Electrostatic interactions at the donor side of the photosynthetic reaction center of Rhodopseudomonas viridis

The photosynthetic reaction center of Rhodopseudomonas viridis, a purple bacterium, contains a tetraheme cytochrome subunit. After its photoinduced oxidation, the primary donor, P, is reduced by the nearby heme (c559) of the tetraheme subunit in about 200 ns. This heme, in turn, is reduced by another heme (c556) of the subunit in about 2 micro(s). The midpoint potentials of P, c559, and c556 are known to be +500, +380, and +320 mV, respectively. The reduction kinetics of P+ are strongly biphasic in living cells, membrane fragments, and isolated reaction centers. We show here that this biphasicity reflects a small equilibrium constant (lower than 10) for the electron-transfer reaction between P and c559, which arises from a significant difference between the operating redox potentials of the P+/P and c559+/c559 couples and their equilibrium midpoint potentials. This difference is partly due to the effect of the permanent transmembrane potential, which arises from the cell metabolism, and to significant electrostatic interactions which develop between the electron carriers of the reaction center. Interestingly, the kinetic parameters of P+ reduction in decoupled cells or membrane fragments are identical to those reported for isolated reaction centers. We estimate an interaction of about 20 mV between c556 and c559 and about 90 mV between c559 and P. Consequently, the operating redox potential of the P+/P couple is 410 mV.     

Acclimation of the photosynthetic response of Chromatium vinosum to light-limiting conditions

The predictive value of the Neonatal Neurological Examination (NNE) adapted from Prechtl (1977) and the Infant Motor Screen (IMS) from Nickel (1989) at 4 months was studied in severely jaundiced infants in Zimbabwe. Fifty infants were examined with the NNE, 41 with the IMS and 43 with the Bayley Scales of Infant Development (BSID) (Bayley 1969). Five infants had choreoathetosis and six had a motor delay at age 1 year. The NNE and IMS proved to be sensitive instruments particularly when two infants who became malnourished after the neonatal period were excluded. Logistic regression was used to investigate the relation between the BSID and five selected predictors from the NNE. This resulted in a correct classification of 93%. By using only the predictors acoustic blink and traction response, 80% of the infants were correctly classified but the number of false negatives was reduced from three to one.     

The atpIBEXF operon coding for the F0 sector of the ATP synthase from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus

BACKGROUND: The P1A1/P1A2 polymorphism of the platelet glycoprotein IIIa has been variably associated with an increased risk of coronary thrombosis. MATERIALS: We investigated the linkage between the P1A1/P1A2 polymorphism and the risk of myocardial infarction in 98 patients who suffered their first myocardial infarction at the age of 45 years or less and 98 well-matched control subjects without coronary artery disease. Lipid parameters were measured using conventional methods of clinical chemistry; P1A genotypes were determined by polymerase chain reaction and restriction enzyme digestion. RESULTS: There was no significant difference in the prevalence of P1A2-positive genotypes (either P1A1/P1A2 or P1A2/P1A2) between patients and control subjects (chi 2 = 0.66, d.f. = 1, P = 0.41). CONCLUSIONS: These results suggest that the P1A2 polymorphism of the platelet glycoprotein IIIa does not contribute to the genetic susceptibility to premature myocardial infarction.     

Effects of temperature and deltaGo on electron transfer from cytochrome c2 to the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides

The kinetics of electron transfer from cytochrome c2 to the primary donor (P) of the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides have been investigated by time-resolved absorption spectroscopy. Rereduction of P+ induced by a laser pulse has been measured at temperatures from 300 K to 220 K in a series of specifically mutated reaction centers characterized by altered midpoint redox potentials of P+/P varying from 410 mV to 765 mV (as compared to 505 mV for wild type). Rate constants for first-order electron donation within preformed reaction center-cytochrome c2 complexes and for the bimolecular oxidation of free cytochrome c2 have been obtained by multiexponential deconvolution of the kinetics. At all temperatures the rate of the fastest intracomplex electron transfer increases by more than two orders of magnitude as the driving force -deltaGo is varied over a range of 350 meV. The temperature and deltaGo dependences of the rate constant fit the Marcus equation well. Global analysis yields a reorganization energy lambda = 0.96 +/- 0.07 eV and a set of electronic matrix elements, specific for each mutant, ranging from 1.2 10(-4) eV to 2.5 10(-4) eV. Analysis in terms of the Jortner equation indicates that the best fit is obtained in the classical limit and restricts the range of coupled vibrational modes to frequencies lower than approximately 200 cm(-1). An additional slower kinetic component of P+ reduction, attributed to electron transfer from cyt c2 docked in a nonoptimal configuration of the complex, displays a Marcus type dependence of the rate constant upon deltaGo, characterized by a similar value of lambda (0.8 +/- 0.1 eV) and by an average electronic matrix element smaller by more than one order of magnitude. In all of the mutants, as the temperature is decreased below 260 K, both intracomplex reactions are abruptly inhibited, their rate being negligible at 220 K. The free energy dependence of the second-order rate constant for oxidation of cyt c2 in solution suggests that the collisional reaction is partially diffusion controlled, reaching the diffusion limit at exothermicities between 150 and 250 meV over the temperature range investigated.     

Molecular genetic evidence for extracytoplasmic localization of sulfur globules in Chromatium vinosum

Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope. We cloned the genes sgpA, sgpB, and sgpC, which encode the three different proteins that constitute the sulfur globule envelope of Chromatium vinosum D (DSMZ 180(T)). Southern hybridization analyses and nucleotide sequencing showed that these three genes are not clustered in the same operon. All three genes are preceded by sequences resembling sigma70-dependent promoters, and hairpin structures typical for rho-independent terminators are found immediately downstream of the translational stop codons of sgpA, sgpB, and sgpC. Insertional inactivation of sgpA in Chr. vinosum showed that the presence of only one of the homologous proteins SgpA and SgpB suffices for formation of intact sulfur globules. All three sgp genes encode translation products which - when compared to the isolated proteins - carry amino-terminal extensions. These extensions meet all requirements for typical signal peptides indicating an extracytoplasmic localization of the sulfur globule proteins. A fusion of the phoA gene to the sequence encoding the proposed signal peptide of sgpA led to high specific alkaline phosphatase activities in Escherichia coli, further supporting the envisaged targeting process. Together with electron microscopic evidence these results provide strong indication for an extracytoplasmic localization of the sulfur globules in Chr. vinosum and probably in other Chromatiaceae. Extracytoplasmic formation of stored sulfur could contribute to the transmembranous Deltap that drives ATP synthesis and reverse electron flow in Chr. vinosum.     

Shortcut of the photosynthetic electron transfer in a mutant lacking the reaction center-bound cytochrome subunit by gene disruption in a purple bacterium, Rubrivivax gelatinosus

OBJECTIVE: The purpose of our study was to determine the aetiological factors of uterine rupture during labour, and propose preventive measures. METHODS: This retrospective study was performed between February 1989 and July 1994, to analyze the cases of rupture uterus in relation to causes, age, parity, maternal and fetal mortality and morbidity. RESULTS: There were 37 cases of uterine rupture at our institutions. Obstructed labour by malpresentation and disproportion was the main cause. The presence of previous caesarean section scar, dysfunctional labour, injudicious use of uterine stimulant, were the other causes. There was no maternal death and the fetal loss was 17 (46%). CONCLUSIONS: The high incidence of uterine rupture is attributed to lack of prenatal care, labour in high risk patients outside the hospital because of declining economy, and more patients with 2 or more previously scarred uterus with many of them labouring more than 14 hours. Maternal and neonatal complications have remained very high in the developing countries.     

Purification and characterization of pyruvate oxidoreductase from the photosynthetic bacterium Rhodobacter capsulatus

The effects of the addition of a calcium channel blocker, verapamil (20 mg/kg/day) to an ACE inhibitor, trandolapril (0.7 mg/kg/day) in a 6-month treatment on renal insufficiency development in rats with 5/6th nephrectomy, were studied. Every month we measured heart rate and arterial pressure by the tail-cuff method. Renal function studies were performed in metabolic cages. At the end of the study, renal tissue was prepared for light microscope analysis. Renal lesions were assessed by semiquantitative scores in a blind fashion. Corpuscular section area, intraglomerular and tubulointerstitial fibrosis were determined by digital image analysis with a specific software (Fibrosis HR) on syrium red-stained renal sections. Trandolapril markedly increased the survival ratio that after 6 months reached 87% in comparison with 61% in untreated rats. No mortality was observed in rats treated with the combination of verapamil and trandolapril. Trandolapril treatment prevented the development of hypertension. The combination verapamil-trandolapril did not induce further reduction on blood pressure. The untreated group showed a marked proteinuria, that in the trandolapril group showed an important reduction. The verapamil + trandolapril group showed a proteinuria significantly smaller than that of all the other groups. Light microscopy semiquantitative studies of the renal injury showed that the trandolapril and verapamil + trandolapril groups had a marked reduction in glomerular and tubulointerstitial alterations, compared with untreated animals. Quantitative determinations of glomerular and interstitial fibrosis performed on syrium red-stained renal sections demonstrated that fibrosis was reduced when rats when treated with trandolapril and even more with verapamil + trandolapril when they were compared to untreated animals' values. In conclusion, long-term treatment with verapamil given in addition to trandolapril produces additional protection against progressive renal injury associated to subtotal nephrectomy.     

Analysis of subfossil molecular remains of purple sulfur bacteria in a lake sediment

Molecular remains of purple sulfur bacteria (Chromatiaceae) were detected in Holocene sediment layers of a meromictic salt lake (Mahoney Lake, British Columbia, Canada). The carotenoid okenone and bacteriophaeophytin a were present in sediments up to 11,000 years old. Okenone is specific for only a few species of Chromatiaceae, including Amoebobacter purpureus, which presently predominates in the chemocline bacterial community of the lake. With a primer set specific for Chromatiaceae in combination with denaturing gradient gel electrophoresis, 16S rRNA gene sequences of four different Chromatiaceae species were retrieved from different depths of the sediment. One of the sequences, which originated from a 9, 100-year-old sample, was 99.2% identical to the 16S rRNA gene sequence of A. purpureus ML1 isolated from the chemocline. Employing primers specific for A. purpureus ML1 and dot blot hybridization of the PCR products, the detection limit for A. purpureus ML1 DNA could be lowered to 0.004% of the total community DNA. With this approach the DNA of the isolate was detected in 7 of 10 sediment layers, indicating that A. purpureus ML1 constituted at least a part of the ancient purple sulfur bacterial community. The concentrations of A. purpureus DNA and okenone in the sediment were not correlated, and the ratio of DNA to okenone was much lower in the subfossil sediment layers (2.7 . 10(-6)) than in intact cells (1.4). This indicates that degradation rates are significantly higher for genomic DNA than for hydrocarbon cell constituents, even under anoxic conditions and at the very high sulfide concentrations present in Mahoney Lake.     

Temperature dependence of the Qy resonance Raman spectra of bacteriochlorophylls, the primary electron donor, and bacteriopheophytins in the bacterial photosynthetic reaction center

Qy-excited resonance Raman spectra of the accessory bacteriochlorophylls (B), the bacteriopheophytins (H), and the primary electron donor (P) in the bacterial photosynthetic reaction center (RC) of Rhodobacter sphaeroides have been obtained at 95 and 278 K. Frequency and intensity differences are observed in the low-frequency region of the P vibrational spectrum when the sample is cooled from 278 to 95 K. The B and H spectra exhibit minimal changes of frequencies and relative intensities as a function of temperature. The mode patterns in the Raman spectra of B and H differ very little from Raman spectra of the chromophores in vitro. The Raman scattering cross sections of B and H are 6-7 times larger than those for analogous modes of P at 278 K. The cross sections of B and of H are 3-4 times larger at 95 K than at 278 K, while the cross sections of P are approximately constant with temperature. The temperature dependence of the Raman cross sections for B and H suggests that pure dephasing arising from coupling to low-frequency solvent/protein modes is important in the damping of their excited states. The weak Raman cross sections of the special pair suggest that the excited state of P is damped by very rapid (<30 fs) electronic relaxation processes. These resonance Raman spectra provide information for developing multimode vibronic models of the excited-state structure and dynamics of the chromophores in the RC.     

Excited states and trapping in reaction center complexes of the green sulfur bacterium Prosthecochloris aestuarii

The excited states of bacteriochlorophyll (BChl) a were studied by pump-probe transient absorption spectroscopy in reaction center core (RCC), Fenna-Matthews-Olson (FMO) and FMO-RCC complexes of the green sulfur bacterium Prosthecochloris aestuarii. Excitation at 790 or 835 nm resulted in rapid equilibration of the energy between the BChl a molecules of the RCC complex: within 1 ps, most of the excitations had relaxed to the lowest energy level (835 nm), as a result of strong interactions between the BChls. Excitation of chlorophyll a 670 resulted in energy transfer to BChl a with a time constant of 1.2 ps, followed by thermal equilibration. Independent of the wavelength of excitation, the decay at 835 nm could be fitted with a time constant of about 25 ps, comparable to the 30 ps measured earlier with membrane fragments, which is ascribed to trapping in the reaction centers. Similar results were obtained with the FMO-RCC complex upon excitation at 835 or 670 nm, but the results upon 790 nm excitation were quite different. Again an equilibrium was rapidly reached, but now most of the excitations remained within the FMO complex, with a maximum bleaching at 813 nm, the same as observed in the isolated FMO. Even after 100 ps there was no bleaching at 835 nm and no evidence for charge separation. We conclude that there is no equilibration of the energy between the FMO and the RCC complex and that the efficiency of energy transfer from FMO to the reaction center core is low.     

A permanent hole burning study of the FMO antenna complex of the green sulfur bacterium Prosthecochloris aestuarii

A permanent hole burning study on the Fenna-Matthews-Olson, or FMO, antenna complex of the green sulfur bacterium Prosthecochloris aestuarii was carried out at 6 K. Excitation resulted not only in relatively sharp features resonant with the burn wavelength but also in broad absorbance changes in the wavelength region of 800-820 nm. The shape of the latter changes was almost independent of the wavelength of excitation. Evidence is given that they are induced by a different mechanism than that which causes the resonant holes and that they may be due to a conformational change of the protein. The original spectrum was restored upon warming to 60 K. The effective dephasing times T2, as obtained from the homogeneous line widths, increased from about 0.5 ps at 803 nm to >/=20 ps at 830 nm and are in good agreement with recent measurements of accumulated photon-echo and time-resolved absorbance changes.     

Triplet properties and interactions of the primary electron donor and antenna chromophores in membranes of Heliobacterium chlorum, studied with ADMR spectroscopy

The triplet states of antenna and reaction center bacteriochlorophyll (BChl) g in membranes of Heliobacterium chlorum were studied by optically detected magnetic resonance in zero magnetic field, using absorbance detection. A variety of triplet states was detected, which were all localized on single BChl g chromophores as concluded from a comparison with the triplet state of monomeric BChl g in organic solvents. With the aid of the microwave-induced absorbance difference spectra, we assign a triplet state with zero-field splitting parameters |D| = 727.5 and |E| = 254. 5 MHz to that of the primary donor. The low |E| value indicates that the BChls of the primary donor are monoligated. The intensities of the zero-field transitions were strongly dependent on the redox state of the secondary electron acceptors. A triplet state with |D| = 690-705 MHz and |E| =230 MHz, present under all redox conditions, is associated with antenna BChl g absorbing at 814 nm. Its triplet yield was independent of the redox conditions; we conclude therefore that the antenna chromophores absorbing at 814 nm are not connected with the reaction center at cryogenic temperatures (1.2 K). In addition, relatively strong signals were detected belonging to triplet states with |D| and |E| of 663-680 and 220-227 MHz, respectively, whose amplitudes were dependent on the redox conditions. Triplet states with these zero-field splitting parameters are located on antenna chromophores absorbing between 798-814 nm; their zero-field transitions and absorbance difference spectra indicate a considerable heterogeneity. The concentration of triplet states of antenna chromophores absorbing around 800 nm decreased markedly upon prolonged excitation at 1.2 K. This phenomenon is attributed to quenching of excitations on antenna pigments by stable charge separation in the closely connected reaction center, possibly involving a low-quantum yield menaquinone electron acceptor.     

The triplet state of the FMO complex of the green sulfur bacterium Prosthecochloris aestuarii studied with single-crystal EPR

Triplet-electron paramagnetic resonance (EPR) spectra were obtained of single crystals of the FMO complex of the green sulfur bacterium Prostecochloris aestuarii. The experiments support the results presented in a previous paper (Louwe et al., J. Phys. Chem. 101 (1997) 11280), which showed that the experimental optical spectra of this pigment-protein complex are best reproduced by assuming that one bacteriochlorophyll (BChl 3) is energetically isolated and that this BChl is the triplet-carrying BChl of the FMO complex at cryogenic temperatures for low excitation density. When comparing the experimental and simulated data sets of the triplet-EPR spectra in single crystals, the best fit is obtained for two triplet states, one localized at BChl 3 and the other at BChl 1. The existence of two different triplet states is traced to the relatively high excitation power necessary to observe the small triplet-EPR signal of the FMO single crystals.     

Isolation and characterization of the lipopolysaccharide of Chromatium vinosum

Lipolysaccharide was isolated from Chromatium vinosum by phenol/water extraction. The lipopolysaccharide is found exclusively in the phenol phase and can be cleaved into a sugar moiety and a lipid A fraction by hydrolysis in 10% acetic acid at 100 degrees C for 3-4 h. The sugar moiety contains the neutral sugars 3-O-methyl-D-ribose, D-ribose, L-arabinose, mannosamine and glucose, and smaller quantities of D-rhamnose, D-glycero-D-manno-heptose (tentatively identified), quinovosamine and 2-keto-3-deoxyoctonate. L-glycero-D-manno-heptose was not detected. The 2-keto-3-deoxyoctonate linkage in C. vinosum lipopolysaccharide is more resistant to acid hydrolysis than that of Escherichia coli. The lipid A fraction contains glucosamine, mannose and the fatty acids of the lipopolysaccharide. The major fatty acid is beta-hydroxymyristic acid, with smaller amounts of lauric and palmitic acids as well as 14-carbon mono-unsaturated fatty acid, also being present. The phosphorus content of the C. vinosum lipopolysaccharide was found to be approximately 0.1%. Erythrocytes sensitized with alkali-treated C. vinosum lipopolysaccharide were agglutinated by antisera prepared against heat-killed cells. Untreated or heat-treated lipopolysaccharide did not sensitize erythrocytes. The lethal toxicity to mice of the C. vinosum lipopolysaccharide is about one-tenth as that from Salmonella abortus equi.     

Complete assimilation of cysteine by a newly isolated non-sulfur purple bacterium resembling Rhodovulum sulfidophilum (Rhodobacter sulfidophilus)

A rod-shaped, motile, phototrophic bacterium, strain SiCys, was enriched and isolated from a marine microbial mat, with cysteine as sole substrate. During phototrophic anaerobic growth with cysteine, sulfide was produced as an intermediate, which was subsequently oxidized to sulfate. The molar growth yield with cysteine was 103 g mol-1, in accordance with complete assimilation of electrons from the carbon and the sulfur moiety into cell material. Growth yields with alanine and serine were proportionally lower. Thiosulfate, sulfide, hydrogen, and several organic compounds were used as electron donors in the light, whereas cystine, sulfite, or elemental sulfur did not support phototrophic anaerobic growth. Aerobic growth in the dark was possible with fructose as substrate. Cultures of strain SiCys were yellowish-brown in color and contained bacteriochlorophyll a, spheroidene, spheroidenone, and OH-spheroidene as major photosynthetic pigments. Taking the morphology, photosynthetic pigments, aerobic growth in the dark, and utilization of sulfide for phototrophic growth into account, strain SiCys was assigned to the genus Rhodovulum (formerly Rhodobacter) and tentatively classified as a strain of R. sulfidophilum. In cell-free extracts in the presence of pyridoxal phosphate, cysteine was converted to pyruvate and sulfide, which is characteristic for cysteine desulfhydrase activity (l-cystathionine gamma-lyase, EC 4.4. 1.1).     

Site-directed mutations affecting the spectroscopic characteristics and midpoint potential of the primary donor in photosystem I

Photosystem I is a member of the iron-sulfur center or type I reaction centers. The primary electron donor in photosystem I is a chlorophyll a dimer termed P700. The biophysical properties of P700 are well understood, but the protein environment that gives it such unique properties is unknown. We have characterized site-directed mutants of the photosystem I reaction center protein PsaB and identified an amino acid, His-656, that interacts closely with one of the P700 chlorophylls. Mutation of His-656 to Asn or Ser increases the oxidation midpoint potential of P700/P700+. by 40 mV. The P700/P700+. optical difference spectra show the appearance of a new bleaching band at 667 nm. Electron nuclear double resonance spectroscopy indicates a significant increase in the hyperfine coupling corresponding to methyl protons at position 12 of the spin carrying chlorophyll a of P700+. The implication of these results to current structural models of the photosystem I reaction center is discussed.     

Effects of hydrogen bonds on the redox potential and electronic structure of the bacterial primary electron donor

The primary donor, P, of photosynthetic bacterial reaction centers (RCs) is a dimer of excitonically interacting bacteriochlorophyll (BChl) molecules. The two constituents are named PL and PM to designate their close association with the L- and M-subunits, respectively, of the RC protein. A series of site-directed mutants of RCs from Rhodobacter sphaeroides has been constructed in order to model the effects of hydrogen bonding on the redox midpoint potential and electronic structure of P. The leucine residue at position M160 was genetically replaced with eight other amino acid residues capable of donating a hydrogen bond to the C9 keto carbonyl group of the PM BChl a molecule of P. Fourier transform (FT) (pre)resonance Raman spectroscopy with 1064 nm excitation was used to (i) determine the formation and strengths of hydrogen bonds on this latter keto carbonyl group in the reduced, neutral state (PO), and (ii) determine the degree of localization of the positive charge on one of the two constituent BChl molecules of P in its oxidized, radical cation state (P*+). A correlation was observed between the strength of the hydrogen bond and the increase in PO/P*+ redox midpoint potential. This correlation is less pronounced than that observed for another series of RC mutants where hydrogen bonds to the four pi-conjugated carbonyl groups of P were broken or formed uniquely involving histidinyl residues [Mattioli, T. A., Lin, X., Allen, J. P. and Williams, J. C. (1995) Biochemistry 34, 6142-6152], indicating that histidinyl residues are more effective in raising the PO/P*+ redox midpoint potential via hydrogen bond formation than are other hydrogen bond-forming residues. In addition, an increase in positive charge localization is correlated with the strength of the hydrogen bond and with the PO/P*+ redox midpoint potential. This latter correlation was analyzed using an asymmetric bacteriochlorophyll dimer model based on Hückel-type molecular orbitals in order to obtain estimates of certain energetic parameters of the primary donor. Based on this model, the correlation is extrapolated to the case of complete localization of the positive charge on PL and gives a predicted value for the P/P+ redox midpoint potential similar to that experimentally determined for the Rb. sphaeroides HL(M202) heterodimer. The model yields parameters for the highest occupied molecular orbital energies of the two BChl a constituents of P which are typical for the oxidation potential of isolated BChl a in vitro, suggesting that the protein, as compared to many solvents, does not impart atypical redox properties to the BChl a constituents of P.     

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.