Photosynthetic Reaction Center

91125

Preface

The study of photosynthesis entered a new era in 1984 with the publication of the detailed X-ray structure of the photosynthetic reaction center from the bacterium Rhodopseudomonas viridis. No longer could researchers speculate so easily on the connection between function and structure. Rather, they were faced with a known entity for explaining mechanism and function. Considerable and rapid progress resulted in understanding the details of the operation of the photosynthetic membrane in both photosynthetic bacteria and green plants. The protein reaction center is not only interesting for its own sake; it is also the only current model for membrane-bound proteins for which the atomic structure is accurately known. Except for the reaction center protein, textbooks show only atomic structures of water soluble proteins.

These two volumes present various aspects of our progress in understanding how photosynthesis converts light energy into stored chemical energy. Volume I is devoted mostly to overall general features and implications of the bacterial reaction center in green plant research. Volume II primarily deals with specific details of the electron transfer process. This division in classification is not always so clear cut; thus most of the contributing authors could have written for either volume.

Volume I covers chemical and biochemical descriptions of photosynthesis, including green plant photosynthesis. This volume starts with a novel new description of the structure of the reaction center (Chapter 1) followed by three chapters (2–4) on the antenna and light functions of the reaction center. The manipulation of the reaction center is discussed in Chapters 5–7, in which both chemical and genetic modifications of the reaction center are covered in detail. The reaction center provides reducing power via electron transfer chemistry coupled to proton uptake and release, described in considerable detail in Chapter 8. The coupling of electron transport between the oxidized reaction center and the aqueous periplasm is accomplished via either bound or unbound cytochrome (Chapter 9). Both Chapters 8 and 9 have relevance to the general operation of membrane-bound proteins, including those involved in mitochondrial processes. An important consequence of the first detailed X-ray structure of a membrane-bound protein has been a tremendous increase in the understanding of green plant photosynthesis, especially photosystem II. And because of the importance of green plant photosynthesis in future research, four chapters (10–13) are devoted to these nonbacterial systems.

Volume II is oriented to the physical aspects of the process of photosynthesis. It thus deals primarily with either bacterial photosynthesis or model compounds. The first two chapters (1 and 2) in Volume II treat the very complex and rapidly evolving issues associated with the theory of electron transfer in the bacterial reaction center. Intimately connected with the theory of electron transfer and the mechanisms for the primary events of bacterial photosynthesis is the subject of time domain spectroscopy. These primary events of charge separation are explored by a variety of techniques, including picosecond and femtosecond spectroscopy, in Chapters 3 – 6. Closely coupled to the picosecond events is hole-burning spectroscopy, described in Chapter 7. Chapter 8 discusses the primary events of bacterial photosynthesis with emphasis placed on the application of large, external electric fields designed to manipulate and to probe the mechanism of the initial chemistry. The role of accessory carotenoid pigments is discussed in Chapter 9 while the relatively new field of infrared spectroscopy as applied to photosynthesis is covered in Chapter 10. The technique of magnetic resonance, which has been employed in the study of photosynthesis for about 35 years, is covered in Chapters 11–12. Finally, Chapters 13–16 present the interplay between natural and artificial photosynthesis. Chapters 14–15 stress a synthetic approach to understanding photoinduced charge separation. In Chapter 13 electron paramagnetic resonance of model compounds is reviewed. Chapter 16 complements Chapters 1 and 17 by describing X-ray structures of many important porphyrins and related model compounds. The X-ray data on porphyrin model compounds were quite important in solving the X-ray structure of the reaction center protein as well as to the unraveling of the mechanism of photoinduced charge separation in both natural and artifical photosynthesis. The last chapter of this two-volume set, Chapter 17, completes a full circle of discussion by returning to the X-ray structure of the bacterial reaction center protein. Without the work and the authors of Chapter 17 this two-volume set would not exist.

Although the understanding of the primary events of bacterial and green plant photosynthesis is at an advanced level, it is by no means complete. Thus in the study of photosynthesis one can find problems that are of genuine interest to quite diverse audiences. The interests of the authors of the chapters of these two volumes represent this diversity. We are grateful to our authors for their participation and patience.

Johann Deisenhofer and James R. Norris

1

Electron-Transfer Mechanisms in Reaction Centers: Engineering Guidelines

Christopher C. Moser, Jonathan M. Keske, Kurt Warncke, Ramy S. Farid and P. Leslie Dutton,     The Johnson Research Foundation University of Pennsylvania, Philadelphia

Publisher Summary

This chapter discusses electron transfer mechanisms in reaction centers. Biological electron transfer has been made possible by the solution of the X-ray diffraction structure of two photosynthetic reaction centers and by extensive measurements on the free-energy dependence of photosynthetic electron transfer reactions. However, a relatively simple form of electron transfer theory is sufficient to define the rate of the majority of intraprotein electron transfer reactions within the uncertainty currently associated with measuring the fundamental parameters of distance, free energy, and reorganization energy. This chapter presents a first-order description of the important parameters of intraprotein electron transfer and presents a list of basic principles of electron transfer protein design. A simple equation derived from quantum mechanical perturbation theory, known as Fermi’s Golden Rule, provides a good first-order description of the rate of nonadiabatic electron transfer. The extensive free-energy dependence of the quinone electron transfer reactions in the bacterial photosynthetic reaction centers provides the best illustration of how the Franck–Condon factors and the optimal rates of intraprotein electron transfer are determined experimentally.

I. Introduction

II. General electron-transfer theory

III. Free-energy dependence of electron transfer

A. Electron transfers with extensive empirical free-energy dependence

B. Other photosynthetic electron transfers

IV. Relationship of optimal rate and distance

V. Electron-transfer engineering

A. Empirical relationship for intraprotein electron transfer

B. Engineering distance

C. Engineering Franck-Condon factors

VI. Alternative photosynthetic charge-separating designs

References

I

Introduction

In the years since the seminal experiments of Chance and Nishimura and DeVault and Chance demonstrated light-activated photosynthetic electron transfer at cryogenic temperatures (Chance and Nishimura, 1960; DeVault and Chance, 1966), electron tunneling has been clearly determined to be common in biological electron-transfer proteins. These early experiments helped stimulate the development of a general theory of long distance electron tunneling. (For an introduction to and review of this topic, see DeVault, 1980.) The simple form of the initial theoretical description has been enhanced continuously to consider the many molecular details that might influence the rate of biological electron transfer. Dramatic progress in understanding which factors are, in practice, important in modulating biological electron transfer has been made possible by the solution of the X-ray diffraction structure of two photosynthetic reaction centers (Chang et al., 1986; Michel et al., 1986; Allen et al., 1987) and by extensive measurements on the free-energy dependence of photosynthetic electron-transfer reactions (Gunner and Dutton, 1989). The evidence that we present supports the view that some important parameters that influence electron-transfer rates are relatively constant for a majority of biological electron-transfer systems. Further, a relatively simple form of electron-transfer theory is sufficient to define the rate of the majority of intraprotein electron-transfer reactions within the uncertainty currently associated with measuring the fundamental parameters of distance, free energy, and reorganization energy (Moser et al., 1992). This simplification permits us to address, in a practical way, some basic engineering concerns faced by intraprotein electron-transfer systems in general and by the photosynthetic reaction centers in particular.

II General electron-transfer theory

Because biological electron transfer generally takes place between redox centers that are separated by relatively large distances (many atomic diameters), the coupling between redox centers is small. A simple equation derived from quantum mechanical perturbation theory, known as Fermi’s Golden Rule, provides a good first-order description of the rate of nonadiabatic electron transfer (see DeVault, 1980).

(1)

The two variables that determine the rate, V² and FC, are primarily dependent on electronic and nuclear factors, respectively. V² is the quantum mechanical matrix element that couples the reactant and product electronic states and is critically dependent on the extent of overlap of the reactant and product electronic wavefunctions. FC is the Franck-Condon weighted density of states and reflects the integrated overlap of the reactant and product nuclear wavefunctions.

For biological systems, the most significant variation of V² can be understood as the continuously decreasing overlap of electronic wavefunctions of the redox centers with increasing distance between them. A simple approximation based on the tunneling between two narrow potential wells (representing the redox centers) separated by a distance R through a barrier of constant height gives an exponential decay.

(2)

Marcus derived a simple description of Franck-Condon factors based on approximating the reactant and product nuclear wavefunctions as simple harmonic oscillations with identical frequencies (ħω), but with different equilibrium nuclear positions and free energies (Marcus, 1956; Marcus and Sutin, 1985). In this description, the overlap of the wavefunctions and, hence, the rate will have, overall, an approximately Gaussian dependence on the free energy of the reaction. When all other variables are held constant, the Marcus relation predicts that the rate will rise as the free energy of the reaction increases from zero to some maximal value. The free energy associated with the maximal rate provides the experimental definition of the reorganization energy (λ). Theoretically, the reorganization energy reflects the energy required to distort the equilibrium nuclear geometry of the reactant state into the geometry of the product state while constraining the electron to remain on the donor. As the free energy is increased beyond the reorganization energy, the overlap of the harmonic oscillator nuclear wavefunctions becomes poor so the rate is expected to fall. This behavior of increasing reaction free energy and decreasing rate of electron transfer defines the Marcus inverted region and has been confirmed experimentally (Miller et al., 1984). Here is a form of the Marcus relation for the Franck-Condon factors in an exact quantum mechanical form, as expressed by Jortner (Levich and Dogonadze, 1959; Jortner, 1976; Marcus and Sutin, 1985):

(3)

where S=λ/ħω is the reorganization energy normalized by the oscillator frequency in energy units, P = − ΔG°/ ħω is the free energy of the reaction normalized by the oscillator frequency, and n=1/(eħω−1) is the average harmonic oscillator level populated at the temperature T, using the Boltzman constant k. IP is the modified Bessel function of order P. Under many conditions, this equation behaves like a simpler and strictly Gaussian relation described by Hopfield (1974),

(4)

where the variance σ² = λħω coth(ħω)/2kBT).

Although Eq. 3 is written so the rate is defined for any exothermic free energy, the strict derivation of this relationship requires that the free energy of the reaction be a strict multiple of the quantum energy of the harmonic oscillator that is coupled to the electron transfer (the oscillator that corresponds to the difference in nuclear geometries of the reactant and product). However, the spectrum of valid free energies will be much larger if multiple oscillations are coupled to the electron transfer. All possible combinations of vibrations and energies can be accounted for by mathematically convoluting the energy distribution of each individual vibration with all the others. Since the convolution of two Gaussians is another Gaussian, the rate dependence for multiple oscillators will be similar in form to Eq. 3, but the single oscillator frequency will be replaced by a "characteristic frequency.′′ This characteristic frequency represents a reorganization energy weighted average of all the individual frequencies. Vibrations with energies larger than twice the Boltzman thermal energy (2kT) contribute to the average characteristic frequency with their quantum frequencies, whereas lower energy vibrations will mimic 2kT oscillators.

The factors that determine the width of the Gaussian dependence of the rate on free energy are especially clear in the Hopfield expression. The coth term reduces to a value of 1 at high temperatures and a value of 2kT/ħω) at low temperatures. Thus, when the characteristic frequency is greater than 2kT, , but when characteristic frequencies are much less than 2kT,

The Franck-Condon factors are affected least by particular values of λ, ħω, and T at the rate optima, where the exponential term disappears and only a very modest square root dependence remains. Thus, a comparison of different systems to gain an appreciation of the non-Franck-Condon factors that influence electron-transfer rates is best done by considering the free energy optimal rates for each system. This suggests the strategy that we follow to examine experimental systems. We restrict our attention to systems in which the distance between donor and acceptor is fixed and known to within 2 Å accuracy by X-ray crystallography or molecular modeling. In this group, our attention is focused on systems in which the free energy has been varied extensively and measured under conditions similar to those under which the rate was measured. In these cases, the free-energy dependence defines the optimal rate and the reorganization energy. We supplement this limited data set with systems in which the distances and free energies are known, but the empirically determined free-energy dependence of the rate covers a narrow range and the reorganization energy and optimal rate are, correspondingly, more uncertain. Finally, we draw together a first-order description of the determinants of the rates of intraprotein electron transfer and extract some basic engineering principles for biological electron-transfer systems.

III Free-energy dependence of electron transfer

A Electron transfers with extensive empirical free-energy dependence

The extensive free-energy dependence of the quinone electron-transfer reactions in the bacterial photosynthetic reaction centers provides the best illustration of how the Franck-Condon factors and the optimal rates of intraprotein electron transfer are determined experimentally. The rates, free energies, and X-ray crystal structure distances between redox centers have been determined for photosynthetic reaction centers of two species. The arrangements of cofactors and distances are illustrated in Fig. 1 (Michel et al., 1986). After light excitation of the bacteriochlorophyll dimer (Bchl2), electron transfer progresses apparently sequentially through bacteriochlorophyll monomer (Bchl), bacteriopheophytin (Bphe), primary quinone (QA), and secondary quinone (QB). The resulting positively charged oxidized dimer (Bchl2+) can be rereduced by the nearest of four bound hemes of cytochrome c in Rhodopseudomonas viridis or by a soluble cytochrome c2 in Rhodobac-ter sphaeroides. Reducing equivalents at the QB site are united with protons funneled in from the aqueous phase. In native membranes, quinone and cytochrome reducing and oxidizing equivalents are reunited by interaction with the cytochrome bc1 complex, completing the conversion of light energy into a transmembrane electric field and proton gradient. In Fig. 1, arrows indicate the physiologically productive charge separations and nonproductive charge recombinations of the reaction center considered in this chapter. Vertical lines represent the approximate dimension of the membrane profile, across which the light-driven electron transfer generates transmembrane potential. Another Bchl and Bphe are found symmetric to the Bchl and Bphe already described; these cofactors do not appear to be involved as redox intermediates and their function is uncertain.

FIGURE 1 The arrangement of redox cofactors of the photosynthetic reaction center protein of Rhodopseudomonas viridis as revealed by X-ray crystallography (Chang et al., 1986; Michel et al., 1986; Allen et al., 1987). The amino acids of the protein matrix and the side groups of the various redox centers have been omitted for clarity. Only one of the four cytochrome c hemes, the one closest to the bacteriochlorophyll dimer, has been shown. Arrows indicate the closest edge-to-edge distance between redox centers in electron transfers that are considered in this chapter. The initial charge separation from the excited bacteriochlorophyll dimer (Bchl2) to the bacteriopheophytin (Bphe) is presented as a conventional two-step mechanism in which Bchl2* first reduces a bacteriochlorophyll monomer (Bchl) which in turn reduces a Bphe. A single-step mechanism involving Bchl in superexchange also has been proposed. Bphe then reduces the primary quinone (QA), which in turn transfers an electron to the secondary quinone (QB). Cytochrome c559 can rereduce the oxidized Bchl2*. Under the appropriate conditions, the physiologically unproductive back reactions from Bphe, QA, and QB to the ground state-oxidized dimer can be observed also. Vertical bars on either side of the cofactors represent the approximate thickness of the low dielectric region of the native membrane.

Two extensive studies of the free-energy dependence of intraprotein electron transfer rate as a function of temperature have been performed (Fig. 2) (Gunner and Dutton, 1989). In these studies, native ubiquinone in Rb. sphaeroides was extracted from the QA site of the reaction center and replaced by a series of quinones. The rates for both the Bphe− to QA and QA− to Bchl2+electron transfer as a function of free energy, shown in Fig. 2A, suggest an approximately Gaussian Marcus-like relationship with optima around 700 meV. The temperature dependence of the free-energy relationship shows that the characteristic frequency is close to twice the Boltzman energy at physiological temperatures. The data best accommodate a value of ∼70 meV. This value reflects the relative temperature independence of electron-transfer rates for electron transfers that are several hundred meV from the free energy optimum, and shows the importance of including the influence of high frequency modes that couple to electron transfer. Substitution of nonquinonoid redox cofactors, including dinitrobenzene (Warncke and Dutton, 1990), leads to electron-transfer rates consistent with quinone substitutions (Fig. 2) and suggests that electron-transfer rates are not strongly dependent on the chemical class of the redox cofactors. Parallel quinone substitution experiments have been performed in Rps. viridis(Keske and Dutton, 1991), in which the free-energy drop between Bphe and QA is naturally smaller than in Rb. sphaeroides. Figure 2 shows that the rate versus free energy trend of Rb. sphaeroides is extended to smaller free energies in Rps. viridis, suggesting that the rate versus free energy relationship is approximately similar in both species.

FIGURE 2 The free energy dependence of the rate of intraprotein electron transfers. The rates and free energies are shown for electron transfer from Bphe− to QA and from QA−to Bchl2+ for reaction centers of Rb. sphaeroides ; ) and include m-dinitrobenzene, 2-hydroxy-l-nitrosonaphthalene, and fluorenones (Warncke and Dutton, 1990). Analogous quinone extraction and replacement experiments for the Bphe− to QA electron transfer in Rps. viridis ). Electron transfer between photoexcited metal-substituted heme centers and ruthenated histidines for a myoglobin and several species of cytochrome c are shown (Δ; Cowan et al., 1988; Meade et al., 1989; Therien et al., 1991). The theoretical relationship of Eq. 3 is plotted through the data using characteristic frequencies in the range of 30 to 150 meV. For the reaction center Bphe− to QA and QA− to Bchl2+electron transfers, we show a reorganization energy of 700 meV, whereas for the ruthenated heme proteins, we show published reorganization energies of 1.2 − 1.3 eV. We show the QA_ to Bchl2+ data at 35 K, at which temperature the differences between characteristic frequencies are more obvious. For this temperature, we also compare the quantized expression with a classical Marcus rate/free energy relation.

Artificial intraprotein electron-transfer systems also show an approximately Gaussian Marcus-type free-energy dependence of the rate (Fig. 2). Electron transfer between surface ruthenated histidines and buried heme centers in myoglobin and cytochrome c molecules from several species has been examined, over the free-energy range accessible, by substituting the metal center of the hemes and altering ruthenium ligands (Cowan et al., 1988; Meade et al., 1989; Therien et al., 1991). Reorganization energies of these reactions appear to be larger (∼1.3 eV) than those of the chlorin QA reactions, suggesting that additional energy is required to rearrange the relatively polar environments of the near-surface histidines of these systems to a configuration that favors electron transfer.

The approximately Gaussian free-energy dependence of the electron-transfer rate also has been shown for several fully synthetic systems with rigid covalent links between donors and acceptors (Wasielewski et al., 1985; Joran et al., 1987; Closs and Miller, 1988; Fox et al., 1990; Gaines et al., 1991). In only one of these systems has a low temperature free-energy dependence been examined (Gaines et al., 1991) to complement the room temperature data (Wasielewski et al., 1985). In this case, the optimal rate remained approximately constant as the temperature was lowered to 77 K, as expected, despite the fact that the reorganization energy decreased from about 0.8 to 0.6 eV. The characteristic frequency associated with the width of the parabola appears to be similar to the 70-meV value found for the chlorin/QA reactions of the reaction center. The only other measurements of the free-energy dependence of rates at low temperature (77 K) have been made on donors and acceptors dispersed in methyltetrahydrofuran (MTHF) glass (Miller et al., 1984); this result also reveals an ħω higher than physiological kT of ∼ 100 meV. Thus, reactions in which rates have been measured over an extensive free-energy range suggest that the breadth of the approximately Gaussian dependency of these rates on free-energy curves is, at physiological temperatures, largely due to differences in the value of λ rather than to variations in ħω.

B Other photosynthetic electron transfers

For electron-transfer systems in which the free-energy dependence of the rate is not well denned empirically, we must estimate a range of reasonable values of λ based on the range defined by the experiments just described, in conjunction with any additional data available. The reaction centers of Rb. sphaeroides and Rps. viridis offer several additional reactions for which the distances, free energies, and rates are well described. We will consider these reactions in the order in which they occur in time. Suggested free-energy dependencies are illustrated in Fig. 3.

FIGURE 3 Free energy/rate relations for electron-transfer reactions in the photosynthetic reaction center for which the experimental free energy dependence is limited. The theoretical curves represent Eq. 3 with a characteristic frequency of 70 meV and a range of reorganization energies. A two-step mechanism for the initial charge separation mechanism is considered. Both Bchl2* to Bchl and Bchl− to Bphe electron transfer are shown (Holzapfel et al., ). Charge recombination of the singlet and triplet Bphe-Bchl2+ in Rb. sphaeroides are shown (Schenck et al., 1982; Chidsey et al., 1985) with a common reorganization energy of 0.5 eV. Cytochrome c559 to Bchl2+ electron transfer in Rps. viridis ). QA to QB electron transfer in Rb. sphaeroides ) (Giangiacomo and Dutton, 1989; Giangiacomo et al., ; Paddock et al., 1989, 1991) with a range of reorganization energies spanning 0.7− 1.3 eV. The pH-insensitive electron transfer rate from QB− to Bchl2 + is shown for a mutant of Rb. sphaeroides(Takahashi and Wraight, 1991), with possible values of the reorganization energy ranging from 0.7 to 1.3 eV.

The initial charge separation reaction in photosynthetic reaction centers has been described both as a single-step transfer between Bchl2 and Bphe involving the intervening Bchl monomer as a virtual superexchange intermediate and as a sequential two-step reaction in which the Bchl monomer is a genuine electron acceptor and donor (see Michel-Beyerle et al., 1988, for extended discussion). Experimental evidence has been provided to support both hypotheses. We will consider what appears to us to be the simpler hypothesis of a sequential mechanism that defines two electron-transfer reactions over edge-to-edge distances of approximately 5.4 and 4.6 Å with rates of approximately 3 × 10¹¹ and 1.5 × 10¹² sec¹ (Holzapfel et al., 1989, 1990). The individual free energies of the reactions are unknown, but should be less than the overall free-energy drop of 160 meV. The reorganization energy for this reaction is also unknown, although molecular dynamics simulations have suggested reorganization energies of 200 − 300 meV (Parson et al., 1990), values significantly smaller than those found for the reactions considered so far. The relative insensitivity of the initial charge separation to applied electric field (Lockhart et al., 1990; C. C. Moser, R. Sension, S. Repenic, P. L. Dutton, and R. A. Hochstrasser, unpublished observations) and to changes in temperature also suggest that the reorganization energy of these reactions will be within ∼200 meV of the free energy of these reactions. For these reasons, we expect the rate for these reactions to be within an order of magnitude of the free-energy optimal rate.

The singlet and triplet Bchl2+Bphe− to ground state recombination reactions (Schenck et al., 1982; Chidsey et al., 1985) represent an approximate free-energy dependence for this reaction. Including both these reactions in a single Marcus relationship defines a reorganization energy of ∼500 meV with an optimal rate of about 6 × 10⁹ sec−1.

The QA to QB electron transfer has been explored through quinone extraction and substitution, similar to that performed for the Bphe− to QA and QA−to Bchl2+ reactions (Giangiacomo and Dutton, 1989; Giangiacomo et al., 1990), and through site-directed mutagenesis (Paddock et al., 1989, 1991). The free-energy range explored has been rather limited, although the increase in rate with free energy suggests that a parabolic Marcus relationship may be appropriate. Since this reaction has been studied only at relatively small free energies to date, the reorganization energy and optimal rate cannot be defined. However, if we consider a range of reorganization energies of 0.7-1.3 eV typical of the systems in which the reorganization energy has been defined empirically, then we expect the log optimal rate for the 14.5-Å reaction in Rb. sphaeroides to be about 6.6 ±0.9 sec−1.

The native electron recombination back to the ground state Bchl2 for QB−Bchl2+is complicated by a pH sensitivity and an equilibrium with the Bchl2+QA− state. Whether this reaction is adiabatic or nonadiabatic is questionable. However, a mutant exists in which the recombination rate is pH insensitive and appears to represent a direct recombination between QB− and Bchl2+ (Takahashi and Wraight, 1991). Placing a broad range of reorganization energies from 0.7 to 1.3 eV suggests that the log optimal rate for this reaction will be 0.4 ± 0.7 sec−1.

The electron transfer between cytochrome c559 and Bchl2+ in Rps. viridis takes place over a well-defined distance and free energy (Shopes et al., 1987; Dracheva et al., 1988). A range of reorganization energies of 0.7 − 1.0 eV suggests a log optimal rate of 8.2 ± 0.5 sec−1.

IV Relationship of optimal rate and distance

A comparison of the logarithm of the optimal rates spanning 12 orders of magnitude for intraprotein electron-transfer reactions as a function of the edge-to-edge distance generates a surprisingly linear relationship (Fig. 4). The protein medium appears to act as a relatively uniform barrier to electron tunneling with an average β value of 1.7 Å−1. Because physiological reactions that are expected to feel the effects of natural selection have β values that are indistinguishable from those of competing back reactions or, indeed, of the nonphysiological reactions of semisynthetic systems, β is likely to be a property of the medium and may not be amenable to natural selection. A similar analysis applied to covalently linked systems (including those presented in Fig. 2, as well as others with a less extensive free-energy dependence) shows a somewhat shallower β of ∼0.7 Å−1 (Moser et al., 1992). This result raises a question of connectivity. The wavefunction can propagate through a bond somewhat more effectively than through space between redox cofactors. For comparison, β in a vacuum can be estimated to be ∼2.8 Å−1, based on a simple Gamow model (Gamow, 1928) and using a typical ionization energy for biological redox centers of ∼ 8 eV. The β value for the reaction center and other proteins falls in between the covalently linked value and the vacuum value, and may reflect the average density of covalent type linkages in comparison with gaps found in the typical condensed protein medium between redox centers. Note that the value we find is in the middle of the range of calculations made on a topographical analysis of connectivity of different proteins, calibrated by a few electron-transfer reactions with known structures (Beratan et al., 1991). Also, the relatively large size of redox centers such as chlorins means that the wavefunctions can propagate over many similar distance paths between redox centers, so the effect of a specific bond or gap will tend to be averaged out of the complete effect.

FIGURE 4 ) and excited heme-ruthenium electron transfers in modified myoglobin and cytochromes c are shown (Δ). Filled symbols represent reactions with extensive free energy dependence, presented in Fig. 2, whereas open symbols represent reactions with a smaller experimental free energy dependence and more uncertain optimal rates, presented in Fig. 3. Error bars are associated with uncertainties in rate optima. For cytochrome c to Bchl2+, QA− to QB, and QB− to Bchl2+ electron transfer, this uncertainty represents the X ranges shown in Fig. 3. The fit line represents a simple exponential decay of Eq. 1 with a β of 1.4 Å−1. Distance is denned as center of edge atom of donor to center of edge atom of acceptor; thus, the vertical line at 3.6 Å represents van der Waals contact. Distances were derived from the crystal structures.

Two complementary views of a tunneling reaction picture the particle either passing through a classically inpenetrable barrier, in which case the value of β is dependent on the height of the barrier, or participating in classically inaccessible virtual states that are higher in energy than the starting state. If these virtual states are of uniformly high energy, then a β value can be defined that will depend on the energy gap. On the other hand, if an intermediate cofactor redox center presents a virtual state quite close in energy to the starting state, then this cofactor would assist the propagation of the electronic wavefunction significantly and have the effect of dramatically decreasing the value of β. Although superexchange with intermediate Bchl in the electron transfer between Bchl2 and Bphe cannot be ruled out, the observed rates appear to be consistent with the distance and free-energy dependence of other intraprotein electron-transfer reactions, and do not require superexchange with a relatively low energy intermediate redox center. On the other hand, the electron transfer between QA− and cytochromes c559 and c553 in Rps. viridis take place over distances of 43 and 68 Å, with rates much faster than the millennia rates expected from the linear relationship of Fig. 4. This reaction obviously is complicated by the involvement of Bchl2+, which could be a conventional thermally activated intermediate state. The rates and free energies of these reactions at low temperatures are not certain, but one set of experiments suggests that these rates appear to become relatively temperature independent at low temperatures (about 2.5 and 0.20 sec−1; Gao et al., 1990), implicating some sort of tunneling mechanism. These relatively rapid rates could be viewed as evidence for a superexchanging intermediate such as Bchl2. On the other hand, Bchl2 could act as an intermediate in a tunneling equilibrium, analogous to the tunneling equilibria and temperature-independent equilibrium constants that have been observed in Tutton salts (Trapani and Strauss, 1989). An endothermic extension of the parabolic relationship we have considered already for the electron transfer from cytochrome c559 to Bchl2+ in Fig. 3 can be used to define a reorganization energy-independent tunneling equilibrium constant. This equilibrium constant, in combination with the measured low temperature QA− to Bchl2+ rate, predicts a QA− to cytochrome c559 tunneling rate of 3 sec−1. This value compares well with the 2.5 sec−1 measured in these experiments.

V Electron-transfer engineering

A Empirical relationship for intraprotein electron transfer

We have presented a first-order analysis of intraprotein electron transfer that provides a simple expression that describes the primary parameters influencing rates. This expression allows predictions to be made in arbitrary protein systems with an accuracy compatible with the current uncertainties of measurement of electron-transfer rates, distances, free energies, and reorganization energies. The values of β appear to be generic for many protein systems. The value of ħω does not appear to modulate electron-transfer rates significantly and appears to be, genetically, ∼70 meV. This value of the characteristic frequency coupled to electron transfer may reflect an unremarkable subset of the total spectrum of vibrational modes that contribute to the typical heat capacity of a condensed organic medium. This value of ħω is only slightly greater than twice room temperature Boltzman thermal energy. Thus, for experiments restricted to room temperature, the width of the rate versus free-energy relationship will be only slightly greater than the classical Marcus relationship. This information allows development of a practical, empirical approximation that has a simple Gaussian form of a Marcus-like expression and includes a log relationship between the optimal rate and the edge-to-edge distance, using the apparently generic values of β and ħω.

(5)

The coefficients of this empirical relationship are derived with the following units: sec−1 for the electron transfer rate kct, Å for the edge-to-edge distance R, and eV for the free energy and reorganization energy. With this useful relationship, we can now address the engineering principles that must be followed in the design of intraprotein electron-transfer systems, including photosynthetic reaction centers.

B Engineering distance

Reaction centers, as well as other bioenergetic electron-transfer proteins, operate in a Mitchell-like chemiosmotic system (Mitchell, 1961). One of the principle characteristics of such a system is the presence of a low dielectric membrane that defines two aqueous phases and permits the storage of energy in transmembrane electric field and pH difference. Since distance is the overwhelming determinant of electron-transfer rates, the 35-Å low dielectric profile of the membrane represents a principle constraint on the design of transmembrane electron-transfer proteins. Even under the most favorable conditions of free energy and reorganization energy, it is not possible to have a single electron transfer between two redox centers that can cross this barrier with a rate that approaches the typical submillisecond lifetime of encounter complexes of integral membrane proteins and mobile redox agents such as cytochrome c or quinone, or even the minimum rate of 10³ sec−1 that appears to be characteristic of the overall electron transfer through the chemiosmotic system. Thus, all electron-transfer proteins that work in such a system must have at least two sequential electron-transfer reactions to span the transmembrane distance, involving a minimum of three redox centers. This situation exists for respiratory proteins including the bc1 complex and cytochrome c oxidase.

Photosynthetic reaction centers face the same distance constraints and a much more severe time constraint, because electron transfer must take place not on the leisurely millisecond time scale but on the typical nanosecond time scale of stability of excited electronic states that form the starting point of photosynthesis. Thus, efficient electron transfer in any photosynthetic system must take place in at least three steps involving at least four redox centers. Fig 1 illustrates that the bacterial reaction center involves four steps and five redox centers to stabilize light energy into a transmembrane charge separation.

C Engineering Franck–Condon factors

Despite the dominant control of distance on electron transfer, Franck-Condon factors can be critical, especially when distances of different paths are similar. Manipulation of Franck-Condon factors necessarily involves both free energy and λ changes. Generally speaking, natural systems will want to minimize the expenditure of free energy in forward reactions that lead to stable charge separation to provide the maximum free energy to drive subsequent bioenergetic pathways. Because λ for each reaction reflects the energy required to move the reactant nuclei into a configuration that is favorable for the product charge distribution, λ generally will be largest when electrostatic interactions between the tunneling electron and the redox centers and surrounding medium is largest. This condition is most likely to be met when the redox centers are small, concentrating the charge, and are imbedded in a relatively polar medium. Similarly, λ most likely will be small when the redox centers are large and delocalize the electron, and the surrounding medium is relatively nonpolar. Temperature and time scale of the reaction can influence the reorganizations that are possible and, hence, modify λ. Because a sequence of electron transfers will share intermediate redox centers, λ of a subsequent reaction often will be within several hundred meV of the preceding reaction. From our examination of the limited set of intraprotein electron-transfer reactions with significant free energy dependencies, a typical reorganization energy for an electron transfer involving chlorins or hemes embedded in a protein appears to be 600 − 800 meV. Typically, reactions involving redox centers with access to the aqueous medium will have a larger λ value whereas, in special cases, a redox center in a nonpolar environment may have a smaller λ.

A primary example of the influence of Franck-Condon factors on electron transfer engineering is found in the initial charge separation of any light-induced electron transfer. These reactions will compete with a generally large free-energy back reaction to the ground state over the same distance as the charge separation. Indeed, a number of synthetic systems designed to study electron transfer suffer from a short-lived charge-separated state for this reason. An elegant engineering solution, recognized by Miller and colleagues (Miller et al., 1984) and Warshel (1981), is designing the reorganization energy to be as small as practical, forcing the back reaction into the Marcus inverted region. This strategy is used in the photosynthetic reaction centers. If a typical λ for intraprotein electron transfer is ∼700 meV, then a λ of approximately 300 meV for the initial charge separation in reactions centers may reflect natural selection pressure for a photosynthetic center with a relatively delocalized charge (a dimer of chlorins) in a relatively rigid and non-polar protein environment.

The initial photosynthetic reactions also may provide another illustration of the importance of Franck-Condon factors. The excited Bchl2* is faced with potential electron transfer to similarly close Bchls on either side of the approximate symmetry axis of the reaction center. The function of the Bchl and Bphe on the M subunit side of the reaction center is unknown, but the evidence for asymmetric electron transfer is clear. Electron transfer to the functionally inactive side may be made unfavorable by permitting λ to rise to 600 meV (Moser et al., 1992). Similarly, an inactive side endothermic free energy of 200 meV (Parson et al., 1990) is sufficient to insure that electron transfer to the active site will take place with greater than 99% efficiency.

The reaction center provides one more example of the importance of adjusting Franck-Condon factors. The distances and free energy for the return of the electron from QA− or QB− to Bchl2+ are quite similar, yet the QB− state can be stabilized for seconds if the reorganization energy for this reaction is made large (Okamura and Feher, 1992), perhaps as large as 1300 meV. A large reorganization energy could be associated with a relatively polar QB site that is designed to couple protonation to electron transfer. Alternatively, the presence of protonation reactions at QB may make this reaction adiabatic and outside the scope of the nonadiabatic tunneling analysis presented here. Clearly, adiabatic reactions are found in the relatively slow reactions involving diffusion, such as the interaction of soluble cytochrome c with the reaction center, which can stabilize separated charges essentially indefinitely by translating them physically.

The two-step mechanism for the initial charge separation in reaction centers suggests that another general strategy, independent of Franck-Condon factors, is involved. When a third redox center is placed close to the original acceptor, subsequent charge separation can be rapid, and charge recombination over the longer distance will be slower. Thus, a rapid electron transfer between Bchl and Bphe can insure a charge separation of 10 Å with nanosecond stability.

VI Alternative photosynthetic charge-separating designs

The particular reaction center structures revealed by the X-ray diffraction of crystals of reaction centers from Rps. viridis and Rb. sphaeroides are only one means of fulfilling the engineering constraints we have considered. Obviously, natural protein design follows many constraints including the need for successful and stable folding of the polypeptide and resistance to proteolysis. However, with respect to the electron-transfer reactions, the simple description presented in this chapter suggests many possible successful photosynthetic charge-separating protein designs that differ in terms of distances, free energies, and reorganization energies. In principle, it is possible to create a transmembrane light-activated charge-separating protein design that matches the efficiency of the bacterial reaction center with fewer redox centers but still satisfies the requirements of a transmembrane chemiosmotic system, that is, millisecond stability of charge separation over the dielectric of 48 Å (the heme center to QA center distance in Rps. viridis) and 99% or greater quantum efficiency of charge separation from an absorber with nanosecond fluorescence decay. For simplicity, we assume our terminal redox centers to be a quinone (designated Q) and a cytochrome heme (designated C) that is oriented toward Q for an edge-to-edge distance of 41 A. We choose chlorins (A and B) with internal diameters of 8.4 Å as intermediate redox centers that are oriented toward the heme and Q. We also assume that such a reaction center obeys our generic parameters for electron transfer with a β of 1.7 Å−1 and a 70-meV characteristic frequency. We choose a typical absorbed photon energy of 1.4 eV, corresponding to the long wavelength absorption maximum of Rb. sphaeroides.

Using the useful approximation of Esq. 5, if only one intermediate redox center is used, the electron transfer rate over 16.3 Å is 2 × 10⁵ sec−1 at best, and the quantum efficiency is an extremely poor 0.02%. What is the most efficient reaction center that can be made with two redox centers intermediate between the heme and the Q? Fig 5 provides one possible illustration. An initial charge separation of 10¹¹ sec−1 would insure a quantum efficiency of 99%. This rate can be achieved while conserving free energy by using as small a reorganization energy as possible, for example, 0.3 eV, by using a distance of 6.7 Å. The charge recombination rate to ground state is appropriately slow at 3 × 10⁴ sec−1. Accounting for the width of the chlorins, we divide the remaining 17.6 Å in half for two identical 8.8 Å edge-to-edge reactions. Choosing as small a free energy as possible to avoid thermal back reactions, for example, 0.12 eV each, leads to a rate for each of these reactions of 2 × 10⁸ sec−1. Charge recombination back to ground state from Q over 23.9 Å is less than 5 sec−1, even for the worst case in which the free energy is close to λ. The net result is rapid charge separation across the membrane (about 20 ns), with a consumption of only 0.54 eV of the photon energy to give a chemical potential of better than 0.8 eV, compared with the native 0.4 eV, using one fewer intermediate to build into the reaction center!

FIGURE 5 Possible arrangements of redox centers for efficient charge separation in a hypothetical photosynthetic protein. The overall distance of charge separation between C and Q is represented as the same distance as cytochrome c to QA electron transfer of the photosynthetic reaction center shown in Fig. 1. A and B are represented as chlorin-type redox centers that function as the absorber of light energy and as an electron-transfer intermediate, respectively. Distances and free energies of reactions are chosen according to the first order description of intraprotein electron transfer presented in this chapter, to achieve 99% quantum efficiency in charge separation across a low dielectric membrane with millisecond or better stability and minimum consumption of free energy. Optimum geometry of redox centers is presented in the left half of the figure, whereas an example of the effect of poor orientation of redox center B is considered on the right.

The extraordinary efficiency and speed of this hypothetical reaction center depend on the orientation of the chlorins with the diagonal axes toward the heme and Q redox centers. If one of the chlorin redox centers is oriented unfavorably (for example, perpendicular to the transmembrane vector as in Fig. 5), the edge-to-edge distance of centers A and B remains the same, but the edge-to-edge distance of centers B and Q becomes just long enough to make the rate of the reaction from B to Q 100 times faster than the back reaction from A+B− to ground state, while still using the modest 0.12 eV of free energy. For a distance of 11.8 Å, the rate is 3 × 10⁶ sec−1. The back rate from A+Q− to ground state over 18.5 Å is 4 × 10³ sec−1. The distance between C and A will be 14.4 Å, with a rate of 7 × 10⁴ sec−1 for a modest 0.12-eV expenditure. A system with a poorly oriented chlorin B will perform better than one with a poorly oriented chlorin A. This case still shows great energy efficiency (0.8 eV remain for dark reactions) as well as 99% quantum efficiency, all accomplished in 700 μs. If both redox centers A and B have the worst orientations (perpendicular to the transmembrane direction of electron transfer), 99% quantum efficiency will not be possible without adding an additional redox center, making the hypothetical photounits formally similar to the reaction centers observed in nature.

So far we have not considered the influence of a triplet state decay mechanism, which may be unavoidable or even part of an original primitive mechanism of light-induced charge separation. Using typical energy levels of triplet states for various Bchls and Bphes of about 100 meV below the QY electronic transitions (Weiss, 1978), a charge recombination from triplet A+B− may have a free energy as small as 600 meV and moderately close to the reorganization energy. Quantum efficiency will suffer if singlet-triplet conversion is extremely fast, for example, 2 orders of magnitude faster than the ∼ 10⁸ sec−1rate in reaction centers, and the orientation of cofactors is unfavorable. Nevertheless, with favorable orientations, A to B and B to Q distances of 6.7 Å, and a C to A distance of 10.8 Å, a 99% quantum efficiency is still possible. In principle, it is possible to take advantage of the long lifetime of an excited triplet state and permit the initial charge separation to take place at longer lifetimes, provided the reorganization energy of this separation can be made small. However, the examples provided by protein systems suggest that slow and long-distance electron transfers are associated with modest to large reorganization energies.

We can consider the practical long wavelength limit for photosynthetically active photons that leave ∼ 0.4 eV of energy for subsequent dark redox reactions of the cytochrome bc1 complex. Using the first geometry we considered with an ideal orientation of cofactors, a photon with energy of 0.94 eV or 1300 nm will stabilize charge on a 300 ms timescale. Under a common biological transmembrane electric load of ∼250 mV, each of the reactions will be under an 80-mV load. The B to Q free-energy drop should be increased from 0.12 to 0.2 eV to compensate for a more facile thermal back reaction. Thus, the lowest energy photon for efficient charge separation under load will be ∼1.1 eV or ∼1100 nm. The low energy absorption of the reaction center of Rps. viridis is 960 nm, although antennae proteins in this species have significant absorption out to 1000 nm, surprisingly close to this ideal value.

We have presented evidence in this chapter for a powerful first-order description of the important parameters of intraprotein electron transfer, leading to a list of basic principles of electron-transfer protein design. These engineering concerns suggest that many arrangements of redox cofactors can, in principle, lead to efficient photosynthetic charge separation. Although the arrangement of cofactors in the bacterial reaction centers of Rb. sphaeroides and Rps. viridis are very similar, and ultimately may resemble the arrangement of redox cofactors in green plant photosystems, with respect to intraprotein electron transfer, efficient reaction centers can be constructed with three fewer chlorins than found in these centers, provided the spacing and orientation of the remaining cofactors is moderately favorable. The particular arrangement of cofactors revealed by the bacterial X-ray crystal structures may be determined by concerns that are not related directly to electron transfer, for example, protein folding and stability and gene duplication. Rhodopseudomonas viridis appears to operate with photon energy relatively close to the anticipated 1100-nm engineering limit expected for efficient photosynthetic charge separation, although this report suggests that other species with a long wavelength absorption maximum greater than 960 nm may be found.

Acknowledgment

The authors gratefully acknowledge the support of the National Institutes of Health (Grant R01 GM41048).

References

Allen, J. P., Feher, G., Yeates, T. O., Komiya, H., Rees, D. C. Structure of the reaction center from Rhodobacter sphaeroides R-26: The cofactors. Proc. Natl. Acad. Sci. U.S.A. 1987; 84(16):5730–5734.

Beratan, D. N., Betts, J. N., Onuchic, J. N. Protein electron transfer rates set by the bridging secondary and tertiary structure. Science. 1991; 252:1285–1288.

Chance, B., Nishimura, M. On the mechanism of chlorophyll cytochrome interaction: The temperature insensitivity of light-induced cytochrome oxidation in Chromatium. Proc. Natl. Acad. Sci. U.S.A. 1960; 46:19–24.

Chang, C.-H., Tiede, D., Tang, J., Smith, U., Norris, J., Schiffer, M. Structure of Rhodopseudomonas sphaeroides R-26 reaction center. FEBS Lett. 1986; 205(1):82–86.

Chidsey, C. E.D., Kirmaier, C., Holten, D., Boxer, S. G. Magnetic field dependence of radical-pair decay kinetics and molecular triplet quantum yield in quinone-depleted reaction centers. Biochim. Biophys. Acta. 1985; 766:424–437.

Closs, G. L., Miller, J. R. Intramolecular long-distance electron transfer in organic molecules. Science. 1988; 240(4851):440–447.

Cowan, J. A., Upmacis, R. K., Beratan, D. N., Onuchic, J. N., Gray, H. B. Long-range electron transfer in myoglobin. Ann. N. Y. Acad. Sci. 1988; 550:68–84.

DeVault, D. Quantum mechanical tunnelling in biological systems. Q. Rev. Biophys. 1980; 13:387–564.

DeVault, D., Chance, B. Studies of photosynthesis using a pulsed laser. I. Temperature dependence of cytochrome oxidation rate in Chromatium. Evidence for tunneling. Biophys. J. 1966; 6:825–847.

Dracheva, S. M., Drachev, L. A., Konstantinov, A. A., Semenov, A. Y., Skulachev, V. P., Arutjunjan, A. M., Shuvalov, V. A., Zaberezhnaya, S. M. Electrogenic steps in the redox reactions catalyzed by photosynthetic reaction-centre complex from Rhodopseudomonas viridis. Eur. J. Biochem. 1988; 171(1–2):253–264.

Fox, L. S., Kozik, M., Winkler, J. R., Gray, H. B. Gaussian free-energy dependence of electron-transfer rates in iridium complexes. Science. 1990; 247(4946):1069–1071.

Gaines, G. L., O’Neil, M. P., Svec, W. A., Niemczyk, M. P., Wasielewski, M. R. Photoinduced electron transfer in the solid state: Rate vs. free energy dependence in fixed- distance porphyrin-acceptormolecules. J. Am. Chem. Soc. 1991; 113(2):719–721.

Gamow, G. Zur Quanten Theories des Atom Kernes. Z. Phys. 1928; 51:204–212.

Gao, J.-L., Shopes, R. J., Wraight, C. A. Charge recombination between the oxidized high-potential c-type cytochromes and QA- in reaction centers from Rhodopseudomonas viridis. Biochim. Biophys. Acta. 1990; 1015:96–108.

Giangiacomo, K. M., Dutton, P. L. In photosynthetic reaction centers, the free-energy difference for electron transfer between quinones bound at the primary and secondary quinone-binding sites governs the observed secondary site specificity. Proc. Natl. Acad. Sci. U.S.A. 1989; 86(8):2658–2662.

Giangiacomo, K. M., Gunner, M. R., Dutton, P. L. QA and QB site control of quinone electrochemistry in the photosynthetic reaction center from Rhodobacter sphaeroides. Biophys. J. 1990; 57:566a.

Gunner, M. R., Dutton, P. L. Temperature and -ΔG° dependence of the electron-transfer from BPh− to QA in reaction center protein from Rhodobacter sphaeroides with different quinones as QA. J. Am. Chem. Soc. 1989; 111(9):3400–3412.

Holzapfel, W., Finkele, U., Kaiser, W., Oesterhelt, D., Scheer, H., Stilz, H. U., Zinth, W. Observation of a bacteriochlorophyll anion radical during the primary charge separation in a reaction center. Chem. Phys. Lett. 1989; 160:1–7.

Holzapfel, W., Finkele, U., Kaiser, W., Oesterhelt, D., Scheer, H., Stilz, H. U., Zinth, W. Initial electron-transfer in the reaction center from Rhodobacter sphaeroides. Proc. Natl. Acad. Sci. U.S.A. 1990; 87(13):5168–5172.

Hopfield, J. J. Electron transfer between biological molecules by thermally activated tunneling. Proc. Natl. Acad. Sci. U.S.A. 1974; 71:3640–3644.

Joran, A. D., Dervan, P. B., Felker, P. M., Hopfield, J. J., Leland, B. A., Zewail, A. H. Effect of exothermicity on electron-transfer rates in photosynthetic molecular models. Nature (London). 1987; 327(6122):508–511.

Jortner, J. Temperature dependent activation energy for electron transfer between biological molecules. J. Chem. Phys. 1976; 64(12):4860–4867.

Keske, J. M., Dutton, P. L. Extraction and reconstitution of the QA site in Rhodopseudomonas viridis: Evidence for QA site similarities between Rhodopseudomonas viridis and Rhodobacter sphaeroides. Biophys. J. 1991; 59(2):144a.

Levich, V. G., Dogonadze, R. R. Teiriya bezizluchatelnikh electronnikh perekhodov mezhdu ionami v rastvorakh. Doklady Akad. Nauk. SSSR. 1959;