Probing the contribution of electronic coupling to the directionality of electron transfer in photosynthetic reaction centers

Subpicosecond transient absorption studies are reported for a set of Rhodobacter (R.) capsulatus bacterial photosynthetic reaction centers (RCs) designed to probe the origins of the unidirectionality of charge separation via one of two electron transport chains in the native pigment-protein complex. All of the RCs have been engineered to contain a heterodimeric primary electron donor (D) consisting of a bacteriochlorophyll (BChl) and a bacteriopheophytin (BPh). The BPh component of the M heterodimer (Mhd) or L heterodimer (Lhd) is introduced by substituting a Leu for His M200 or His L173, respectively. Previous work on primary charge separation in heterodimer mutants has not included the Lhd RC from R. capsulatus, which we report for the first time. The Lhd and Mhd RCs are used as controls against which we assess RCs that combine the heterodimer mutations with a second mutation (His substituted for Leu at M212) that results in replacement of the native L-side BPh acceptor with a BChl (beta). The transient absorption spectra reveal clear evidence for charge separation to the normally inactive M-side BPh acceptor (H(M)) in Lhd-beta RCs to form D+H(M)- with a yield of approximately 6%. This state also forms in Mhd-beta RCs but with about one-quarter the yield. In both RCs, deactivation to the ground state is the predominant pathway of D decay, as it is in the Mhd and Lhd single mutants. Analysis of the results indicates an upper limit ofV2L/V2m < or = 4 for the contribution of the electronic coupling elements to the relative rates of electron transfer to the L versus M sides of the wild-type RC. In comparison to the L/M rate ratio (kL/kM) approximately 30 for wild-type RCs, our findings indicate that electronic factors contribute approximately 35% at most to directionality with the other 65% deriving from energetic considerations, which includes differences in free energies, reorganization energies, and contributions of one- and two-step mechanisms on the two sides of the RC.     

Temperature dependence of electron transfer to the M-side bacteriopheophytin in rhodobacter capsulatus reaction centers

Subpicosecond time-resolved absorption measurements at 77 K on two reaction center (RC) mutants of Rhodobacter capsulatus are reported. In the D(LL) mutant the D helix of the M subunit has been substituted with the D helix from the L subunit, and in the D(LL)-FY(L)F(M) mutant, three additional mutations are incorporated that facilitate electron transfer to the M side of the RC. In both cases the helix swap has been shown to yield isolated RCs that are devoid of the native bacteriopheophytin electron carrier HL (Chuang, J. I.; Boxer, S. G.; Holten, D.; Kirmaier, C. Biochemistry 2006, 45, 3845-3851). For D(LL), depending whether the detergent Deriphat 160-C or N-lauryl-N,N-dimethylamine-N-oxide (LDAO) is used to suspend the RCs, the excited state of the primary electron donor (P*) decays to the ground state with an average lifetime at 77 K of 330 or 170 ps, respectively; however, in both cases the time constant obtained from single-exponential fits varies markedly as a function of the probe wavelength. These findings on the D(LL) RC are most easily explained in terms of a heterogeneous population of RCs. Similarly, the complex results for D(LL)-FY(L)F(M) in Deriphat-glycerol glass at 77 K are most simply explained using a model that involves (minimally) two distinct populations of RCs with very different photochemistry. Within this framework, in 50% of the D(LL)-FY(L)F(M) RCs in Deriphat-glycerol glass at 77 K, P* deactivates to the ground state with a time constant of approximately 400 ps, similar to the deactivation of P* in the D(LL) mutant at 77 K. In the other 50% of D(LL)-FY(L)F(M) RCs, P* has a 35 ps lifetime and decays via electron transfer to the M branch, giving P+HM- in high yield (> or =80%). This result indicates that P* --> P(+)H(M)(-) is roughly a factor of 2 faster at 77 K than at 295 K. In alternative homogeneous models the rate of this M-side electron-transfer process is the same or up to 2-fold slower at low temperature. A 2-fold increase in rate with a reduction in temperature is the same behavior found for the overall L-side process P* --> P(+)H(L)(-) in wild-type RCs. Our results suggest that, as for electron transfer on the L side, the M-side electron-transfer reaction P* --> P(+)H(M)(-) is an activationless process.     

Electron transfer kinetics in photosynthetic reaction centers embedded in polyvinyl alcohol films

The coupling between electron transfer and protein dynamics has been studied at room temperature in isolated reaction centers (RCs) from the photosynthetic bacterium Rhodobacter sphaeroides by incorporating the protein in polyvinyl alcohol (PVA) films of different water/RC ratios. The kinetic analysis of charge recombination shows that dehydration of RC-containing PVA films causes reversible, inhomogeneous inhibition of electron transfer from the reduced primary quinone acceptor (Q(A)(-)) to the secondary quinone Q(B). A more extensive dehydration of solid PVA matrices accelerates electron transfer from Q(A)(-) to the primary photooxidized electron donor P(+). These effects indicate that incorporation of RCs into dehydrated PVA films hinders the conformational dynamics gating Q(A)(-) to Q(B) electron transfer at room temperature and slows down protein relaxation which stabilizes the primary charge-separated state P(+)Q(A)(-). A comparison with analogous effects observed in trehalose-coated RCs suggests that protein motions are less severely reduced in PVA films than in trehalose matrices at comparable water/RC ratios.     

Mechanism of carotenoid singlet excited state energy transfer in modified bacterial reaction centers

Ultrafast transient laser spectroscopy has been used to investigate carotenoid singlet excited state energy transfer in various Rhodobacter (Rb.) sphaeroides reaction centers (RCs) modified either genetically or chemically. The pathway and efficiency of energy transfer were examined as a function of the structures and energies of the donor and acceptor molecules. On the donor side, carotenoids with various extents of pi-electron conjugation were examined. RCs studied include those from the anaerobically grown wild-type strain containing the carotenoid spheroidene, which has 10 conjugated carbon-carbon double bonds; the GA strain containing neurosporene, which has nine conjugated double bonds; and aerobically grown wild-type cells, as well as aerobically grown H(M182)L mutant, both containing the carbonyl-containing carotenoid spheroidenone, which has 11 conjugated double bonds. By varying the structure of the carotenoid, we observed the effect of altering the energies of the carotenoid excited states on the rate of energy transfer. Both S(1)- and S(2)-mediated carotenoid-to-bacteriochlorophyll energy transfer processes were observed. The highest transfer efficiency, from both the S(1) and S(2) states, was observed using the carotenoid with the shortest chain. The S(1)-mediated carotenoid-to- bacteriochlorophyll energy transfer efficiencies were determined to be 96%, 84%, and 73% for neurosporene, spheroidene, and spheroidenone, respectively. The S(2)-mediated energy transfer efficiencies follow the same trend but could not be determined quantitatively because of limitations in the time resolution of the instrumentation. The dependence of the energy transfer rate on the energetics of the energy transfer acceptor was verified by performing measurements with RCs from the H(M182)L mutant. In this mutant, the bacteriochlorophyll (denoted B(B)) located between the carotenoid and the RC special pair (P) is replaced by a bacteriopheophytin (denoted phi(B)), where the Q(X) and Q(Y) bands of phi(B) are 1830 and 1290 cm(-1), respectively, higher in energy than those of B(B). These band shifts associated with phi(B) in the H(M182)L mutant significantly alter the spectral overlap between the carotenoid and phi(B), resulting in a significant decrease of the transfer efficiency from the carotenoid S(1) state to phi(B). This leaves energy transfer from the carotenoid S(2) state to phi(B) as the dominant channel. Largely because of this change in mechanism, the overall efficiency of energy transfer from the carotenoid to P decreases to less than 50% in this mutant. Because the spectral signature of phi(B) is different from that of B(A) in this mutant, we were able to demonstrate clearly that the carotenoid-to-P energy transfer is via phi(B). This finding supports the concept that, in wild-type RCs, the carotenoid-to-P energy transfer occurs through the cofactor located at the B(B) position.     

Signals in solid-state photochemically induced dynamic nuclear polarization recover faster than signals obtained with the longitudinal relaxation time

During the photocycle of quinone-blocked photosynthetic reaction centers (RCs), photochemically induced dynamic nuclear polarization (photo-CIDNP) is produced by polarization transfer from the initially totally electron polarized electron pair and can be observed by 13C magic-angle spinning (MAS) NMR as a strong modification of signal intensities. The same processes creating net nuclear polarization open up light-dependent channels for polarization loss. This leads to coherent and incoherent enhanced signal recovery, in addition to the recovery due to light-independent longitudinal relaxation. Coherent mixing between electron and nuclear spin states due to pseudosecular hyperfine coupling within the radical pair state provides such a coherent loss channel for nuclear polarization. Another polarization transfer mechanism called differential relaxation, which is based on the long lifetime of the triplet state of the donor, provides an efficient incoherent relaxation path. In RCs of the purple bacterium Rhodobacter sphaeroides R26, the photochemical active channels allow for accelerated signal scanning by a factor of 5. Hence, photo-CIDNP MAS NMR provides the possibility to drive the NMR technique beyond the T1 limit.     

Local electrostatic field induced by the carotenoid bound to the reaction center of the purple photosynthetic bacterium Rhodobacter sphaeroides

Electroabsorption (EA) spectra were recorded in the region of the reaction center (RC) Qy absorption bands of bacteriochlorophyll (Bchl) and bacteriopheophytin, to investigate the effect of carotenoid (Car) on the electrostatic environment of the RCs of the purple bacterium Rhodobacter (Rb.) sphaeroides. Two different RCs were prepared from Rb. sphaeroides strain R26.1 (R26.1-RC); R26.1 RC lacking Car and a reconstituted RC (R26.1-RC+ Car) prepared by incorporating a synthetic Car (3,4-dihydrospheroidene). Although there were no detectable differences between these two RCs in their near infrared (NIR) absorption spectra at 79 and 293 K, or in their EA spectra at 79 K, significant differences were detected in their EA spectra at 293 K. Three nonlinear optical parameters of each RC were determined in order to evaluate quantitatively these differences; transition dipole-moment polarizability and hyperpolarizability (D factor), the change in polarizability upon photoexcitation (Deltaalpha), and the change in dipole-moment upon photoexcitation (Deltamu). The value of D or Deltaalpha determined for each absorption band of the two RC samples showed similar values at 77 or 293 K. However, the Deltamu values of the special pair Bchls (P) and the monomer Bchls absorption bands showed significant differences between the two RCs at 293 K. X-ray crystallography of the two RCs has revealed that a single molecule of the solubilizing detergent LDAO occupies part of the carotenoid binding site in the absence of a carotenoid. The difference in the value of Deltamu therefore represents the differential effect of the detergent LDAO and the carotenoid on P. The change of electrostatic field around P induced by the presence of Car was determined to be 1.7 x 10(5) [V/cm], corresponding to a approximately 10% change in the electrostatic field around P.     

Femtosecond spectroscopy of the primary charge separation in reaction centers of Chloroflexus aurantiacus with selective excitation in the QY and Soret bands

The primary charge separation and electron-transfer processes of photosynthesis occur in the reaction center (RC). Isolated RCs of the green filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus were studied at room temperature by using femtosecond transient absorption spectroscopy with selective excitation. Upon excitation in the Q(Y) absorbance band of the bacteriochlorophyll (BChl) dimer (P) at 865 nm, a 7.0 +/- 0.5 ps kinetic component was observed in the 538 nm region (Q(X) band of the bacteriopheophytin (BPheo)), 750 nm region (Q(Y) band of the BPheo), and 920 nm region (stimulated emission of the excited-state of P), indicating that this lifetime represents electron transfer from P to BPheo. The same time constant was also observed upon 740 nm or 800 nm excitation. A longer lifetime (300 +/- 30 ps), which was assigned to the time of reduction of the primary quinone, Q(A), was also observed. The transient absorption spectra and kinetics all indicate that only one electron-transfer branch is involved in primary charge separation under these excitation conditions. However, the transient absorption changes upon excitation in the Soret band at 390 nm reveal a more complex set of energy and electron-transfer processes. By comparison to studies on the RCs of the purple bacterium Rhodobacter sphaeroides, we discuss the possible mechanism of electron-transfer pathway dependence on excitation energy and propose a model of the Cf. aurantiacus RC that better explains the observed results.     

Type 1 reaction center of photosynthetic heliobacteria

The reaction center (RC) of heliobacteria contains iron-sulfur centers as terminal electron acceptors, analogous to those of green sulfur bacteria as well as photosystem I in cyanobacteria and higher plants. Therefore, they all belong to the so-called type 1 RCs, in contrast to the type 2 RCs of purple bacteria and photosystem II containing quinone molecules. Although the architecture of the heliobacterial RC as a protein complex is still unknown, it forms a homodimer made up of two identical PshA core proteins, where two symmetrical electron transfer pathways along the C2 axis are assumed to be equally functional. Electrons are considered to be transferred from membrane-bound cytochrome c (PetJ) to a special pair P800, a chlorophyll a-like molecule A0, (a quinone molecule A1) and a [4Fe-4S] center Fx and, finally, to 2[4Fe-4S] centers FA/FB. No definite evidence has been obtained for the presence of functional quinone acceptor A1. An additional interesting point is that the electron transfer reaction from cytochrome c to P800 proceeds in a collisional mode. It is highly dependent on the temperature, ion strength and/or viscosity in a reaction medium, suggesting that a heme-binding moiety fluctuates in an aqueous phase with its amino-terminus anchored to membranes.     

Transmembrane charge transfer in photosynthetic reaction centers: some similarities and distinctions

This mini review presents a general comparison of structural and functional peculiarities of three types of photosynthetic reaction centers (RCs)--photosystem (PS) II, RC from purple bacteria (bRC) and PS I. The nature and mechanisms of the primary electron transfer reactions, as well as specific features of the charge transfer reactions at the donor and acceptor sides of RCs are considered. Comparison of photosynthetic RCs shows general similarity between the core central parts of all three types, between the acceptor sides of bRC and PS II, and between the donor sides of bRC and PS I. In the latter case, the similarity covers thermodynamic, kinetic and dielectric properties, which determine the resemblance of mechanisms of electrogenic reduction of the photooxidized primary donors. Significant distinctions between the donor and acceptor sides of PS I and PS II are also discussed. The results recently obtained in our laboratory indicate in favor of the following sequence of the primary and secondary electron transfer reactions: in PS II (bRC): Р(680)(Р(870)) → Chl(D1)(В(А)) → Phe(bPhe) → Q(A); and in PS I: Р(700) → А(0А)/A(0B) → Q(A)/Q(B).     

Primary processes in plant photosynthesis: photosystem I reaction center

The photosystem I (PSI) pigment-protein complex of plants converts light energy into a transmembrane charge separation, which ultimately leads to the reduction of carbon dioxide. Recent studies on the dynamics of primary energy transfer, charge separation, and following electron transfer of the reaction center (RC) of the PSI prepared from spinach are reviewed. The main results of femtosecond transient absorption and fluorescence spectroscopies as applied to the P700-enchied PSI RC are summarized. This specially prepared material contains only 12–14 chlorophylls per P700, which is a special pair of chlorophyll a and has a significant role in primary charge separation. The P700-enriched particles are useful to study dynamics of cofactors, since about 100 light-harvesting chlorophylls are associated with wild PSI RC and prevent one from observing the elementary steps of the charge separation. In PSI RC energy and electron transfer were found to be strongly coupled and an ultrafast up-hill energy equilibration and charge separation were observed upon preferential excitation of P700. The secondary electron-transfer dynamics from the reduced primary electron acceptor chlorophyll a to quinone are described. With creating free energy differences (ΔG₀) for the reaction by reconstituting various artificial quinones and quinoids, the rate of electron transfer was measured. Analysis of rates versus ΔG₀ according to the quantum theory of electron transfer gave the reorganization energy, electronic coupling energy and other factors. It was shown that the natural quinones are optimized in the photosynthetic protein complexes. The above results were compared with those of photosynthetic purple bacteria, of which the structure and functions have been studied most.     

Structure of the charge separated state P865(+)Q(A)- in the photosynthetic reaction centers of Rhodobacter sphaeroides by quantum beat oscillations and high-field electron paramagnetic resonance: evidence for light-induced Q(A)- reorientation

The structure of the secondary radical pair, P865(+)Q(A)-, in fully deuterated and Zn-substituted reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides R-26 has been determined by high-time resolution and high-field electron paramagnetic resonance (EPR). A computer analysis of quantum beat oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the orientation of the various magnetic tensors of P865(+)Q(A)- with respect to a magnetic reference frame. The orientation of the g-tensor of P865(+) in an external reference system is adapted from a single-crystal W-band (95 GHz) EPR study [Klette, R.; T?rring, J. T.; Plato, M.; M?bius, K.; B?nigk, B.; Lubitz, W. J. Phys. Chem. 1993, 97, 2015-2020]. Thus, we obtain the three-dimensional structure of the charge separated state P865(+)Q(A)- on a nanosecond time scale after light-induced charge separation. Comparison with crystallographic data reveals that the position of the quinone is essentially the same as that in the X-ray structure. However, the head group of Q(A)- has undergone a 60 degrees rotation in the ring plane relative to its orientation in the crystal structure. Analysis suggests that the two different QA conformations are functionally relevant states which control the electron-transfer kinetics from Q(A)- to the secondary quinone acceptor QB. It appears that the rate-limiting step of this reaction is a reorientation of Q(A)- in its binding pocket upon light-induced reduction. The new kinetic model accounts for striking observations by Kleinfeld et al. who reported that electron transfer from Q(A)- to QB proceeds in RCs cooled to cryogenic temperature under illumination but does not proceed in RCs cooled in the dark [Kleinfeld, D.; Okamura, M. Y.; Feher, G. Biochemistry 1984, 23, 5780-5786].     

Conformational control of the Q(A) to Q(B) electron transfer in bacterial reaction centers: evidence for a frozen conformational landscape below -25 degrees C

The competition between the P(+)Q(A)(-) --> PQ(A) charge recombination (P, bacteriochlorophyll pair acting as primary photochemical electron donor) and the electron transfer to the secondary quinone acceptor Q(A)(-)Q(B) --> Q(A)Q(B)(-) (Q(A) and Q(B), primary and secondary electron accepting quinones) was investigated in chromatophores of Rb. capsulatus, varying the temperature down to -65 degrees C. The analysis of the flash-induced pattern for the formation of P(+)Q(A)Q(B)(-) shows that the diminished yield, when lowering the temperature, is not due to a homogeneous slowing of the rate constant k(AB) of the Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer but to a distribution of conformations that modulate the electron transfer rate over more than 3 orders of magnitude. This distribution appears "frozen", as no dynamic redistribution was observed over time ranges > 10 s (below -25 degrees C). The kinetic pattern was analyzed to estimate the shape of the distribution of k(AB), showing a bell-shaped band on the high rate side and a fraction of "blocked" reaction centers (RCs) with very slow k(AB). When the temperature is lowered, the high rate band moves to slower rate regions and the fraction of blocked RCs increases at the expense of the high rate band. The RCs that recombine from the P(+)Q(A)Q(B)(-) state appear temporarily converted to a state with rapid k(AB), indicating that the stabilized state described by Kleinfeld et al. (Biochemistry 1984, 23, 5780-5786) is still accessible at -60 degrees C.     

Influence of cardiolipin on the functionality of the Q(a) site of the photosynthetic bacterial reaction center

The effect of cardiolipin on the functionality of the Q(A) site of a photosynthetic reaction center (RC) was studied in RCs from the purple non-sulfur bacterium Rhodobacter sphaeroides by means of time-resolved absorbance measurements. The binding of the ubiquinone-10 to the Q(A) site of the RC embedded in cardiolipin or lecithin liposomes has been followed at different temperatures and phospholipid loading. A global fit of the experimental data allowed us to get quite reliable values of the thermodynamic parameters joined to the binding process. The presence of cardiolipin does not affect the affinity of the Q(A) site for ubiquinone but has a marked influence on the rate of P+QA(-) --> PQA electron transfer. The P+QA(-) charge recombination kinetics has been examined in liposomes made of cardiolipin/lecithin mixtures and in detergent (DDAO) micelles doped with cardiolipin. The electron-transfer rate constant increases upon cardiolipin loading. It appears that the main effect of cardiolipin on the electron transfer can be ascribed to a destabilization of the charge-separated state. Results obtained in micelles and vesicles follow the same titration curve when cardiolipin concentration evaluated with respect to the apolar phase is used as a relevant variable. The dependence of the P+QA(-) recombination rate on cardiolipin loading suggests two classes of binding sites. In addition to a high-affinity site (compatible with previous crystallographic studies), a cooperative binding, involving about four cardiolipin molecules, takes place at high cardiolipin loading.     

Redox control of photoinduced electron transfer in axial terpyridoxy porphyrin complexes

The photophysical properties of axial-bonding types (terpyridoxy)aluminum(III) porphyrin (Al(PTP)), bis(terpyridoxy)tin(IV) porphyrin (Sn(PTP) 2), and bis(terpyridoxy)phosphorus(V) porphyrin ([P(PTP) 2] (+)) are reported. Compared with their hydroxy analogues, the fluorescence quantum yields and singlet-state lifetimes were found to be lower for Sn(PTP) 2 and [P(PTP) 2] (+), whereas no difference was observed for Al(PTP). At low temperature, all of the compounds show spin-polarized transient electron paramagnetic resonance (TREPR) spectra that are assigned to the lowest excited triplet state of the porphyrin populated by intersystem crossing. In contrast, at room temperature, a triplet radical-pair spectrum that decays to the porphyrin triplet state with a lifetime of 175 ns is observed for [P(PTP) 2] (+), whereas no spin-polarized TREPR spectrum is found for Sn(PTP) 2 and only the porphyrin triplet populated by intersystem crossing is seen for Al(PTP). These results clarify the role of the internal molecular structure and the reduction potential for electron transfer from the terpyridine ligand to the excited porphyrin. It is argued that the efficiency of this process is dependent on the oxidation state of the metal/metalloid present in the porphyrin and the reorganization energy of the solvent.     

Thermal Effects and Structural Changes of Photosynthetic Reaction Centers Characterized by Wide Frequency Band Hydrophone: Effects of Carotenoids and Terbutryn

Photothermal characteristics and light‐induced structural (volume) changes of carotenoid‐containing and noncontaining photosynthetic reaction centers (RCs) were investigated by wide frequency band hydrophone. We found that the presence of carotenoid either does not play considerable role in the light‐induced conformational movements, or these rearrangements are too slow for inducing a photoacoustic (PA) signal. The kinetic component with a few tens of microseconds, exhibited by the carotenoid‐less RCs, appears to be similar to that of triplet state lifetimes, identified by other methods. The binding of terbutryn to the acceptor side is shown to affect the dynamics of the RC. Our results do not confirm large displacements or volume changes induced by the charge movements and by the charge relaxation processes in the RCs in few hundreds of microseconds time scale that accompanies the electron transfer between the primary and secondary electron acceptor quinones.     

Attenuated total reflection Fourier transform infrared spectroscopy of redox transitions in photosynthetic reaction centers: comparison of perfusion- and light-induced difference spectra

Chemically induced Fourier transform infrared difference spectra associated with redox transitions of several primary electron donors and acceptors in photosynthetic reaction centers (RCs) have been compared with the light-induced FTIR difference spectra involving the same cofactors. The RCs are deposited on an attenuated total reflection (ATR) prism and form a film that is enclosed in a flow cell. Redox transitions in the film of RCs can be repetitively induced either by perfusion of buffers poised at different redox potentials or by illumination. The perfusion-induced ATR-FTIR difference spectra for the oxidation of the primary electron donor P in the RCs of the purple bacteria Rb. sphaeroides and Rp. viridis and P700 in the photosystem 1 of Synechocystis 6803, as well as the Q(A)/Q(A) transition of the quinone acceptor (Q(A)) in Rb. sphaeroides RCs are reported for the first time. They are compared with the light-induced ATR-FTIR difference spectra P+Q(A)/PQ(A) for the RCs of Rb. sphaeroides and P700+/P700 for photosystem 1. It is shown that the perfusion-induced and light-induced ATR-FTIR difference spectra recorded on the same RC film display identical signal to noise ratios when they are measured under comparable conditions. The ATR-FTIR difference spectra are very similar to the equivalent FTIR difference spectra previously recorded upon photochemical or electrochemical excitation of these RCs in the more conventional transmission mode. The ATR-FTIR technique requires a smaller amount of sample compared with transmission FTIR and allows precise control of the aqueous environment of the RC films.     

Photo-CIDNP MAS NMR in intact cells of Rhodobacter sphaeroides R26: molecular and atomic resolution at nanomolar concentration

Photochemically induced dynamic nuclear polarization (photo-CIDNP) is observed in photosynthetic reaction centers of the carotenoid-less strain R26 of the purple bacterium Rhodobacter sphaeroides by (13)C solid-state NMR at three different magnetic fields (4.7, 9.4, and 17.6 T). The signals of the donor appear enhanced absorptive (positive) and of the acceptor emissive (negative). This spectral feature is in contrast to photo-CIDNP data of reactions centers of Rhodobacter sphaeroides wildtype reported previously (Prakash, S.; Alia; Gast, P.; de Groot, H. J. M.; Jeschke, G.; Matysik, J. J. Am. Chem. Soc. 2005, 127, 14290-14298) in which all signals appear emissive. The difference is due to an additional mechanism occurring in RCs of R26 in the long-living triplet state of the donor, allowing for spectral editing by different enhancement mechanisms. The overall shape of the spectra remains independent of the magnetic field. The strongest enhancement is observed at 4.7 T, enabling the observation of photo-CIDNP enhanced NMR signals from reaction center cofactors in entire bacterial cells allowing for detection of subtle changes in the electronic structure at nanomolar concentration of the donor cofactor. Therefore, we establish in this paper photo-CIDNP MAS NMR as a method to study the electronic structure of photosynthetic cofactors at the molecular and atomic resolution as well as at cellular concentrations.     

Proton uptake of rhodobacter capsulatus reaction center mutants modified in the primary quinone environment

Flash-induced absorbance spectroscopy was used to analyze the proton uptake and electron transfer properties of photosynthetic reaction centers (RC) of Rhodobacter capsulatus that have been genetically modified near the primary quinone electron acceptor (Q(A)). M246Ala and M247Ala, which are symmetry-related to the positions of two acidic groups, L212Glu and L213Asp, in the secondary quinone electron acceptor (QB) protein environment, have been mutated to Glu and Asp, respectively. The pH dependence of the stoichiometry of proton uptake upon formation of the P+Q(A)- (H+/P+Q(A)-) and PQ(A) (H+/Q(A)-) (P is the primary electron donor, a noncovalently linked bacteriochlorophyll dimer) states have been measured in the M246Ala --> Glu and the M247Ala --> Asp mutant RC, in the M246Ala-M247Ala --> Glu-Asp double mutant and in the wild type (WT). Our results show that the introduction of an acidic group (Glu or Asp) in the QA protein region induces notable additional proton uptake over a large pH region (approximately 6-9), which reflects a delocalized response of the protein to the formation of Q(A)-. This may indicate the existence of a widely spread proton reservoir in the cytoplasmic region of the protein. Interestingly, the pH titration curves of the proton release caused by the formation of P+ (H+/P+: difference between H+/P+Q(A)- and H+/PQ(A)- curves) are nearly superimposable in the WT and the M246Ala --> Glu mutant RC, but substantial additional proton release is detected between pH 7 and 9 in the M247Ala --> Asp mutant RC. This effect can be accounted for by an increased proton release by the P+ environment in the M247Ala --> Asp mutant. The M247Ala --> Asp mutation reveals the existence of an energetic and conformational coupling between donor and acceptor sides of the RC at a distance of nearly 30A.     

1D radical motion in protein pocket: proton-coupled electron transfer in human serum albumin

Photoinduced, proton-coupled electron transfer (ET) between 9,10-anthraquinone-2,6-disulfonate (ADQS) and an amino acid residue of tryptophan in human serum albumin (HSA) was observed using time-resolved electron paramagnetic resonance (TREPR). The ET reaction reduces the protein binding affinity of the ligand. TREPR chemically induced dynamic electron polarization (CIDEP) spectra establish that photoinduced ET takes place from the tryptophan residue (W214) to the excited triplet state of AQDS2- while bound in subdomain IIA, a protein cleft of HSA. The TREPR CIDEP signals also reveal that the anion radical of the ligand escapes toward the bulk water region by a one-dimensional translation diffusion process within the protein's pocket area. This pilot study of HSA demonstrates how TREPR CIDEP can provide significant means to investigate dynamic characteristics of protein-surface reactions.