Impact of A90P, F106L and H64V mutations on neuroglobin stability and ligand binding kinetics.

Human neuroglobin (Ngb) is a hexacoordinated globin which binds some small ligands. Its function is still not well-established, even though Ngb seems to be implicated in the protection against neurodegenerative diseases. It has been shown by molecular dynamics and crystallography that ligand binding could occur thanks to a haem sliding mechanism specific to Ngb. In this paper, we studied some regions which could participate in this mechanism. We used UV-visible spectroscopy, CD and NMR to have a look on the protein structure and NMR and stopped-flow to study the ligand binding properties of the proteins. In the haem environment we mutated the distal histidine H64, the alanine A90 which is on the proximal F helix and the phenylalanine F106 which is close to the haem. We showed that both H64V and A90P variants, which affect the haem coordination, seemed to be important to haem and protein secondary structure stabilities whereas F106L mutation did not affect those properties. Then we confirmed that the cyanide binding kinetics were isomer dependent on wild-type Ngb and A90P and F106L variants. H64V Ngb variant had a behavior similar to wild-type Mb or Hb with a loss of the haem kinetic differentiation. Moreover, our results suggested that one haem isomer was more sensitive to A90P and F106L mutations. Those results brought some evidence that the haem sliding mechanism could occur for the cyanide binding and could be haem isomer dependent. The isomer forms may play distinct roles for the potential function of Ngb in vivo.

The dynamics of the non-heme iron in bacterial reaction centers from Rhodobacter sphaeroides.

We investigate the dynamical properties of the non-heme iron (NHFe) in His-tagged photosynthetic bacterial reaction centers (RCs) isolated from Rhodobacter (Rb.) sphaeroides. Mössbauer spectroscopy and nuclear inelastic scattering of synchrotron radiation (NIS) were applied to monitor the arrangement and flexibility of the NHFe binding site. In His-tagged RCs, NHFe was stabilized only in a high spin ferrous state. Its hyperfine parameters (IS=1.06±0.01mm/s and QS=2.12±0.01mm/s), and Debye temperature (θ(D0)~167K) are comparable to those detected for the high spin state of NHFe in non-His-tagged RCs. For the first time, pure vibrational modes characteristic of NHFe in a high spin ferrous state are revealed. The vibrational density of states (DOS) shows some maxima between 22 and 33meV, 33 and 42meV, and 53 and 60meV and a very sharp one at 44.5meV. In addition, we observe a large contribution of vibrational modes at low energies. This iron atom is directly connected to the protein matrix via all its ligands, and it is therefore extremely sensitive to the collective motions of the RC protein core. A comparison of the DOS spectra of His-tagged and non-His-tagged RCs from Rb. sphaeroides shows that in the latter case the spectrum was overlapped by the vibrations of the heme iron of residual cytochrome c(2), and a low spin state of NHFe in addition to its high spin one. This enabled us to pin-point vibrations characteristic for the low spin state of NHFe.

Key role of proline L209 in connecting the distant quinone pockets in the reaction center of Rhodobacter sphaeroides.

Photosynthetic bacterial reaction centers convert light excitation into chemical free energy. The initial electron transfer leads to the consecutive semireductions of the primary (Q(A)) and secondary (Q(B)) quinone acceptors. The Q(A)(-) and Q(B)(-) formations induce proton uptake from the bulk. Their magnitudes (H(+)/Q(A)(-) and H(+)/Q(B)(-), respectively) probe the electrostatic interactions within the complex. The pH dependence of H(+)/Q(A)(-) and H(+)/Q(B)(-) were studied in five single mutants modified at the L209 site (L209P-->F,Y,W,E,T). This residue is situated at the border of a continuous chain of water molecules connecting Q(B) to the bulk. In the wild type (WT), a proton uptake band is present at high pH in the H(+)/Q(A)(-) and H(+)/Q(B)(-) curves and is commonly attributed to a cluster of acidic groups situated nearby Q(B). In the H(+)/Q(A)(-) curves of the L209 variants, this band is systematically absent but remains in the H(+)/Q(B)(-) curves. Moreover, notable increase of H(+)/Q(B)(-) is observed in the L209 mutants at neutral pH as compared with the WT. The large effects observed in all L209 mutants are not associated with significant structural changes (Kuglstatter, A., Ermler, U., Michel, H., Baciou, L. & Fritzsch, G. Biochemistry (2001) 40, 4253-4260). Our data suggest that, in the L209 mutants, the Q(B) cluster does not respond to the Q(A)(-) formation as observed in the WT. We propose that, in the mutants, removal of the rigid proline L209 breaks a necessary hydrogen bonding connection between the quinone sites. These findings suggest an important role for structural rigidity in ensuring a functional interaction between quinone binding sites.

Revealing the involvement of extended hydrogen bond networks in the cooperative function between distant sites in bacterial reaction centers.

In reaction center proteins of photosynthetic bacteria, the amplitude of proton uptake induced by the one-electron reduction of either of the two quinone electron acceptors (Q(A) and Q(B)) is an intrinsic observable of the electrostatic interactions associated with the redox function of the complex. We report here that, in Rhodobacter capsulatus, complete restoration of proton uptake (upon formation of Q(A)(-) and Q(B)(-)) to the level found in the wild type is observed in a mutant reaction center in which a tyrosine substitution in the Q(A) environment (Ala(M274) --> Tyr) is coupled with mutations of acidic residues near Q(B) (Glu(L212) --> Ala/Asp(L213) --> Ala) that initially cancel the proton uptake above pH 8. This result demonstrates that proton uptake occurs by strong cooperation between structural motifs, such as hydrogen-bonded networks, that span the 18 A distance between the two quinone acceptors.

Modeling the effects of mutations on the free energy of the first electron transfer from QA- to QB in photosynthetic reaction centers.

Numerical calculations of the free energy of the first electron transfer in genetically modified reaction centers from Rhodobacter (Rb.) sphaeroides and Rb. capsulatus were carried out from pH 5 to 11. The multiconformation continuum electrostatics (MCCE) method allows side chain, ligand, and water reorientation to be embedded in the calculations of the Boltzmann distribution of cofactor and amino acid ionization states. The mutation sites whose effects have been modeled are L212 and L213 (the L polypeptide) and two in the M polypeptide, M43(44) and M231(233) in Rb. capsulatus (Rb. sphaeroides). The results of the calculations were compared to the experimental data, and very good agreement was found especially at neutral pH. Each mutation removes or introduces ionizable residues, but the protein maintains a net charge close to that in native RCs through ionization changes in nearby residues. This reduces the effect of mutation and makes the changes in state free energy smaller than would be found in a rigid protein. The state energy of QA-QB and QAQB- states have contributions from interactions among the residues as well as with the quinone which is ionized. For example, removing L213Asp, located in the QB pocket, predominantly changes the free energy of the QA-QB state, where the Asp is ionized in native RCs rather than the QAQB- state, where it is neutral. Side chain, hydroxyl, and water rearrangements due to each of the mutations have also been calculated showing water occupancy changes during the QA- to QB electron transfer.

Structure of the photosynthetic reaction centre from Rhodobacter sphaeroides reconstituted with anthraquinone as primary quinone Q(A).

In the photosynthetic reaction centre (RC) from the purple bacterium Rhodobacter sphaeroides, the primary quinone, a ubiquinone-10 (Q(A)), has been substituted by anthraquinone. Three-dimensional crystals have been grown from the modified RC and its structure has been determined by X-ray crystallography to 2.4 A resolution. The bindings of the head-group from ubiquinone-10 and of the anthraquinone ring are very similar. In particular, both rings are parallel to each other and the hydrogen bonds connecting the native ubiquinone-10 molecule to AlaM260 and HisM219 are conserved in the anthraquinone containing RC. The space of the phytyl tail missing in the anthraquinone exchanged RC is occupied by the alkyl chain of a detergent molecule. Other structural changes of the Q(A)-binding site are within the limit of resolution. Our structural data bring strong credit to the very large amount of spectroscopic data previously achieved in anthraquinone-replaced RCs and which have participated in the determination of the energetics of the quinone system in bacterial RCs.

Proton uptake by bacterial reaction centers: the protein complex responds in a similar manner to the reduction of either quinone acceptor.

In bacterial photosynthetic reaction centers, the protonation events associated with the different reduction states of the two quinone molecules constitute intrinsic probes of both the electrostatic interactions and the different kinetic events occurring within the protein in response to the light-generated introduction of a charge. The kinetics and stoichiometries of proton uptake on formation of the primary semiquinone Q(A)(-) and the secondary acceptor Q(B)(-) after the first and second flashes have been measured, at pH 7.5, in reaction centers from genetically modified strains and from the wild type. The modified strains are mutated at the L212Glu and/or at the L213Asp sites near Q(B); some of them carry additional mutations distant from the quinone sites (M231Arg --> Leu, M43Asn --> Asp, M5Asn --> Asp) that compensate for the loss of L213Asp. Our data show that the mutations perturb the response of the protein system to the formation of a semiquinone, how distant compensatory mutations can restore the normal response, and the activity of a tyrosine residue (M247Ala --> Tyr) in increasing and accelerating proton uptake. The data demonstrate a direct correlation between the kinetic events of proton uptake that are observed with the formation of either Q(A)(-) or Q(B)(-), suggesting that the same residues respond to the generation of either semiquinone species. Therefore, the efficiency of transferring the first proton to Q(B) is evident from examination of the pattern of H(+)/Q(A)(-) proton uptake. This delocalized response of the protein complex to the introduction of a charge is coordinated by an interactive network that links the Q(-) species, polarizable residues, and numerous water molecules that are located in this region of the reaction center structure. This could be a general property of transmembrane redox proteins that couple electron transfer to proton uptake/release reactions.

In Rhodobacter sphaeroides reaction centers, mutation of proline L209 to aromatic residues in the vicinity of a water channel alters the dynamic coupling between electron and proton transfer processes.

The X-ray crystallographic structure of the photosynthetic reaction center from Rhodobacter sphaeroides obtained at high resolution has revealed a number of internal water molecules (Ermler, U., Fritzsch, G., Buchanan, S. K., and Michel, H. (1994) Structure 2, 925-936; Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E., and Feher, G. (1997) Science 276, 812-816). Some of them are organized into distinct hydrogen-bonded water chains that connect Q(B) (the terminal quinone electron acceptor of the reaction center) to the aqueous phase. To investigate the role of the water chains in the proton conduction process, proline L209, located immediately adjacent to a water chain, was mutated to the following residues: F, Y, W, E, and T. We have first analyzed the effects of the mutations on the kinetic and thermodynamic properties of the rate constants of the second electron transfer (k(AB)(2)) and of the coupled proton uptake (k(H)+) at the second flash. In all aromatic mutants, k(AB)(2) and k(H)+ are notably and concomitantly decreased compared to the wild-type, while no effect is observed in the other mutants. The temperature dependence of these rates shows activation energy values (DeltaH) similar for the proton and electron-transfer processes in the wild-type and in most of the mutants, except for the L209PW and L209PF mutants. The analysis of the enthalpy factors related to the electron and proton-transfer processes in the L209PF and the L209PW mutants allows to distinguish the respective effects of the mutations for both transfer reactions. It is noteworthy that in the aromatic mutants a substantial increase of the free energies of activation is observed (DeltaG(L209PY) < DeltaG(L209PF) < DeltaG(L209PW)) for both proton and electron-transfer reactions, while in the other mutants, DeltaG is not affected. The salt concentration dependence of k(AB)(2) shows, in the L209PF and L209PW mutants, a higher screening of the protein surface potential experienced by Q(B). Our data suggest that residues F and W in position L209 increase the polarizability of the internal water molecules and polar residues by altering the organization of the hydrogen-bond network. We have also analyzed the rates of the first electron-transfer reaction (k(AB)(1)), in the 100 micros time domain. These kinetics have previously been shown to reflect protein relaxation events possibly including proton uptake events (Tiede, D. M., Vazquez, J., Cordova, J., and Marone, P. M. (1996) Biochemistry 35, 10763-10775). Interestingly, in the L209PF and L209PW mutants, k(AB)(1) is notably decreased in comparison to the wild type and the other mutants, in a similar way as k(AB)(2) and k(H)+. Our data imply that the dynamic organization of this web is tightly coupled to the electron transfer process that is kinetically limited by protonation events and/or conformational rearrangements within the protein.

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.

Flash-induced changes in buffering capacity of reaction centers from photosynthetic bacteria reveal complex interaction between quinone pockets.

A novel method was applied to determine light-induced protonation in reaction centers from photosynthetic purple bacteria. Changes in buffering capacities upon flash excitation were detected in (0.03% Triton X-100) detergent solution of reaction centers from Rhodobacter (Rb.) sphaeroides and Rb. capsulatus wild type and mutant strains with empty or occupied secondary quinone (QB) binding sites in the presence of an external electron donor. The light-induced differences in buffering capacities between pH 4 and 11 were analyzed in terms of protonatable residues. Due to its differential nature, this method is more sensitive to the position and shift of pKa of the individual groups than the direct method based on proton uptake measurements. Out of the four different ionizable residues which were used to fit the curves, the two groups with apparent (dark) pKa values between 8.4-8.8 and 9.5-10.0 (depending on the species and conditions) disappeared when the native ubiquinone10 was replaced by menadione at the primary quinone (QA) binding site of Rb. sphaeroides or when the key protonatable residues (L212Glu and L213Asp) were replaced by non-protonatable alanines in the QB binding site of the AA+M43D mutant from Rb. capsulatus. The experimentally observed acidic and neutral residues remained unchanged. These results obtained from modifications in both quinone sites reveal the origin of the alkaline pH groups: they reflect the interaction of QA- and the cluster of ionizable residues around L212Glu in the QB binding pocket. The involvement of two residues with close pKa values reflects the complex titration of the cluster. The interaction between the quinone pockets is best described qualitatively as a network of ionizable residues extending from the QA site to the QB site.

In bacterial reaction centers, a key residue suppresses mutational blockage of two different proton transfer steps.

In reaction centers of Rhodobacter (Rb.) capsulatus, the M43Asn --> Asp substitution is capable of restoring rapid rates for delivery of the second proton to QB in a mutant that lacks L212Glu. Flash-induced absorbance spectroscopy was used to show a nearly native rate for transfer of the second proton to QB (approximately 700 s-1) in the L212Gln+M43Asp double-mutant reaction center; this rate was shown to decrease more than 1000-fold in the photoincompetent L212Glu --> Gln mutant [Miksovska, J., Kálmán, L., Maróti, P., Schiffer, M., Sebban, P., and Hanson, D.K. (1997) Biochemistry 36, 12216-12226]. In Rb. sphaeroides, the equivalent M44Asn --> Asp mutation was reported to restore the rate of transfer of the first proton to a wild-type level when it is added to the L213Asp --> Asn photoincompetent mutant [Rongey, S.H., Paddock, M.L., Feher, G., and Okamura, M.Y. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1325-1329]. It is remarkable that the same second-site mutation can compensate for both of these mutations which severely impair reaction center function by blocking two different proton-transfer reactions. We suggest that residue M43Asp is situated in a key position which can link pathways for delivery of both the first and second protons (involving structured water molecules) to QB.

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.

A native electrostatic environment near Q(B) is not sufficient to ensure rapid proton delivery in photosynthetic reaction centers.

Flash-induced absorption spectroscopy has been used to characterize Rhodobacter capsulatus reaction centers mutated in the secondary quinone acceptor site (Q(B). We compared the wild-type, the L212Glu-L213Asp --> Ala-Ala photosynthetically incompetent double mutant (DM), and two photocompetent revertants, the DM+L217Arg --> Cys and the DM+M5Asn- --> Asp strains. The electrostatic environment for Q(B)- is different in the two revertant strains. Only the L217Arg --> Cys mutation nearly restores the native electrostatic environment of Q(B)-. However, the level of recovery of the reaction center function, measured by the rates of second electron transfer and cytochrome c turnover, is quite incomplete in both strains. This shows that a wild-type-like electrostatic environment of Q(B)- cannot ensure on its own, rapid and efficient proton transfer to Q(B)-.

pH-metric study of reaction centers from photosynthetic bacteria in micellular solutions: protonatable groups equilibrate with the aqueous bulk phase.

Hydrogen ion equilibria of the reaction center protein from photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus dissolved in micellular solution were studied by acid-base titration to estimate the water accessibility of protonatable residues of the protein determined from structural data. The ionizable amino acids of the reaction center underwent protonation-deprotonation with protons from the interfacial layer, which, however, exchanged protons from the aqueous bulk phase. The equilibrium was described in terms of the buffering capacity of the multiphase system. The detergents decreased the proton activity coefficient (increased the buffering capacity) of the aqueous solution by a factor of 0.33 (in 0.03% Triton X-100 and LDAO) and 0.12 (0.04% dodecyl beta-D-maltoside). The observed buffering capacities of the reaction center protein were large and detergent-dependent. However, corrections for proton activities made the pH dependence of buffering capacities in different detergents uniform and similar to that expected from the number and pK values of protonatable groups of the protein. The vast majority of protonatable amino acids of the reaction center are in protonation equilibria with the aqueous bulk phase on an extended time scale.

Distant electrostatic interactions modulate the free energy level of QA- in the photosynthetic reaction center.

In the reaction centers from the purple photosynthetic bacterium Rhodobacter capsulatus, we have determined that residue L212Glu, situated near the secondary quinone acceptor QB, modulates the free energy level of the reduced primary quinone molecule QA- at high pH. Even though the distance between L212Glu and QA is 17 A, our results indicate an apparent interaction energy between them of 30 +/- 18 meV. This interaction was measured by quantitating the stoichiometry of partial proton uptake upon formation of QA- as a function of pH in four mutant strains which lack L212Glu, in comparison with the wild type. These strains are the photosynthetically incompetent site-specific mutants L212Glu -->Gln and L212Glu-L213Asp-->Ala-Ala and the photocompetent strains L212Glu-->Ala and L212Ala-L213Ala-M43Asn-->Ala-Ala-Asp. Below pH 7.5, the stoichiometry of proton uptake from all strains is nearly superimposable with that of the wild type. However, at variance with the wild type, reaction centers from all strains that lack L212Glu fail to take up protons above pH 9. The lack of a change in the free energy level of QA- at high pH in the L212Glu-modified strains is confirmed by the determination of the pH dependence of the rate (kAP) of P+QA- charge recombination in the reaction centers where the native QA is replaced by quinones having low redox potentials. Contrary to the wild-type reaction centers where kAP increases at high pH, almost no pH dependence could be detected in the strains that lack L212Glu. Our data show that the ionization state of L212Glu, either on its own or via interactions with closely associated ionizable groups, is mainly involved in the proton uptake at high pH by reaction centers in the PQA- state. This suggests that the formation of the QA- semiquinone state induces shifts in pKas of residues in the QB proteic environment. This long-distance influence of ionization states is a mechanism which would facilitate electron transfer from QA to QB on the first and second flashes. The functional communication between the two quinone protein pockets may involve the iron-ligand complex which spans the distance between them.

Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer.

Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15±0.1) than in Rhodobacter capsulatus (1.2±0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary quinone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.

Energetics of the quinone electron acceptor complex in Rubrivivax gelatinosus.

The pH and temperature dependences of the free energy stabilization of the Q-A and Q-B semiquinone anions (QA and QB are respectively the primary and secondary quinone electron acceptors) were studied in antenna-reaction centre complex from Rubrivivax (R.) gelatinosus. This was achieved by measuring the rate constants of the P+Q-A (kAP) and P+Q-B (kBP) (P is the primary electron donor) charge recombination processes by flash-induced absorption spectroscopy. Despite the high primary sequence analogies of the QA and QB protein pockets between R. gelatinosus and the much more studied species as Rps. viridis, Rb. sphaeroides and Rb. capsulatus, the energetic behaviour of the quinone complex of R. gelatinosus appears to be somewhat different: (i) above pH 10, kAP decreases, whereas it increases in Rps. viridis; this suggests the presence of a protonatable group that stabilizes I- (I is a bacteriopheophytin electron acceptor) rather than Q-A; (ii) the pH dependence of kBP is unusually flat in the range 4-7.5, possibly reflecting that a substantial part of the P+Q-B charge recombination proceeds via the direct route through the protein by an electron tunnelling mechanism, at variance to what is observed in the three species mentioned above; (iii) the very substantial increase of kBP observed above pH 7.5 is reasonably well described by the presence of two apparent protonatable groups: pK1QB = 9.4, pK1Q-B = 11 and pK2QB = 8.5, pK2Q-B = 9.4. The latter group was not reported in Rps. viridis, Rb. sphaeroides or Rb. capsulatus. We conclude that the apparent pK values measured here in R. gelatinosus may reflect the contribution as a whole of several and/or distant groups rather than of well-defined residues.

Electrostatic dominoes: long distance propagation of mutational effects in photosynthetic reaction centers of Rhodobacter capsulatus.

Two point mutants from the purple bacterium Rhodobacter capsulatus, both modified in the M protein of the photosynthetic reaction center, have been studied by flash-induced absorbance spectroscopy. These strains carry either the M231Arg --> Leu or M43ASN --> Asp mutations, which are located 9 and 15 A, respectively, from the terminal electron acceptor QB. In the wild-type Rb. sphaeroides structure, M231Arg is involved in a conserved salt bridge with H125Glu and H232Glu and M43Asn is located among several polar residues that form or surround the QB binding site. These substitutions were originally uncovered in phenotypic revertants isolated from the photosynthetically incompetent L212Glu-L213Asp --> Ala-Ala site-specific double mutant. As second-site suppressor mutations, they have been shown to restore the proton transfer function that is interrupted in the L212Ala-L213Ala double mutant. The electrostatic effects that are induced in reaction centers by the M231Arg --> Leu and M43Asn --> Asp substitutions are roughly the same in either the double-mutant or wild-type backgrounds. In a reaction center that is otherwise wild type in sequence, they decrease the free energy gap between the QA- and QB- states by 24 +/- 5 and 45 +/- 5 meV, respectively. The pH dependences of K2, the QA-QB <--> QAQB- equilibrium constant, are altered in reaction centers that carry either of these substitutions, revealing differences in the pKas of titratable groups compared to the wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Electron and proton transfer to the quinones in bacterial photosynthetic reaction centers: insight from combined approaches of molecular genetics and biophysics.

We present here new results together with an overview of the current knowledge on the coupled processes of electron and proton transfer in bacterial reaction centers. The importance of a multidisciplinary approach associating molecular genetics, structural biology, biochemistry and spectroscopy is underlined. We emphasize the electrostatic role of the protein to maintain a negative electrostatic potential near the second quinone electron acceptor in order to: i) accelerate the overall rate of proton transfer from the cytoplasm to this acceptor by increasing the pKs of some groups involved in this process; ii) increase the local proton concentration near this acceptor. We also point out the possibility of long distance propagation of the electrostatic effects through the protein associated with relaxation processes triggered by the formation of the semiquinone anions on the first flash.