bond to HB. For each set of mutants, the results of a rapid millisecond screening assay that probes the yield of P(+)QB(-) are compared among that set and to the other mutants reported here or previously. For a subset of eight mutants, the rate constants and yields of the individual B-side electron transfer processes are determined via transient absorption measurements spanning 100 fs to 50 μs. The resulting ranking of mutants for their yield of P(+)QB(-) from ultrafast experiments is in good agreement with that obtained from the millisecond screening assay, further validating the efficient, high-throughput screen for B-side transmembrane charge separation. Results from mutants that individually show progress toward optimization of P(+)HB(-)→P(+)QB(-) electron transfer or initial P*→P(+)HB(-) conversion highlight unmet challenges of optimizing both processes simultaneously.

Topics: Amino Acid Motifs; Bacteriochlorophylls; Electron Transport; Gene Expression; Hydrogen Bonding; Kinetics; Light; Light-Harvesting Protein Complexes; Models, Molecular; Molecular Sequence Data; Mutagenesis; Mutation; Pheophytins; Photosynthesis; Rhodobacter capsulatus; Static Electricity; Structure-Activity Relationship; Ubiquinone

Time-resolved spectroscopic studies of recombination of the P(+)HA(-) radical pair in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides give an opportunity to study protein dynamics triggered by light and occurring over the lifetime of P(+)HA(-). The state P(+)HA(-) is formed after the ultrafast light-induced electron transfer from the primary donor pair of bacteriochlorophylls (P) to the acceptor bacteriopheophytin (HA). In order to increase the lifetime of this state, and thus increase the temporal window for the examination of protein dynamics, it is possible to block forward electron transfer from HA(-) to the secondary electron acceptor QA. In this contribution, the dynamics of P(+)HA(-) recombination were compared at a range of temperatures from 77 K to room temperature, electron transfer from HA(-) to QA being blocked either by prereduction of QA or by genetic removal of QA. The observed P(+)HA(-) charge recombination was significantly slower in the QA-deficient RCs, and in both types of complexes, lowering the temperature from RT to 77 K led to a slowing of charge recombination. The effects are explained in the frame of a model in which charge recombination occurs via competing pathways, one of which is thermally activated and includes transient formation of a higher-energy state, P(+)BA(-). An internal electrostatic field supplied by the negative charge on QA increases the free energy levels of the state P(+)HA(-), thus decreasing its energetic distance to the state P(+)BA(-). In addition, the dielectric response of the protein environment to the appearance of the state P(+)HA(-) is accelerated from ∼50-100 ns in the QA-deficient mutant RCs to ∼1-16 ns in WT RCs with a negatively charged QA(-). In both cases, the temperature dependence of the protein dynamics is weak.

Topics: Bacterial Proteins; Bacteriochlorophylls; Electron Transport; Mutation; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Recombinant Proteins; Rhodobacter sphaeroides; Spectrometry, Fluorescence; Static Electricity; Temperature; Time Factors; Ubiquinone

The tunneling mechanisms of electron transfers (ETs) in photosynthetic reaction center of Blastochloris viridis are studied by the ab initio fragment molecular orbital (FMO) method combined with the generalized Mulliken-Hush (GMH) and the bridge Green function (GF) calculations of the electronic coupling T(DA) and the tunneling current method for the ET pathway analysis at the fragment-based resolution. For the ET from batctriopheophytin (H(L)) to menaquinone (MQ), a major tunneling current through Trp M250 and a minor back flow via Ala M215, Ala M216, and His M217 are quantified. For the ET from MQ to ubiquinone, the major tunneling pathway via the nonheme Fe(2+) and His L190 is identified as well as minor pathway via His M217 and small back flows involving His L230, Glu M232, and His M264. At the given molecular structure from X-ray experiment, the spin state of the Fe(2+) ion, its replacement by Zn(2+), or its removal are found to affect the T(DA) value by factors within 2.2. The calculated T(DA) values, together with experimentally estimated values of the driving force and the reorganization energy, give the ET rates in reasonable agreement with experiments.

Topics: Electron Transport; Hyphomicrobiaceae; Models, Molecular; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Protein Conformation; Quantum Theory; Ubiquinone; Vitamin K 2

The replacement of tyrosine by aspartic acid at position M210 in the photosynthetic reaction center of Rhodobacter sphaeroides results in the generation of a fast charge recombination pathway that is not observed in the wild-type. Apparently, the initially formed charge-separated state (cation of the special pair, P, and anion of the A-side bacteriopheophytin, H(A)) can decay rapidly via recombination through the neighboring bacteriochlorophyll (B(A)) soon after formation. The charge-separated state then relaxes over tens of picoseconds and recombination slows to the hundreds-of-picoseconds or nanosecond timescale. This dielectric relaxation results in a time-dependent blue shift of B(A)(-) absorption, which can be monitored using transient absorbance measurements. Protein dynamics also appear to modulate the electron transfer between H(A) and the next electron carrier, Q(A) (a ubiquinone). The kinetics of this reaction are complex in the mutant, requiring two kinetic terms, and the spectra associated with the two terms are distinct; a red shift of the H(A) ground-state bleaching is observed between the shorter and longer H(A)-to-Q(A) electron-transfer phases. The kinetics appears to be pH-independent, suggesting a negligible contribution of static heterogeneity originating from protonation/deprotonation in the ground state. A dynamic model based on the energy levels of the two early charge-separated states, P(+)B(A)(-) and P(+)H(A)(-), has been developed in which the energetics of these states is modulated by fast protein dielectric relaxations and this in turn alters both the kinetic complexity of the reaction and the reaction pathway.

Topics: Amino Acid Sequence; Aspartic Acid; Bacterial Proteins; Bacteriochlorophylls; Electron Transport; Hydrogen-Ion Concentration; Kinetics; Light; Molecular Sequence Data; Mutation, Missense; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Static Electricity; Ubiquinone

The calculation of radiationless transition rates and of their temperature dependence from first principles is addressed by combining reliable electronic computations of the normal modes of the two electronic states with Kubo's generating function approach for the evaluation of the Franck-Condon weighted density of states. The whole sets of normal modes of the involved cofactors have been employed, taking into account the effects of nuclear equilibrium position displacements, of vibrational frequency changes, and of mixing of the normal modes. Application to the case of the elementary electron transfer step between bacteriopheophytin and ubiquinone cofactors of bacterial photosynthetic reaction centers yields a temperature dependence of the electron transfer rates in very good agreement with the experimental data.

Topics: Electron Transport; Electrons; Kinetics; Models, Molecular; Molecular Conformation; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Quantum Theory; Reproducibility of Results; Rhodobacter sphaeroides; Temperature; Ubiquinone

Energy transfer and the primary charge separation process are studied as a function of excitation wavelength in membrane-bound reaction centers of Rhodobacter sphaeroides in which the excitonically coupled bacteriochlorophyll homodimer is converted to a bacteriochlorophyll-bacteriopheophytin heterodimer, denoted D [Bylina, E. J., and Youvan, D. C. (1988) Proc. Natl. Acad. Sci. U.S. A. 85, 7226]. In the HM202L heterodimer reaction center, excitation of D using 880 nm excitation light results in a 43 ps decay of the excited heterodimer, D. The decay of D results for about 30% in the formation of the charge separated state D+QA- and for about 70% in a decay directly to the ground state. Upon excitation of the monomeric bacteriochlorophylls using 798 nm excitation light, approximately 60% of the excitation energy is transferred downhill to D, forming D. Clear evidence is obtained that the other 40% of the excitations results in the formation of D+QA- via the pathway BA --> BA+HA- --> D+HA- --> D+QA-. In the membrane-bound "reversed" heterodimer reaction center HL173L, the simplest interpretation of the transient absorption spectra following B excitation is that charge separation occurs solely via the slow D-driven route. However, since a bleach at 812 nm is associated with the spectrum of D in the HL173L reaction center, it cannot be excluded that a state including BB is involved in the charge separation process in this complex.

Topics: Bacteriochlorophylls; Dimerization; Energy Transfer; Kinetics; Leucine; Light-Harvesting Protein Complexes; Methionine; Models, Chemical; Mutagenesis, Site-Directed; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Spectrometry, Fluorescence; Temperature; Ubiquinone

Electron transfer from P+QA-QB to form P+QAQB- was measured in Rhodobacter sphaeroides R-26 reaction centers (RCs) where the native primary quinone, ubiquinone-10 (UQA), was replaced by 2-methyl-3-phytyl-1,4-naphthoquinone (MQA). The native secondary quinone, UQ-10, was retained as UQB. The difference spectrum of the semiquinone MQA- minus UQB- absorption is very similar to that of MQ- minus UQ- in solution (398-480 nm). Thus, the absorption change provides a direct monitor of the electron transfer from MQA- to UQB. In contrast, when both QA and QB are UQ-10 the spectral difference between UQA- and UQB- arises from electrochromic responses of RC chromophores. Three kinetic processes are seen in the near UV (390-480 nm) and near-IR (740-820 nm). Analysis of the time-correlated spectra support the conclusion that the changes at tau1 approximately 3 micros are mostly due to electron transfer, electron transfer and charge compensation are mixed in tau2 approximately 80 micros, while little or no electron transfer occurs at 200-600 micros (tau3) in MQAUQB RCs. The 80-micros rate has been previously observed, while the fast component has not. The fast phase represents 60% of the electron-transfer reaction (398 nm). The activation energy for electron transfer is DeltaG approximately 3.5 kcal/mol for both tau1 and tau2 between 0 and 30 degrees C. In isolated RCs with UQA, if there is any fast component, it appears to be faster and less important than in the MQA reconstituted RCs.

Topics: Electron Transport; Kinetics; Models, Chemical; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Ubiquinone; Vitamin K; Vitamin K 1

Seven site-directed mutants of the bacterial photosynthetic reaction center (RC) from the 2.4.1 and WS 231 wild-type strains of Rhodobacter sphaeroides have been crystallized and their X-ray diffraction analyzed to resolutions between 3.0 and 4.0 A. The mutations can be divided into four distinct categories: (1) mutations altering cofactor composition that affect electron transfer and quantum yield, His M202-->Leu (M202HL), His L173-->Leu (L173HL), and Leu M214-->His (M214LH); (2) a mutation in the proposed pathway of electron transfer altering electron-transfer kinetics, Tyr M210-->Phe (M210YF); (3) a mutation around the non-heme iron resulting in an iron-less reaction center, His M219-->Cys (M219HC); and (4) mutations around the secondary electron acceptor, a ubiquinone, affecting proton transfer and quinone turnover, Glu L212-->Gln (L212EQ) and Asp L213-->Asn (L213DN). Residues L173 and M202 are within bonding distance of the respective magnesiums of the two bacteriochlorophylls of the BChl special pair, while M214 is close to the bacteriopheophytin on the active A branch of the RC. The L173HL and M202HL crystal structures show that the respective bacteriochlorophylls are replaced with bacteriopheophytins (i.e., loss of magnesium) without significant structural perturbations to the surrounding main-chain or side-chain atoms. In the M214LH mutant, the bacteriopheophytin has been replaced by a bacteriochlorophyll, and the side chain of His M214 is within ligand distance of the magnesium. The M210YF, L212EQ, and L213DN mutants show no significant tertiary structure changes near the mutation sites. The M219HC diffraction data indicate that the overall tertiary structure of the reaction center is maintained in the absence of the non-heme iron.

Topics: Chemical Phenomena; Chemistry, Physical; Crystallization; Crystallography, X-Ray; Electron Transport; Fourier Analysis; Kinetics; Light-Harvesting Protein Complexes; Magnesium; Molecular Structure; Mutagenesis, Site-Directed; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Protons; Rhodobacter sphaeroides; Ubiquinone

The native ubiquinone-10 redox cofactor has been removed from the QA site of the isolated reaction center protein from Rhodobacter sphaeroides and reconstitution attempted with 28 non-quinone molecules in order to identify factors governing cofactor function and the selectivity displayed by the site in the electron transfers that it catalyzes. Equilibrium binding, in situ electrochemistry, and the kinetics of electron transfer to and from the QA site occupant were examined. Four classes of non-quinone molecules are distinguished according to their ability to occupy the QA site and conduct intraprotein electron transfers. The minimal requirements for occupancy of the QA site are at least one ring and a heteroatom hydrogen bond acceptor. Thus, binding at the site is not highly selective. The rates of electron transfers to and from the class of non-quinone molecules (four) that satisfy the criteria for cofactor function at the QA site compare well with rates previously determined from 14 to 295 K for 14 quinone replacements with comparable values of the reaction free energy. This indicates that the rates are relatively insensitive to variations in exotic and quinone cofactor reorganization energy and the vibrational frequencies coupled to the electron transfers, and that the exotic and quinone cofactors are bound in the QA site in comparable positions. It appears that any variation in rate is determined predominantly by the value of the reaction free energy. The QA site protein-cofactor solvation contribution to the in situ electrochemical potential is roughly constant for 12 rigid quinone and 2 exotic cofactors (average value-61 +/- 2 kcal/mol). Favorable electrostatic contributions governing the reaction free energy are therefore also relatively insensitive to cofactor structure. However, flexible molecules appear to encounter in situ steric constraints that lower the electron affinity by destabilizing the reduced cofactor species. This is a strong determinant of whether a molecule, once in the QA site, will function. These findings compare well with those from studies of electron transfers in synthetic systems.

Topics: Bacteriochlorophylls; Binding Sites; Electron Transport; Kinetics; Light-Harvesting Protein Complexes; Oxidation-Reduction; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Structure-Activity Relationship; Thermodynamics; Ubiquinone

The rate of the electron-transfer reaction between bacteriopheophytin and the first quinone in isolated reaction centers of Rhodopseudomonas sphaeroides has an unusual temperature dependence. The rate increases about threefold with decreasing temperature between 300 and 25 K, and decreases abruptly at temperatures below 25 K. Partial deuteration of the reaction centers alters the temperature dependence of the rate constant. Qualitative features of the temperature dependence can be understood in the context of a theory of nonadiabatic electron transfer (Sarai, 1980. Biochim. Biophys. Acta 589:71-83). We conclude that very low-energy (10-50 cm-1) processes, perhaps skeletal vibrations of the protein, are important to electron transfer. Higher-energy vibrations, possibly involving the pyrrolic N--H bonds of bacteriopheophytin, also are important in this process.

Topics: Chlorophyll; Deuterium; Electron Transport; Kinetics; Pheophytins; Protons; Rhodobacter sphaeroides; Temperature; Ubiquinone

Topics: Bacterial Proteins; Bacteriochlorophylls; Dimethylamines; Iron; Light-Harvesting Protein Complexes; Mathematics; Molecular Weight; Pheophytins; Phospholipids; Photosynthetic Reaction Center Complex Proteins; Protein Binding; Protein Conformation; Rhodobacter sphaeroides; Ubiquinone