Charge separation in bacterial photosynthetic reaction centers

The physical aspects of the primary charge separation process in bacterial photosynthesis are discussed. The donor-acceptor model of electron tranfer through proteins is used. The kinetics of the processes of the photosynthetic reaction centers are considered and their energetic scheme is constructed by means of the nonequilibrium density matrix method. It is shown that the theory is in good agreement with experiment if one takes into account the influence of vibrational sublevels of states which take part in transitions.     

A new metal-binding site in photosynthetic bacterial reaction centers that modulates QA to QB electron transfer

Isolated reaction centers (RCs) from Rhodobacter sphaeroides were found to bind Zn(II) stoichiometrically and reversibly in addition to the 1 equiv of non-heme Fe(II). Metal and EPR analyses confirm that Zn(II) is ligated to a binding site that is distinct from the Fe site. When Zn(II) is bound to this site, electron transfer between the quinones QA and QB (QA-QB --> QAQB-) is slowed and the room-temperature kinetics become distributed across the microsecond to millisecond time domain. This effect of metal binding on the kinetics is similar to the more global effect of cooling RCs to 2 degreesC in the absence of Zn(II). This suggests that Zn(II) binding alters localized protein motions that are necessary for rapid QA-QB --> QAQB- electron transfer. Inspection of the RC crystal structure suggests a cluster of histidine ligands located beneath the QB binding pocket as a potential binding site.     

Catalytic role of monovalent cations in the mechanism of proton transfer which gates an interprotein electron transfer reaction

Within the methylamine dehydrogenase (MADH)-amicyanin protein complex, long range intermolecular electron transfer (ET) occurs between tryptophan tryptophylquinone (TTQ) of MADH and the type I copper of amicyanin. The reoxidations of two chemically distinct reduced forms of TTQ were studied, a quinol (O-quinol) generated by reduction by dithionite and the physiologically relevant aminoquinol (N-quinol) generated by reduction by methylamine. The latter contains a substrate-derived amino group which displaces the C6 carbonyl oxygen on TTQ. ET from N-quinol MADH to amicyanin is gated by the transfer of a solvent exchangeable proton [Bishop, G. R., & Davidson, V. L. (1995) Biochemistry 34, 12082-12086]. The factors which influence this proton transfer (PT) reaction have been examined. The rate of PT increases with increasing pH and with increasing salt concentration. The salt effect is due to specific monovalent cations and is not a general ionic strength effect. The rate enhancements by pH and cations do not reflect an elimination of the PT step that gates ET. Over the range of pH from 5.5 to 9.0 and with cation concentrations from 0 to 200 mM, the observed rate of the redox reaction is still that of PT. This is proven by kinetic solvent isotope effect studies which show that a primary isotope effect persists even at the highest values of pH and cation concentration. A model is presented to explain how specific cations contribute to catalysis and influence the rate of PT in this reaction. The pH dependence is attributed to an ionizable group that is involved in cation binding. The effect of the cation is stabilization of a negatively charged reaction intermediate that is formed during the deprotonation of the N-quinol, and from which rapid ET to the copper of amicyanin occurs. The relevance of these findings to other enzymes which exhibit reaction rates that are influenced by monovalent cations is also discussed.     

Mutations in the environment of the primary quinone facilitate proton delivery to the secondary quinone in bacterial photosynthetic reaction centers

In Rhodobacter capsulatus, we constructed a quadruple mutant that reversed a structural asymmetry that contributes to the functional asymmetry of the two quinone sites. In the photosynthetically incompetent quadruple mutant RQ, two acidic residues near QB, L212Glu and L213Asp, have been mutated to Ala; conversely, in the QA pocket, the symmetry-related residues M246Ala and M247Ala have been mutated to Glu and Asp. We have selected photocompetent phenotypic revertants (designated RQrev3 and RQrev4) that carry compensatory mutations in both the QA and QB pockets. Near QA, the M246Ala --> Glu mutation remains in both revertants, but M247Asp is replaced by Tyr in RQrev3 and by Ala in RQrev4. The engineered L212Ala and L213Ala substitutions remain in the QB site of both revertants but are accompanied by an additional electrostatic-type mutation. To probe the respective influences of the mutations occurring near the QA and QB sites on electron and proton transfer, we have constructed two additional types of strains. First, "half" revertants were constructed that couple the QB site of the revertants with a wild-type QA site. Second, the QA sites of the two revertants were linked with the L212Glu-L213Asp --> Ala-Ala mutations of the QB site. We have studied the electron and proton-transfer kinetics on the first and second flashes in reaction centers from these strains by flash-induced absorption spectroscopy. Our data demonstrate that substantial improvements of the proton-transfer capabilities occur in the strains carrying the M246Ala --> Glu + M247Ala --> Tyr mutations near QA. Interestingly, this is not observed when only the M246Ala --> Glu mutation is present in the QA pocket. We suggest that the M247Ala --> Tyr mutation in the QA pocket, or possibly the coupled M246Ala --> Glu + M247Ala --> Tyr mutations, accelerates the uptake and delivery of protons to the QB anions. The M247Tyr substitution may enable additional pathways for proton transfer that are located near QA.     

Influence of iron-removal procedures on sequential electron transfer in photosynthetic bacterial reaction centers studied by transient EPR spectroscopy

Electron spin polarized electron paramagentic resonance (ESP EPR) spectra were obtained with deuterated iron-removed photosynthetic bacterial reaction centers (RCs) to specifically investigate the effect of the rate of primary charge separation, metal-site occupancy, and H-subunit content on the observed P865+QA- charge-separated state. Fe-removed and Zn-substituted RCs from Rb. sphaeroides R-26 were prepared by refined procedures, and specific electron transfer rates (kQ) from the intermediate acceptor H- to the primary acceptor QA of (200 ps)-1 vs (3-6 ns)-1 were observed. Correlation of the transient EPR and optical results shows that the observed slow kQ rate in Fe-removed RCs is H-subunit-independent, and, in some cases, independent of Fe-site occupancy as Zn2+ substitution does not ensure retention of the native kQ. In addition, shifts in the optical spectrum of P865 and differences in the high-field region of the Q-band ESP spectrum for Fe-removed RCs with slow kQ indicate possible structural changes near P865. The experimental X-band and Q-band spin-polarized EPR spectra for deuterated Fe-removed RCs where kQ is at least 15-fold slower at room temperature than the (200 ps)-1 rate observed for native Fe-containing RCs have different relative amplitudes and small g-value shifts compared to the spectra of Zn-RCs which have a kQ unchanged from native RCs. These differences reflect the trends in polarization predicted from the sequential electron transfer polarization (SETP) model [Morris et al. (1995) J. Phys. Chem. 99, 3854-3866; Tang et al. (1996) Chem. Phys. Lett. 253, 293-298]. Thus, SETP modeling of these highly resolved ESP spectra obtained with well-characterized proteins will provide definitive information about any light-induced structural changes of P865, H, and QA that occur upon formation of the P865+QA- charge-separated state.     

Triplet energy transfer in bacterial photosynthetic reaction centres

[3-vinyl]-132-OH-bacteriochlorophyll a has been selectively exchanged against native bacteriochlorophyll a in the monomer binding sites at the A- and B-branch of the photosynthetic reaction centre from Rhodobacter sphaeroides. Transient absorption difference measurements were performed on these samples over a temperature range from 4.2 to 300 K with 20 ns time resolution. Specifically the decay of the primary donor triplet state, 3P870, as well as the rise and decay rates of the carotenoid triplet state, 3Car (spheroidene), were measured. The observed rates revealed a thermally activated carotenoid triplet formation corresponding to the decay of the primary donor triplet state. The activation energies for the triplet energy transfer process were 100(+/-10) cm-1 for reaction centers from wild-type Rhodobacter sphaeroides 2.4.1, with and without exchange of the monomeric bacteriochlorophyll on the electron transfer-active branch, BA. For reaction centers from Rhodobacter sphaeroides R26.1 with both monomers exchanged against [3-vinyl]-132-OH-bacteriochlorophyll a, and subsequent spheroidene reconstitution the activation energy was 460(+/-20) cm-1. These activation energies correspond to the energy difference between the triplet states of the accessory BChl monomer, BB, and the primary donor when native BChl a or [3-vinyl]-132-OH-BChl a is present in the BB binding site. In all samples the 3Car formation rates were bi-phasic over a large temperature range. A fast temperature-independent rate was observed on the wavelength of the carotenoid triplet-triplet absorption which dominated at very low temperatures. Additionally, a slower temperature-independent 3Car formation rate was observed at low temperatures which could be explained with the assumption of heterogeneity in the energy barrier (3BB) and/or the primary donor triplet state (3P870). A tunneling mechanism as proposed earlier by Kolaczkowski (PhD thesis, Brown University, 1989) is not only unnecessary but also incompatible with the available experimental data.     

In bacterial reaction centers rapid delivery of the second proton to QB can be achieved in the absence of L212Glu

In the reaction center (RC) of Rhodobacter capsulatus, residue L212Glu is a component of the pathway for proton transfer to the reduced secondary quinone, QB. We isolated phenotypic revertants of the photosynthetically incompetent (PS-) L212Glu-->Gln mutant; all of them retain the L212Glu-->Gln substitution and carry a second-site mutation: L227Leu-->Phe, L228Gly-->Asp, L231Arg-->Cys, or M231Arg-->Cys. We also characterized the L212Ala strain, which is a phenotypic revertant of the PS- L212Glu-L213Asp-->Ala-Ala mutant. The activities of the RCs of these strains--all of which lack L212Glu--were studied by flash-induced absorption spectroscopy. At pH 7.5, the rate of second electron transfer in the L212Q mutant is comparable to the wild-type rate. However, this mutant shows a marked decrease in the rate of cytochrome oxidation under strong continuous illumination and a very slow phase (0.66 s-1) of the proton transfer kinetics following the second flash, indicating that transfer of the second proton to QB is slowed more than 1000-fold. The levels of recovery of the functional capabilities in the revertant RCs vary widely; their rates of cytochrome oxidation were intermediate between those of the wild-type and the L212Q mutant. The kinetics of proton transfer following the second flash show a significant recovery in the L212Q + M231C and L212A RCs (330-540 s-1), but the L212Q + L227F RCs recover this function only partially. Compensation for the lack of L212Glu in revertant RCs is discussed in terms of (i) conformational changes that could allow water molecules to approach closer to QB and/or (ii) the increase in the negative electrostatic environment and the resultant rise in the free energy level of QB- that is induced by the mutations. The stoichiometries of H+/QB- proton uptake below pH 7.5 in the L212Q mutant, the L212Q + M231C revertant, and the wild-type strains are essentially equivalent, suggesting that L212Glu is protonated at neutral pH in wild-type RCs. This is also supported by the P+QB- charge recombination data. Comparison of H+/QB- proton uptake data with those obtained previously for the stoichiometries of H+/QA- proton uptake [Miksovska, J., Maróti, P., Tandori, J., Schiffer, M., Hanson, D. K., Sebban, P. (1996) Biochemistry 35, 15411-15417] suggests that L212Glu is the key to the electrostatic and perhaps structural interaction between the two quinone sites.     

Conformational gating of the electron transfer reaction QA-.QB --> QAQB-. in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay

The mechanism of the electron transfer reaction, QA-.QB --> QAQB-., was studied in isolated reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides by replacing the native Q10 in the QA binding site with quinones having different redox potentials. These substitutions are expected to change the intrinsic electron transfer rate by changing the redox free energy (i.e., driving force) for electron transfer without affecting other events that may be associated with the electron transfer (e.g., protein dynamics or protonation). The electron transfer from QA-. to QB was measured by three independent methods: a functional assay involving cytochrome c2 to measure the rate of QA-. oxidation, optical kinetic spectroscopy to measure changes in semiquinone absorption, and kinetic near-IR spectroscopy to measure electrochromic shifts that occur in response to electron transfer. The results show that the rate of the observed electron transfer from QA-. to QB does not change as the redox free energy for electron transfer is varied over a range of 150 meV. The strong temperature dependence of the observed rate rules out the possibility that the reaction is activationless. We conclude, therefore, that the independence of the observed rate on the driving force for electron transfer is due to conformational gating, that is, the rate limiting step is a conformational change required before electron transfer. This change is proposed to be the movement, controlled kinetically either by protein dynamics or intermolecular interactions, of QB by approximately 5 A as observed in the x-ray studies of Stowell et al. [Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E. & Feher, G. (1997) Science 276, 812-816].     

Superoxide anion reacts with enzyme intermediate in the bacterial luciferase reaction competitive with intramolecular electron transfer

The aims of this study were to identify the sexual problems of elderly people institutionalised in 2 nursing-homes and to find out how much the caregivers employed at these homes know about the sexuality-related problems affecting the elderly people they care for. Two questionnaires were issued: one to the residents of the home, the other to the caregivers. Results showed that the caregivers had very little awareness of sexuality in elderly people and of the particular problems and discomfort of those living in institutions. Discomfort was mainly due to loneliness, difficulty in communicating with caregivers about sentimental and sexual needs, finding new partners and lack of communication between the elderly person and his relatives regarding sexuality-related problems.     

Low frequency vibrational modes in proteins: changes induced by point-mutations in the protein-cofactor matrix of bacterial reaction centers

As a step toward understanding their functional role, the low frequency vibrational motions (<300 cm-1) that are coupled to optical excitation of the primary donor bacteriochlorophyll cofactors in the reaction center from Rhodobacter sphaeroides were investigated. The pattern of hydrogen-bonding interaction between these bacteriochlorophylls and the surrounding protein was altered in several ways by mutation of single amino acids. The spectrum of low frequency vibrational modes identified by femtosecond coherence spectroscopy varied strongly between the different reaction center complexes, including between different mutants where the pattern of hydrogen bonds was the same. It is argued that these variations are primarily due to changes in the nature of the individual modes, rather than to changes in the charge distribution in the electronic states involved in the optical excitation. Pronounced effects of point mutations on the low frequency vibrational modes active in a protein-cofactor system have not been reported previously. The changes in frequency observed indicate a strong involvement of the protein in these nuclear motions and demonstrate that the protein matrix can increase or decrease the fluctuations of the cofactor along specific directions.     

Electron transfer dynamics of Rhodopseudomonas viridis reaction centers with a modified binding site for the accessory bacteriochlorophyll

Femtosecond spectroscopy in combination with site-directed mutagenesis was used to study the influence of histidine L153 in primary electron transfer in the reaction center of Rhodopseudomonas viridis. Histidine was replaced by cysteine, glutamate, or leucine. The exchange to cysteine did not lead to significant changes in the primary reaction dynamics. In the case of the glutamate mutation, the decay of the excited electronic level of the special pair P* is slowed by a factor of 3. The exchange to leucine caused the incorporation of a bacteriopheophytin b instead of a bacteriochlorophyll b molecule at the BA site. As a consequence of this chromophore exchange, the energy level of the electron transfer state P+BA- is lowered to such an extent that repopulation from the next electron transfer intermediate state P+HA- takes place, resulting in a long-lasting P+BA- population. The observed differences in time constants are discussed in the scope of nonadiabatic electron transfer theory considering the influence of the amino acids at position L153 and the chromophore exchange on the energy level of the intermediate state P+BA-. The results show that the high efficiency of primary electron transfer is reduced substantially, if the energy level of P+BA- is lowered or raised by several hundred wave numbers.     

Large kinetic isotope effects in enzymatic proton transfer and the role of substrate oscillations

We propose an interpretation of the experimental findings of Klinman and coworkers [Cha, Y., Murray, C. J. & Klinman, J. P. (1989) Science 243, 1325-1330; Grant, K. L. & Klinman, J. P. (1989) Biochemistry 28, 6597-6605; and Bahnson, B. J. & Klinman, J. P. (1995) Methods Enzymol. 249, 373-397], who showed that proton transfer reactions that are catalyzed by bovine serum amine oxidase proceed through tunneling. We show that two different tunneling models are consistent with the experiments. In the first model, the proton tunnels from the ground state. The temperature dependence of the kinetic isotope effect is caused by a thermally excited substrate mode that modulates the barrier, as has been suggested by Borgis and Hynes [Borgis, D. & Hynes, J. T. (1991) J. Chem. Phys. 94, 3619-3628]. In the second model, there is both over-the-barrier transfer and tunneling from excited states. Finally, we propose two experiments that can distinguish between the possible mechanisms.     

Kinetic phases in the electron transfer from P+QA-QB to P+QAQB- and the associated processes in Rhodobacter sphaeroides R-26 reaction centers

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.     

Multiple isotope effects as a probe of proton and hydride transfer in the 6-phosphogluconate dehydrogenase reaction

Primary solvent deuterium, primary substrate deuterium, multiple solvent deuterium/substrate deuterium, and multiple solvent deuterium/13C isotope effects on V/K6PG have been measured for the Candida utilis and sheep liver 6-phosphogluconate dehydrogenases (6PGDH). Proton inventory data suggest the presence of a significant medium effect in a step preceding hydride transfer and the presence of a kinetic solvent deuterium isotope effect on hydride transfer. Multiple isotope effect data confirm the presence of multiple solvent deuterium sensitive steps, likely including a conformational change preceding hydride transfer, hydride transfer, and decarboxylation.     

Characterization of bacterial reaction centers having mutations of aromatic residues in the binding site of the bacteriopheophytin intermediary electron carrier

We report the initial characterization of a series of reaction centers (RCs) from the photosynthetic bacterium Rhodobacter capsulatus having single or double mutations of phenylalanines 97 and 121 on the L polypeptide. Substitution of these aromatic amino acids, which may interact with the photoactive bacteriopheophytin associated with the L polypeptide (BPhL), was carried out to examine their possible roles in electron transfer, charge stabilization, and/or BPhL binding. In some mutant RCs, the wild-type pigment content is obtained while in certain others a bacteriochlorophyll (BChL) replaces BPhL. The mutant RCs with wild-type pigment content are found to have overall photochemistry effectively identical to that of wild-type RCs. This indicates aromatic residues at L97 and L121 are not critical factors in the charge separation process, although an approximate 2-fold increase in the rate of electron transfer from BPhL- to QA is observed in two mutants where residue L121 is leucine. In two double mutants where L121 is histidine and L97 is either valine or cysteine, BPhL is replaced with a BChl (denoted beta). This pigment content is surprising since in the native RC structure amino acid L121 is not in optimum geometry for coordination to the Mg in the center of the pigment macrocycle. Charge separation takes place in the beta-containing mutants with an approximately 70% yield of P+QA- at 285 K compared to approximately 100% for wild-type. The photochemistry of these new beta-type RCs is very similar to that reported previously for the beta RC from Rhodobacter sphaeroides wherein the same pigment change was induced by a mutation in the M polypeptide.     

Heat transfer and chemical reaction models for reduction of iron oxide pellets in countercurrent moving bed reactors

Mass, energy and pressure balances are performed in a volume slice of a HyL III reactor in order to build a mathematical simulator for the different reduction steps. Three iron ore feed mixtures were simulated finding, in general, good agreement between plant data for metallization of DRI and the corresponding mathematical simulations. The main differences between these two types of data are explained as a consequence of the low efficiency of the contact between the reducing gases, H₂ and CO, and the bed of solids due to the formation of clusters in the different ore feeds. This tendency observes a remarkable increase with rise of the total iron content in the ore feed. The existence of clusters promotes fluid flow malfunctions affecting the uniformity of the DRI quality expressed as it final metallization. This leads to the need of a 3D simulator which should be able to quantify this effect. Finally it was found, in the present research, that carbon monoxide plays a minor role in the dynamic behaviour of the reduction for the feed mixtures analyzed with the mathematical simulator.     

A new pathway for transmembrane electron transfer in photosynthetic reaction centers of Rhodobacter sphaeroides not involving the excited special pair

It is generally accepted that electron transfer in bacterial photosynthesis is driven by the first singlet excited state of a special pair of bacteriochlorophylls (P*). We have examined the first steps of electron transfer in a mutant of the Rhodobacter sphaeroides reaction center in which charge separation from P* is dramatically slowed down. The results provide for the first time clear evidence that excitation of the monomeric bacteriochlorophyll in the active branch of the reaction center (B(A)) drives ultrafast transmembrane electron transfer without the involvement of P*, demonstrating a new and efficient mechanism for solar energy transduction in photosynthesis. The most abundant charge-separated intermediate state probably is P+B(A)-, which is formed within 200 fs from B(A)* and decays with a lifetime of 6.5 ps into P+H(A)-. We also see evidence for the involvement of a B(A)+H(A)- state in the alternative pathway.     

Septic polyarthritis associated with bacterial endocarditis in two dogs

Two dogs were examined because of chronic shifting lameness. In each case, lameness was attributed to septic polyarthritis, as documented by synovial fluid analysis and culture. In 1 dog, antemortem diagnosis of bacterial endocarditis was verified by clinical and culture data. Treatment of both dogs was unsuccessful, and necropsy of each dog revealed bacterial endocarditis with coexistent septic polyarthritis. Bacteriologic blood cultures yielded an anaerobic Streptococcus sp (dog 1) and Pasteurella multocida (dog 2).