M.E. Michel-Beyerle

Unidirectionality of charge separation in reaction centers of photosynthetic bacteria

Abstract Time-resolved spectroscopy in conjunction with X-ray structural data for reaction centers of Rhodopseudomonas viridis and Rhodobacter sphaeroides reveal a branching ratio a > 5 for the primary electron-transfer rates, favouring one of the two, almost symmetrical pigment/protein branches, L and M. In this paper we explore the origins of this unidirectionality of electron transfer between the excited singlet state of the bacteriochlorophyll dimer ( 1 P∗) and the bacteriopheophytin (H) along the L protein subunit. Nonadiabatic electron-transfer theory is applied to analyze the asymmetry of the electron-transfer rates, k L and k M across the L and M branches. The asymmetry originates from the cumulative contributions of the nuclear Franck-Condon factor and the electronic coupling, both of which enhance the electron transfer rate across the L branch. The nuclear Frank-Condon factors are modified by the energy difference ΔE LM between the states P + H − L and P + H − M , which is induced by the electrostatic interactions of these ion-pair states with the protein polar groups, as well as by asymmetric Coulomb and medium polarization interactions. The computation results in ΔE LM = −(0.09 ± 0.04) eV, which yields a nuclear enhancement contribution at 300 K of 1.5 (+0.8, −0.3) to k L k M and therefore is insufficient to explain alone the observed asymmetry in reaction centers of Rps. viridis . Another contribution to the unidirectionality originates from electronic superexchange coupling for 1 P∗-B-H via the virtual states of the accessory bacteriochlorophyll (B). The ratio of the intermolecular 1 P∗-B L and 1 P∗-B M electronic interaction terms was evaluated utilizing the tight-binding approximation with SCF-MO wavefunctions, together with the structural data for the prosthetic groups and for the polar amino acid side chains of the protein in reaction centers of Rps. viridis . The contribution to the enhancement of k L k M by the electronic superexchange is approx. 8 ± 4. This asymmetry was traced to the combination of an excess negative charge density on the M-dimer component P M , together with structural asymmetry, which enhances the P M -B L electronic overlap. Consequently, the 1 P∗-B L -H L superexchange is favoured over the 1 P∗-B M -H M interaction. The combined effects of asymmetric nuclear Franck-Condon factors and electronic couplings yield a branching ratio of the electron-transfer rates along the two pigment branches in reaction centers of Rps. viridis of an approx. 12 (−7, +15). This is sufficiently large to explain the experimentally observed unidirectionality.

Time-resolved spectroscopy of wild-type and mutant Green Fluorescent Proteins reveals excited state deprotonation consistent with fluorophore-protein interactions

Abstract Recently steady-state and picosecond time-resolved absorption and fluorescence spectroscopy on the Green Fluorescent Protein (GFP) have been interpreted by a mechanism where the key process is an excited state deprotonation of the chromophore (M. Chattoraij, B.A. King, G.U. Bublitz and S.G. Boxer, Proc. Natl. Acad. Sci. USA, 93 (1996) 8362–8367). Such a conclusion was borne out by the mirror image of the picosecond decay of the protonated species RH∗ in the blue and the concomitant picosecond rise of the green fluorescence of the deprotonated fluorophore R−∗ as well as the significant slowing of both kinetic features upon deuteration. We report similar experiments confirming this mechanism. The results of ultrafast spectroscopy on wild-type GFP together with two important mutants combined with the recent crystal structures are shown to shed more light on the interplay between absorption and emission phenomena in GFP. Beyond some differences with previous results pertaining, for instance, to the assignment of vibronic progressions in absorption spectra and the temperature dependence of excited state deprotonation, several new features have been identified. These concern the deprotonated ground state R− in equilibrium as well as the excited state RH∗. In particular, we have studied the distributed fluorescence kinetics in the time and frequency domain, excited state absorption features observed in femtosecond time-resolution, and the dependence of excited state proton transfer kinetics on the aggregational state of the protein.

On the mechanism of the primary charge separation in bacterial photosynthesis

In the light of recent experimental work on femtosecond electron transfer kinetics in the reaction center (RC) we explore the mechanism for the primary process. We focus on the special role of the bacteriochlorophyll monomer (B) located between the primary donor ( 1 P*), a bacteriochlorophyll dimer (P), and a bacteriopheophytin (H), considering a kinetic scheme which combines two parallel pathways of electron transfer: a unistep superexchange channel mediated via electronic interactions with P + B − H, and a two-step sequential channel involving a P + B − H chemical intermediate. In this kinetic scheme we used microscopic nonadiabatic electron transfer rates, which were extended to incorporate the effects of medium-controlled dynamics. The results of the kinetic modelling are presented as a function of the free-energy gap Δ G 1 between the equilibrium nuclear configurations of the donor 1 P* BH and the (physically and/or chemically) mediating state P + B − H. The parallel sequential-superexchange mechanism reduces to the limit of nearly pure sequential pathway for large negative Δ G 1 at all temperatures and to the limit of almost pure superexchange pathway for large positive Δ G 1 at all temperatures and for moderate Δ G 1 at low temperatures. The existing femtosecond kinetic data at room temperature are consistent with either the superposition of sequential and superexchange at all temperatures or to a superposition of superexchange and sequential at room temperature and superexchange at low temperatures. The available femtosecond data at 10 K raise the possibility that the mechanism involves the superposition of superexchange and sequential at 300 K and the dominance of superexchange at low temperatures. Auxiliary experimental information regarding magnetic data, i.e., the singlet-triplet splitting of the radical pair P + BH − , the kinetics of the charge separation in mutagenetically altered RCs, with tyrosine M208 being replaced by phenylalanine, and the unidirectionality of charge separation across the A branch of the RC are analysed in terms of the proposed mechanism. The prevalence of the parallel sequential and superexchange electron transfer routes for the primary charge separation would introduce an element of redundancy, which insures the occurrence of an efficient process which is stable with respect to the variation energetic parameters in different photosynthetic RCs.

On the Mechanism of Long-Range Electron Transfer through DNA

Hopping between bases of similar redox potentials is the mechanism by which charge transport occurs through DNA. This was shown by rate measurements performed with double strands 1-3. This mechanism explains why hole transfer displays a strong sequence dependence, and postulates that electron transfer in unperturbed DNA should not be dependent on the sequence.

The role of the accessory bacteriochlorophyll in reaction centers of photosynthetic bacteria: intermediate acceptor in the primary electron transfer?

Time-resolved spectroscopy in conjunction with magnetic-field-dependent recombination dynamics of the primary radical ion pair in reaction centers of Rb, sphaeroides R26, were used to analyze the mechanism of electron transfer from the bacteriochlorophyll dimer in its excited singlet state (1P∗) to bacteriopheophytin (H). This analysis provides evidence against the participation of the accessory bacteriochlorophyll (B) as a kinetic intermediate and thus favours a single-step electron transfer, which is mediated by superexchange electronic interactions.

A kinetic analysis of the primary charge separation in bacterial photosynthesis. Energy gaps and static heterogeneity

Abstract We consider the energetics, the mechanism and the implications of static heterogeneity for the primary electron transfer (ET) from the electronically excited singlet state of the bacteriochlorophyll dimer (1O∗) in the bacterial photosynthetic reaction center (RC) and some of its mutants. The energetics of the primary ET was inferred from an analysis of the experimental free energy relation (at T = 300 K) between the short-time decay rates of 1P∗ and the oxidation potentials of the dimer (P) for a series of single site “good” mutants, for which geometrical changes are minimized and perturbations of the prosthetic groups of the accessory bacteriochlorophyll (B) and of the bacteriopheophytin (H) by the mutants are minor. This analysis resulted in the reasonable value of λ1 = 800 ± 250 cm−1 for the (mutant invariant) medium reorganization energy and ΔG10(N) = −480 ± 180 cm−1 for the energy gap for the native (N) RC. The low value of ΔG10(N) implies that the dominant room temperature ET mechanism for the native RC involves sequential ET. Next, we have explored the effects of heterogeneity on the primary ET by model calculations for the parallel sequential-superexchange mechanism, which is subjected to Gaussian energy distributions of the energies of the P+B−H and P+BH− ion pair states (with a width parameter of σ = 400 cm−1). The modelling of the heterogeneous kinetics by varying the (mean) energy gap ΔG1 between P+B−H and 1P∗ was performed to elucidate the temporal decay of 1P∗ and the ET quantum yield in “good” mutants, to explore the gross feature of primary ET in a triple hydrogen bonded mutant and to characterize some of the temperature dependence of the primary ET. The most pronounced manifestations of heterogeneity within the native RC and its single site mutants (ΔG1 = −900 to 300 cm−1) are the nonexponential temporal decay probabilities for 1P∗, which exhibit long-time tails, with heterogeneity effects being marked (in the classical limit) when σ(ΔG1 + λ1) > λ1kBT. When ΔG1 ⪢ σ (i.e., ΔG1 ⩾ 1000 cm−1), the relaxation rate of 1P∗ is slow, being dominated by the dimer internal conversion rate, with the effects of heterogeneity being less marked, as is the case for the triple hydrogen bond mutant. Regarding mechanistic issues, our kinetic modelling implies that at room temperature, primary ET in the native RC and its single site mutants is dominated by the sequential route and only the triple mutant exhibits a marked contribution of the superexchange route. At low temperature (T = 20 K), ET in the native RC is still dominated by the sequential route (with a small (i.e., ∼ 10%) superexchange contribution being manifested in its long-time decay), for single site mutants there is an interplay between sequential and superexchange routes, while superexchange dominates ET in the triple mutant. The heterogeneous parallel sequential-superexchange mechanism is of intrinsic significance to insure the stability of primary photosynthetic ET for different native and mutagenetically modified RCs over a broad temperature domain.

A superexchange mechanism for the primary charge separation in photosynthetic reaction centers

We analyse the superexchange model for the primary charge separation from the electronically excited singlet state ( 1 P * ) of the bacteriochlorophyll dimer (P) to the bacteriopheophytin (H) across the A branch of the bacterial photosynthetic reaction centers, which is mediated by the accessory bacteriochlorophyll (B). The dominant contribution to the superexchange electronic interaction between the initial 1 P * BH and the final P + BH − states originates from the mixing with the mediating electronic state P + P − H, the energy of which is above 1 P * . The superexchange electronic interaction is V = V PB V BH /δ E , where V PB and V BH are the electronic couplings of 1 P * BH with P + B − H and P + B − H with P + BH − , respectively, while δ E is the vertical energy difference. The nonadiabatic electron-transfer rate is proportional to V 2 F , where F is the nuclear Franck-Condon factor, which is determined by the (free) energy gap Δ G =−2000 cm −1 , the medium reorganization energy λ (λ^lt;2500 cm −1 ) and the medium characteristic frequency ω≈100 cm −1 . Indirect information on the constituents of the effective electronic coupling V ≈25 cm −1 was inferred from the ration | V BH / V PB | calculated from the intermolecular overlap approximation in conjunction with an activated sequential channel and the utilization of kinetic constraints on the dynamics of the primary electron transfer, which result in V PB ≥60 cm −1 , V BH ≥360 cm −1 and δ E ≥1100 cm −1 . We discuss several physical phenomena and observables, i.e., electric field effects on the prompt fluorescence, the unidirectionality of charge separation across the A branch and magnetic interactions in the primary radial pair in the framework of the superexchange mechanism. The electric field (∈) dependence of the fluorescence quantum yield ( Y f (∈)) for isotropic samples at 75 K predicts Y f (∈)=5 mV/A)/ Y f (0)=1.39 and Y f (∈=9 mV/A)/ Y f (0)=3.5. The fluorescence polarization data at constant field (Lock-hart, D.J., Goldstein R.F. and Boxer, S.G. (1988) J. Chem. Phys. 89, 1408–1415) can be well accounted for in terms of the energetic parameters λ=1600 cm −1 and Δ G =−2000 cm −1 together with the value ψ=61 o for the angle between the dipole P + H − and the transition moment of P. The unidirectionality of the charge separation across the A branch originates predominantly from structural symmetry breaking, which affects the electronic coupling, while the contribution of the nuclear contribution has been shown to be small. The predicted ratio of the electronic transfer rates k (A)/ k (B)=82(+190; −70) at T =80 K is consistent with the recent experimental result k (A)/ k (B)≥25 at this temperature. Finally we examined magnetic interactions of the primary P + H − radical pair, establishing the interrelationship between the singlet energy shifts and the triplet energy shift with the primary electron transfer rate, k , and the triplet recombination rate k T whereupon the singlet-triplet splitting of P + H − is J=αk−βk T where the coefficients α and β depend on energetic parametes and Franck-Condon factors. The estimate of J within the superexchange mechanism rests on the incorporation of an assumed configurational relaxation and essential cancellation effects.

Determination of free energies in reaction centers of Rb. sphaeroides

Abstract Magnetic field-dependent recombination measurements together with magnetic field-dependent triplet lifetimes (Chidsey, E.D., Takiff, L., Goldstein, R.A. and Boxer, S.G. (1985) Proc. Natl. Acad. Sci USA 82, 6850–6854) yield a free energy change ΔG( P + H − − 3 P ∗) = 0.165 eV ±0.008 at 290 K. This does not depend on whether nuclear spin relaxation in the state 3P∗ is assumed to be fast or slow compared to the lifetime of this state. This value, being (almost) temperature independent, indicates ΔG( P + H − − 3 P ∗) ⋍ ΔH( P + H − − 3 P ∗) and is consistent with ΔG( 1 P ∗ − P + H − ) and ΔH( 1 P ∗ − 3 P ∗) from previous delayed fluorescence and phosphorescence data, implying ΔG ⋍ ΔH for all combinations of these states.

Observation of acttvationless recombination in reaction centers of R. Sphaeroides: A new key to the primary electron-transfer mechanism

Abstract Activationless recombination of the primary radical pair of R. Sphaeroides in its triplet state supports the involvement of superexchange electronic coupling in the primary electron-transfer step in bacterial reaction centers.

DRAMATIC REDUCTION IN FLUORESCENCE QUANTUM YIELD IN MUTANTS OF GREEN FLUORESCENT PROTEIN DUE TO FAST INTERNAL CONVERSION

Steady-state absorption and fluorescence excitation spectra together with ps-fluorescence and fs-absorption measurements have identified an important relaxation channel of excited states in Green Fluorescent Protein (GFP). GFP derivatives with (1) shortened lifetimes of the protonated chromophore RH* state and deprotonated chromophore R−* state, and (2) rapid recovery of the RH or R− ground state have been isolated and characterized. These shortened excited state lifetimes and fast ground state recovery are interpreted in terms of internal conversion induced by torsional motion within the chromophore.