Investigation of B-branch electron transfer by femtosecond time resolved spectroscopy in a Rhodobacter sphaeroides reaction centre that lacks the Q(A) ubiquinone

The dynamics of electron transfer in a membrane-bound Rhodobacter sphaeroides reaction centre containing a combination of four mutations were investigated by transient absorption spectroscopy. The reaction centre, named WAAH, has a mutation that causes the reaction centre to assemble without a Q(A) ubiquinone (Ala M260 to Trp), a mutation that causes the replacement of the H(A) bacteriopheophytin with a bacteriochlorophyll (Leu M214 to His) and two mutations that remove acidic groups close to the Q(B) ubiquinone (Glu L212 to Ala and Asp L213 to Ala). Previous work has shown that the Q(B) ubiquinone is reduced by electron transfer along the so-called inactive cofactor branch (B-branch) in the WAAH reaction centre (M.C. Wakeham, M.G. Goodwin, C. McKibbin, M.R. Jones, Photo-accumulation of the P(+)Q(B)(-) radical pair state in purple bacterial reaction centres that lack the Q(A) ubiquinone, FEBS Letters 540 (2003) 234-240). In the present study the dynamics of electron transfer in the membrane-bound WAAH reaction centre were studied by femtosecond transient absorption spectroscopy, and the data analysed using a compartmental model. The analysis indicates that the yield of Q(B) reduction via the B-branch is approximately 8% in the WAAH reaction centre, consistent with results from millisecond time-scale kinetic spectroscopy. Possible contributions to this yield of the constituent mutations in the WAAH reaction centre and the membrane environment of the complex are discussed.     

Charge recombination and protein dynamics in bacterial photosynthetic reaction centers entrapped in a sol-gel matrix

Many proteins can be immobilized in silica hydrogel matrices without compromising their function, making this a suitable technique for biosensor applications. Immobilization will in general affect protein structure and dynamics. To study these effects, we have measured the P(+)Q(A)(-) charge recombination kinetics after laser excitation of Q(B)-depleted wild-type photosynthetic reaction centers from Rhodobacter sphaeroides in a tetramethoxysilane (TMOS) sol-gel matrix and, for comparison, also in cryosolvent. The nonexponential electron transfer kinetics observed between 10 and 300 K were analyzed quantitatively using the spin boson model for the intrinsic temperature dependence of the electron transfer and an adiabatic change of the energy gap and electronic coupling caused by protein motions in response to the altered charge distributions. The analysis reveals similarities and differences in the TMOS-matrix and bulk-solvent samples. In both preparations, electron transfer is coupled to the same spectrum of low frequency phonons. As in bulk solvent, charge-solvating protein motions are present in the TMOS matrix. Large-scale conformational changes are arrested in the hydrogel, as evident from the nonexponential kinetics even at room temperature. The altered dynamics is likely responsible for the observed changes in the electronic coupling matrix element.     

Residual water modulates QA- -to-QB electron transfer in bacterial reaction centers embedded in trehalose amorphous matrices

The role of protein dynamics in the electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B), was studied at room temperature in isolated reaction centers (RC) from the photosynthetic bacterium Rhodobacter sphaeroides by incorporating the protein in trehalose water systems of different trehalose/water ratios. The effects of dehydration on the reaction kinetics were examined by analyzing charge recombination after different regimes of RC photoexcitation (single laser pulse, double flash, and continuous light) as well as by monitoring flash-induced electrochromic effects in the near infrared spectral region. Independent approaches show that dehydration of RC-containing matrices causes reversible, inhomogeneous inhibition of Q(A)(-)-to-Q(B) electron transfer, involving two subpopulations of RCs. In one of these populations (i.e., active), the electron transfer to Q(B) is slowed but still successfully competing with P(+)Q(A)(-) recombination, even in the driest samples; in the other (i.e., inactive), electron transfer to Q(B) after a laser pulse is hindered, inasmuch as only recombination of the P(+)Q(A)(-) state is observed. Small residual water variations ( approximately 7 wt %) modulate fully the relative fraction of the two populations, with the active one decreasing to zero in the driest samples. Analysis of charge recombination after continuous illumination indicates that, in the inactive subpopulation, the conformational changes that rate-limit electron transfer can be slowed by >4 orders of magnitude. The reported effects are consistent with conformational gating of the reaction and demonstrate that the conformational dynamics controlling electron transfer to Q(B) is strongly enslaved to the structure and dynamics of the surrounding medium. Comparing the effects of dehydration on P(+)Q(A)(-)-->PQ(A) recombination and Q(A)(-)Q(B)-->Q(A)Q(B)(-) electron transfer suggests that conformational changes gating the latter process are distinct from those stabilizing the primary charge-separated state.     

The reconstitution of energy transfer in membranes from a bacteriochlorophyll-less mutant of Rhodopseudomonas sphaeroides by addition of light-harvesting and reaction centre pigment-protein complexes.

Antenna and reaction centre complexes purified from photosynthetically-grown cells of Rhodopseudomonas sphaeroides have been mixed with cytoplasmic membranes prepared from an aerobically-grown bacteriochlorophyll-less mutant of Rp. sphaeroides (designated 01) in the presence of 1% sodium cholate. After removal of the cholate by dislysis, the dislysate was subjected to isopycnic centrifugation. Reconstituted cytochrome c2 photooxidation and cytochrome b photoreduction was demonstrated in a pigmented fraction recovered from the sucrose gradient, suggesting that the pigment-proteins were incorporated into the 01 membrane. The fluorescence properties of the system were examined. The appearance of a variable component after the initial fast fluorescence rise indicated that energy transfer occurred between the antenna and reaction centre proteins in the presence of 01 membrane. The order in which the system was assembled was important. Reconstituted energy transfer with a pre-dialysed reaction centre-antenna complex was more effective than when all the components were mixed at once. Energy transfer was also reconstituted between added reaction centre protein and the endogenous antenna present in membranes from the pigmented, but aerobically-grown reaction centre-less mutant PM8dp of Rp. sphaeroides. Preparations of 01 membranes reconstituted with reaction centre exhibited a light intensity dependent cytochrome c2 photooxidation. At low exciting light intensities, preparations containing reconstituted antenna protein in addition to reaction centres showed greated membrane cytochrome c2 photooxidation than preparations with the antenna omitted; this improvement was maximal when a pre-dialysed antenna-reaction centre complex was used.     

Fourier transform infrared evidence of proton uptake by glutamate L212 upon reduction of the secondary quinone QB in the photosynthetic reaction center from Rhodobacter capsulatus

The photoreduction of the secondary quinone Q(B) in native reaction centers (RCs) of Rhodobacter capsulatus and in RCs from the GluL212 --> Gln and GluL212 --> Ala mutants has been investigated at pH 7 in (1)H(2)O and (2)H(2)O by light-induced Fourier transform infrared (FTIR) difference spectroscopy. The Q(B)(-)/Q(B) FTIR difference spectra reflect changes of quinone-protein interactions and of protonation state of carboxylic acid groups as well as reorganization of the protein upon electron transfer. Comparison of Q(B)(-)/Q(B) spectra of native and mutant RCs indicates that the interactions between Q(B) or Q(B)(-) and the protein are similar in all RCs. A differential signal at approximately 1650/1640 cm(-1), which is common to all the spectra, is associated with a movement of a peptide carbonyl or a side chain following Q(B) reduction. On the other hand, Q(B)(-)/Q(B) spectra of native and mutant RCs display several differences, notably between 1700 and 1650 cm(-1) (amide I and side chains), between 1570 and 1530 cm(-1) (amide II), and at 1728-1730 cm(-1) (protonated carboxylic acid groups). In particular, the latter region in native RCs is characterized by a main positive band at 1728 cm(-1) and a negative signal at 1739 cm(-1). In the L212 mutants, the amplitude of the positive band is strongly decreased leading to a differential signal at 1739/1730 cm(-1) that is insensitive to (1)H/(2)H isotopic exchange. In native RCs, only the 1728 cm(-1) band is affected in (2)H(2)O while the 1739 cm(-1) signal is not. The effects of the mutations and of (1)H/(2)H exchange on the Q(B)(-)/Q(B) spectra concur in the attribution of the 1728 cm(-1) band in native RCs to (partial) proton uptake by GluL212 upon the first electron transfer to Q(B), as previously observed in Rhodobacter sphaeroides RCs [Nabedryk, E., Breton, J., Hienerwadel, R., Fogel, C., M?ntele, W., Paddock, M. L., and Okamura, M. Y. (1995) Biochemistry 34, 14722-14732]. More generally, strong homologies of the Q(B) to Q(B)(-) transition in the RCs from Rb. sphaeroides and Rb. capsulatus are detected by differential FTIR spectroscopy. The FTIR data are discussed in relation with the results from global proton uptake measurements and electrogenic events concomitant with the reduction of Q(B) and with a model of the Q(B) turnover in Rb. sphaeroides RCs [Mulkidjanian, A. Y. (1999) FEBS Lett. 463, 199-204].     

Cu2+ site in photosynthetic bacterial reaction centers from Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas viridis

The interaction of metal ions with isolated photosynthetic reaction centers (RCs) from the purple bacteria Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas viridis has been investigated with transient optical and magnetic resonance techniques. In RCs from all species, the electrochromic response of the bacteriopheophytin cofactors associated with Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer is slowed in the presence of Cu(2+). This slowing is similar to the metal ion effect observed for RCs from Rb. sphaeroides where Zn(2+) was bound to a specific site on the surface of the RC [Utschig et al. (1998) Biochemistry 37, 8278]. The coordination environments of the Cu(2+) sites were probed with electron paramagnetic resonance (EPR) spectroscopy, providing the first direct spectroscopic evidence for the existence of a second metal site in RCs from Rb. capsulatus and Rps. viridis. In the dark, RCs with Cu(2+) bound to the surface exhibit axially symmetric EPR spectra. Electron spin echo envelope modulation (ESEEM) spectral results indicate multiple weakly hyperfine coupled (14)N nuclei in close proximity to Cu(2+). These ESEEM spectra resemble those observed for Cu(2+) RCs from Rb. sphaeroides [Utschig et al. (2000) Biochemistry 39, 2961] and indicate that two or more histidines ligate the Cu(2+) at the surface site in each RC. Thus, RCs from Rb. sphaeroides, Rb. capsulatus, and Rps. viridis each have a structurally analogous Cu(2+) binding site that is involved in modulating the Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron-transfer process. Inspection of the Rps. viridis crystal structure reveals four potential histidine ligands from three different subunits (M16, H178, H72, and L211) located beneath the Q(B) binding pocket. The location of these histidines is surprisingly similar to the grouping of four histidine residues (H68, H126, H128, and L211) observed in the Rb. sphaeroides RC crystal structure. Further elucidation of these Cu(2+) sites will provide a means to investigate localized proton entry into the RCs of Rb. capsulatus and Rps. viridis as well as locate a site of protein motions coupled with electron transfer.     

Histidine is involved in coupling proton uptake to electron transfer in photosynthetic proteins

In photosynthesis, the central step in transforming light energy into chemical energy is the coupling of light-induced electron transfer to proton uptake and release. Despite intense investigations of different photosynthetic protein complexes, including the photosystem II (PS II) in plants and the reaction center (RC) in bacteria, the molecular details of this fundamental process remain incompletely understood. In the RC of Rhodobacter (Rb.) sphaeroides, fast formation of the charge separated state, P(+)Q(A)(-), is followed by a slower electron transfer from the primary acceptor, Q(A), to the secondary acceptor, Q(B), and the uptake of a proton from the cytoplasm. The proton transfer to Q(B) takes place via a protonated water chain. Mutation of the amino acid AspL210 to Asn (L210DN mutant) near the entry of the proton pathway can disturb this water chain and consequently slow down proton uptake. Time-resolved step-scan Fourier transform infrared (FTIR) measurements revealed an intermediate X in the Q(A)(-)Q(B) to Q(A)Q(B)(-) transition. The nature of this transition remains a matter of debate. In this study, we investigated the role of the iron-histidine complex located between Q(A) and Q(B). We used time-resolved fast-scan FTIR spectroscopy to characterize the Rb. sphaeroides L210DN RC mutant marked with isotopically labeled histidine. FTIR marker bands of the intermediate X between 1120 cm(-1) and 1050 cm(-1) are assigned to histidine vibrations and indicate the protonation of a histidine, most likely HisL190, during the disappearance of the intermediate. Based on these results we propose a novel mechanism of the coupling of electron and proton transfer in photosynthesis.     

Probing light-induced conformational transitions in bacterial photosynthetic reaction centers embedded in trehalose-water amorphous matrices

The coupling between electron transfer and protein dynamics has been studied in photosynthetic reaction centers (RC) from Rhodobacter sphaeroides by embedding the protein into room temperature solid trehalose-water matrices. Electron transfer kinetics from the primary quinone acceptor (Q(A)(-)) to the photoxidized donor (P(+)) were measured as a function of the duration of photoexcitation from 20 ns (laser flash) to more than 1 min. Decreasing the water content of the matrix down to approximately 5x10(3) water molecules per RC causes a reversible four-times acceleration of P(+)Q(A)(-) recombination after the laser pulse. By comparing the broadly distributed kinetics observed under these conditions with the ones measured in glycerol-water mixtures at cryogenic temperatures, we conclude that RC relaxation from the dark-adapted to the light-adapted state and thermal fluctuations among conformational substates are hindered in the room temperature matrix over the time scale of tens of milliseconds. When the duration of photoexcitation is increased from a few milliseconds to the second time scale, recombination kinetics of P(+)Q(A)(-) slows down progressively and becomes less distributed, indicating that even in the driest matrices, during continuous illumination, the RC is gaining a limited conformational freedom that results in partial stabilization of P(+)Q(A)(-). This behavior is consistent with a tight structural and dynamical coupling between the protein surface and the trehalose-water matrix.     

Characterization of the bonding interactions of Q(B) upon photoreduction via A-branch or B-branch electron transfer in mutant reaction centers from Rhodobacter sphaeroides

In Rhodobacter sphaeroides reaction centers (RCs) containing the mutation Ala M260 to Trp (AM260W), transmembrane electron transfer along the full-length of the A-branch of cofactors is prevented by the loss of the Q(A) ubiquinone, but it is possible to generate the radical pair P(+)H(A)(-) by A-branch electron transfer or the radical pair P(+)Q(B)(-) by B-branch electron transfer. In the present study, FTIR spectroscopy was used to provide direct evidence for the complete absence of the Q(A) ubiquinone in mutant RCs with the AM260W mutation. Light-induced FTIR difference spectroscopy of isolated RCs was also used to probe the neutral Q(B) and the semiquinone Q(B)(-) states in two B-branch active mutants, a double AM260W-LM214H mutant, denoted WH, and a quadruple mutant, denoted WAAH, in which the AM260W, LM214H, and EL212A-DL213A mutations were combined. The data were compared to those obtained with wild-type (Wt) RCs and the double EL212A-DL213A (denoted AA) mutant which exhibit the usual A-branch electron transfer to Q(B). The Q(B)(-)/Q(B) spectrum of the WH mutant is very close to that of Wt RCs indicating similar bonding interactions of Q(B) and Q(B)(-) with the protein in both RCs. The Q(B)(-)/Q(B) spectra of the AA and WAAH mutants are also closely related to one another, but are very different to that of the Wt complex. Isotope-edited IR fingerprint spectra were obtained for the AA and WAAH mutants reconstituted with site-specific (13)C-labeled ubiquinone. Whilst perturbations of the interactions of the semiquinone Q(B)(-) with the protein are observed in the AA and WAAH mutants, the FTIR data show that the bonding interaction of neutral Q(B) in these two mutants are essentially the same as those for Wt RCs. Therefore, it is concluded that Q(B) occupies the same binding position proximal to the non-heme iron prior to reduction by either A-branch or B-branch electron transfer.     

Electron transfer between the quinones in the photosynthetic reaction center and its coupling to conformational changes

The electron transfer between the two quinones Q(A) and Q(B) in the bacterial photosynthetic reaction center (bRC) is coupled to a conformational rearrangement. Recently, the X-ray structures of the dark-adapted and light-exposed bRC from Rhodobacter sphaeroides were solved, and the conformational changes were characterized structurally. We computed the reaction free energy for the electron transfer from to Q(B) in the X-ray structures of the dark-adapted and light-exposed bRC from Rb. sphaeroides. The computation was done by applying an electrostatic model using the Poisson-Boltzmann equation and Monte Carlo sampling. We accounted for possible protonation changes of titratable groups upon electron transfer. According to our calculations, the reaction energy of the electron transfer from to Q(B) is +157 meV for the dark-adapted and -56 meV for the light-exposed X-ray structure; i.e., the electron transfer is energetically uphill for the dark-adapted structure and downhill for the light-exposed structure. A common interpretation of experimental results is that the electron transfer between and Q(B) is either gated or at least influenced by a conformational rearrangement: A conformation in which the electron transfer from to Q(B) is inactive, identified with the dark-adapted X-ray structure, changes into an electron-transfer active conformation, identified with the light-exposed X-ray structure. This interpretation agrees with our computational results if one assumes that the positive reaction energy for the dark-adapted X-ray structure effectively prevents the electron transfer. We found that the strongly coupled pair of titratable groups Glu-L212 and Asp-L213 binds about one proton in the dark-adapted X-ray structure, where the electron is mainly localized at Q(A), and about two protons in the light-exposed structure, where the electron is mainly localized at Q(B). This finding agrees with recent experimental and theoretical studies. We compare the present results for the bRC from Rb. sphaeroides to our recent studies on the bRC from Rhodopseudomonas viridis. We discuss possible mechanisms for the gated electron transfer from to Q(B) and relate them to theoretical and experimental results.     

Investigation of ubiquinol formation in isolated photosynthetic reaction centers by rapid-scan Fourier transform IR spectroscopy

Light-induced formation of ubiquinol-10 in Rhodobacter sphaeroides reaction centers was followed by rapid-scan Fourier transform IR difference spectroscopy, a technique that allows the course of the reaction to be monitored, providing simultaneously information on the redox states of cofactors and on protein response. The spectrum recorded between 4 and 29 ms after the second flash showed bands at 1,470 and 1,707 cm(-1), possibly due to a QH(-) intermediate state. Spectra recorded at longer delay times showed a different shape, with bands at 1,388 (+) and 1,433 (+) cm(-1) characteristic of ubiquinol. These spectra reflect the location of the ubiquinol molecule outside the Q(B) binding site. This was confirmed by Fourier transform IR difference spectra recorded during and after continuous illumination in the presence of an excess of exogenous ubiquinone molecules, which revealed the process of ubiquinol formation, of ubiquinone/ubiquinol exchange at the Q(B) site and between detergent micelles, and of Q(B)(-) and QH(2) reoxidation by external redox mediators. Kinetics analysis of the IR bands allowed us to estimate the ubiquinone/ubiquinol exchange rate between detergent micelles to approximately 1 s. The reoxidation rate of Q(B)(-) by external donors was found to be much lower than that of QH(2), most probably reflecting a stabilizing/protecting effect of the protein for the semiquinone form. A transient band at 1,707 cm(-1) observed in the first scan (4-29 ms) after both the first and the second flash possibly reflects transient protonation of the side chain of a carboxylic amino acid involved in proton transfer from the cytoplasm towards the Q(B) site.     

Specific triazine resistance in bacterial reaction centers induced by a single mutation in the QA protein pocket

We report here the first example of a reaction center mutant from Rhodobacter sphaeroides, where a single mutation (M266His --> Leu) taking place in the primary quinone protein pocket confers selective resistance to triazine-type inhibitors (terbutryn, ametryn, and atrazine), which bind in the secondary quinone protein pocket, at about 13 A from the mutation site. The M266His --> Leu mutation involves one of the iron atom ligands. Interestingly, neither the secondary quinone nor the highly specific inhibitor stigmatellin binding affinities are affected by the mutation. It is noticeable that in the M266His --> Ala mutant a nativelike behavior in observed. We suggest that the long side chain of Leu in position M266 may lack space to accommodate in the Q(A) pocket therefore transferring its hindrance to the Q(B) pocket. This may occur via the structural feature formed by the Q(A)-M219His-Fe-L190His-inhibitor (or Q(B)) connection, pushing L189Leu and/or L229Ile in closer contact to the triazine molecules, therefore decreasing their bindings. This opens the possibility to finely tune, in reaction center proteins, the affinity for herbicides by designing mutations distant from their binding sites.     

Low-temperature interquinone electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides and Blastochloris viridis: characterization of Q(B)- states by high-frequency electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR)

High-frequency electron paramagnetic resonance (HF EPR) techniques have been employed to look for localized light-induced conformational changes in the protein environments around the reduced secondary quinone acceptor (Q(B)(-)) in Rhodobacter sphaeroides and Blastochloris viridis RCs. The Q(A)(-) and Q(B)(-) radical species in Fe-removed/Zn-replaced protonated RCs substituted with deuterated quinones are distinguishable with pulsed D-band (130 GHz) EPR and provide native probes of both the low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron-transfer event and the structure of trapped conformational substates. We report here the first spectroscopic evidence that cryogenically trapped, light-induced changes enable low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer in the B. viridis RC and the first observation of an inactive, trapped P(+)Q(B)(-) state in both R. sphaeroides and B. viridis RCs that does not recombine at 20 K. The high resolution and orientational selectivity of HF electron-nuclear double resonance (ENDOR) allows us to directly probe protein environments around Q(B)(-) for distinct P(+)Q(B)(-) kinetic RC states by spectrally selecting specific nuclei in isotopically labeled samples. No structural differences in the protein structure near Q(B)(-) or reorientation (within 5 degrees ) of Q(B)(-) was observed with HF ENDOR spectra of two states of P(+)Q(B)(-): "active" and "inactive" states with regards to low-temperature electron transfer. These results reveal a remarkably enforced local protein environment for Q(B) in its reduced semiquinone state and suggest that the conformational change that controls reactivity resides beyond the Q(B) local environment.     

Control of electron transfer by protein dynamics in photosynthetic reaction centers

Abstract

Trehalose and glycerol are low molecular mass sugars/polyols that have found widespread use in the protection of native protein states, in both short- and long-term storage of biological materials, and as a means of understanding protein dynamics. These myriad uses are often attributed to their ability to form an amorphous glassy matrix. In glycerol, the glass is formed only at cryogenic temperatures, while in trehalose, the glass is formed at room temperature, but only upon dehydration of the sample. While much work has been carried out to elucidate a mechanistic view of how each of these matrices interact with proteins to provide stability, rarely have the effects of these two independent systems been directly compared to each other. This review aims to compile decades of research on how different glassy matrices affect two types of photosynthetic proteins: (i) the Type II bacterial reaction center from Rhodobacter sphaeroides and (ii) the Type I Photosystem I reaction center from cyanobacteria. By comparing aggregate data on electron transfer, protein structure, and protein dynamics, it appears that the effects of these two distinct matrices are remarkably similar. Both seem to cause a “tightening” of the solvation shell when in a glassy state, resulting in severely restricted conformational mobility of the protein and associated water molecules. Thus, trehalose appears to be able to mimic, at room temperature, nearly all of the effects on protein dynamics observed in low temperature glycerol glasses.     

Electron transfer kinetics in photosynthetic reaction centers embedded in trehalose glasses: trapping of conformational substates at room temperature.

We report on room temperature electron transfer in the reaction center (RC) complex purified from Rhodobacter sphaeroides. The protein was embedded in trehalose-water systems of different trehalose/water ratios. This enabled us to get new insights on the relationship between RC conformational dynamics and long-range electron transfer. In particular, we measured the kinetics of electron transfer from the primary reduced quinone acceptor (Q(A)(-)) to the primary photo oxidized donor (P(+)), by time-resolved absorption spectroscopy, as a function of the matrix composition. The composition was evaluated either by weighing (liquid samples) or by near infrared spectroscopy (highly viscous or solid glasses). Deconvolution of the observed, nonexponential kinetics required a continuous spectrum of rate constants. The average rate constant ( = 8.7 s(-1) in a 28% (w/w) trehalose solution) increases smoothly by increasing the trehalose/water ratio. In solid glasses, at trehalose/water ratios > or = 97%, an abrupt increase is observed ( = 26.6 s(-1) in the driest solid sample). A dramatic broadening of the rate distribution function parallels the above sudden increase. Both effects fully revert upon rehydration of the glass. We compared the kinetics observed at room temperature in extensively dried water-trehalose matrices with the ones measured in glycerol-water mixtures at cryogenic temperatures and conclude that, in solid trehalose-water glasses, the thermal fluctuations among conformational substates are inhibited. This was inferred from the large broadening of the rate constant distribution for electron transfer obtained in solid glasses, which was due to the free energy distribution barriers having become quasi static. Accordingly, the RC relaxation from dark-adapted to light-adapted conformation, which follows primary charge separation at room temperature, is progressively hindered over the time scale of P(+)Q(A)(-) charge recombination, upon decreasing the water content. In solid trehalose-water glasses the electron transfer process resulted much more affected than in RC dried in the absence of sugar. This indicated a larger hindering of the internal dynamics in trehalose-coated RC, notwithstanding the larger amount of residual water present in comparison with samples dried in the absence of sugar.     

X-ray crystal structure of the YM210W mutant reaction centre from Rhodobacter sphaeroides

The X-ray crystal structure of a reaction centre from Rhodobacter sphaeroides with a mutation of tyrosine M210 to tryptophan (YM210W) has been determined to a resolution of 2.5 A. Structural conservation is very good throughout the body of the protein, with the tryptophan side chain adopting a position in the mutant complex closely resembling that of the tyrosine in the wild-type complex. The spectroscopic properties of the YM210W reaction centre are discussed with reference to the structural data, with particular focus on evidence that the introduction of the bulkier tryptophan in place of the native tyrosine may cause a small tilt of the macrocycle of the B(L) monomeric bacteriochlorophyll.     

Photoreduction of the quinone pool in the bacterial photosynthetic membrane: identification of infrared marker bands for quinol formation

The photoreduction of the quinone (Q) pool in the photosynthetic membrane of the purple bacterium Rhodobacter sphaeroides was investigated by steady-state and time-resolved Fourier transform infrared difference spectroscopy. The results are consistent with the existence of a homogeneous Q pool inside the chromatophore membrane, with a size of around 20 Q molecules per reaction center. IR marker bands for the quinone/quinol (Q/QH(2)) redox couple were recognized. QH(2) bands are identified at 1491, 1470, 1433 and 1388-1375 cm(-1). The 1491 cm(-1) band, which is sensitive to (1)H/(2)H exchange, is assigned to a C-C ring mode coupled to a C-OH mode. A feature at approximately 1743/1720 cm(-1) is tentatively related to a perturbation of the carbonyl modes of phospholipid head groups induced by QH(2) formation. Complex conformational changes of the protein in the amide I and II spectral ranges are also apparent during reduction and reoxidation of the Q pool.     

Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to Q(B) in Rhodobacter sphaeroides reaction centers

Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction Q(A-)Q(B)-->Q(A)Q(B-) (k(AB)(1)) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k(AB)(1)), attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to Q(B) [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4-29 ms following a flash showed peaks at 1670 (-), 1601 (-) and 1467 (+) cm(-1), characteristic of Q(A) reduction. The time evolution of the spectra shows reoxidation of Q(A-) and concomitant reduction of Q(B) with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of Q(B-) occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm(-1) [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of Q(B) in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from Q(A-) to Q(B) and the identification of Glu-L212 as the main proton acceptor in the state Q(A)Q(B-).     

Probing the flexibility of the bacterial reaction center: the wild-type protein is more rigid than two site-specific mutants

Experimental and theoretical studies have stressed the importance of flexibility for protein function. However, more local studies of protein dynamics, using temperature factors from crystallographic data or elastic models of protein mechanics, suggest that active sites are among the most rigid parts of proteins. We have used quasielastic neutron scattering to study the native reaction center protein from the purple bacterium Rhodobacter sphaeroides, over a temperature range of 4-260 K, in parallel with two nonfunctional mutants both carrying the mutations L212Glu/L213Asp --> Ala/Ala (one mutant carrying, in addition, the M249Ala --> Tyr mutation). The so-called dynamical transition temperature, Td, remains the same for the three proteins around 230 K. Below Td the mean square displacement, u2, and the dynamical structure factor, S(Q,omega), as measured respectively by backscattering and time-of-flight techniques are identical. However, we report that above Td, where anharmonicity and diffusive motions take place, the native protein is more rigid than the two nonfunctional mutants. The higher flexibility of both mutant proteins is demonstrated by either their higher u2 values or the notable quasielastic broadening of S(Q,omega) that reveals the diffusive nature of the motions involved. Remarkably, we demonstrate here that in proteins, point genetic mutations may notably affect the overall protein dynamics, and this effect can be quantified by neutron scattering. Our results suggest a new direction of investigation for further understanding of the relationship between fast dynamics and activity in proteins. Brownian dynamics simulations we have carried out are consistent with the neutron experiments, suggesting that a rigid core within the native protein is specifically softened by distant point mutations. L212Glu, which is systematically conserved in all photosynthetic bacteria, seems to be one of the key residues that exerts a distant control over the rigidity of the core of the protein.