Photosynthetic charge separation is one of the most important light-induced reactions in the world. This reaction utilizes the energy coming from the Sun to produce electrochemical potential (proton gradient). The primary charge separation is the electron transfer across the photosynthetic membrane (in the case of bacterial photosynthesis, it is also the cell membrane). This reaction takes place in a specized pigment-protein complex called photosynthetic reaction center. The crystall structure of reaction center found in Rhodopseudomonas viridis was determined in 1986 by H.Michel with coworkers , and that of Rhodobacter sphaeroides was revealed shortly thereafter . Pigment arrangement in the bacterial reaction center is shown in Fig. 29A. Obviously, the reaction center exhibits approximate mirror symmetry. The pigments participating in charge separation are shown in different colors. Two closely spaced bacteriochlorophylls shown in red are called special pair P. Slightly ‘lower’, two ‘accesory’ bacteriochlorphylls B are shown in blue, bacteriopheophytins H are in green, and the molecules colored in magenta and cyan are quinons. Electron transfer starts, when the excitation reaches the special pair (the excited state energy of P is the lowest among all pigments). The electron first hops onto the accessory bacteriochlorophyll, then onto bacteriopheophytin, and then – onto the first quinon QA and the second one QB. It is interesting to note that despite apparent symmetry, the electron transfer only occurs via the branch involving the pigments with subscript A (right hand side in Fig. 29A). All the transfer steps, especially the initial ones are ultrafast and only relatively recently – in 1990, W.Zinth with coworkers performed femtosecond pump-probe experiments that revealed the final details of this process . To be able to understand the details of PP experiment, first let us discuss the absorption spectrum of the reaction center shown in Fig. 29B. Different pigments of the reaction center exhibit nicely separated absorption bands (i.e. their excitation energies differ appreciably): the bacteriochlorophylls of the special pair P absorb at 870 nm, accessory bacteriochlorophylls B have their lowest absorption band at 800 nm, bacteriopheophytins H – at 760 nm. These bands correspond to the lowest energy transitions to the states called Qy. Higher electronic excited states of bacteriochlorophylls called Qx absorb at 600 nm (Fig. 29B), and those of pheophytins – at 545 nm. It is natural to expect that the bleaching of these bands will occur at the precise instance, when the electron hops onto the pigment responsible for any particular band. However, we must keep in mind that PP signals also contain other contributions (SE, ESA) and the interpretations of the data have to include all of them.
Fig. (1) A. Pigment arrangement in the photosynthetic reaction center of bacterium Rhodobacter sphaeroides. Bacteriochlorophylls forming the so-called special pair (P) are colored in red, accessory bacteriochlorophylls (B) are shown in blue, bacteriopheophytins (H) – in green, and quinons (Q) – in magenta and cyan. The arrows indicate the pathway of electron transfer and the rates of separate steps. The rates were deterimined from PP spectroscopy by W.Zinth and coworkers . B: Absorption spectrum of Rhodobacter sphaeroides . Absorption band of the lowest excited state of the special pair P (the transition is denoted Qy) is at 870 nm, accessory bacteriochlorphylls absorb at 800 nm, bacteriopheophytins H – at 760 nm. Higher excited state of bacteriochlorophylls (both P and B) is denoted Qx and its absorption band is at 600 nm, whereas bacteriopheophytin absorbs at 545 nm.
Transient absorption kinetics of reaction center excited at the special pair P absorption band (860 nm pulses were used) are shown in Fig. 30. The curve measured at 920 nm probe wavelength (Fig. 30a) is mainly determined by the stimulated emission of the excited special pair P. The negative signal appears instantaneously, when P is excited and decays in approximately 3.5 ps. This suggests that the excited special pair P* disappears within this time frame. At later times (when t>10 ps) the signal slightly decays again, which indicates that the dynamics is not over at 3.5 ps. The kinetic trace at 920 nm is well fitted using a three component model (consisting of three decaying exponential functions with lifetimes 3.5 ps, 220 ps and infinity, i.e. a component that persists within the time window of the experiment).
The absorption change at 785 nm is positive at all times (Fig. 30b). Induced absorption appears immediately after the excitation, decays slightly during the first two picoseconds (the corresponding time constant is approximately 0.9 ps) and starts growing again afterwards. The second phase of signal growth is two-exponential, with time constants of 3.5 ps and 220 ps. The trace at 785 nm cannot be described by just three exponents: if the 0.9 ps component is left out, a clearly unsatisfactory fit is produced, shown in dashed line in Fig. 30b. However, when four-component model is used, the fit is excellent (solid line).
Fig. (2) Transient absorption kinetic traces measured in the reaction center of Rhodobacter sphaeroides, exciting at 860 nm and probing at 920 nm (a), 785 nm (b), and 545 nm (c). The time scale is linear up to 1 ps and logarithmic thereafter. The symbols show experimental data, solid lines – results of modeling obtained using 4 kinetic components, dashed lines – results of 3-component model. Reproduced from .
At 545 nm, where bacteriopheophytin H has its absorption peak, complex kinetics is observed: immediately after the excitation, induced absorption is observed, which persists until 2 ps. Later, DA starts decreasing and becomes negative (2 ps to 20 ps), and subsequently increases again approaching the zero line (~200 ps times). Again, this kinetic trace cannot be satisfactorily described by three kinetic components, at least four are necessary (compare dashed and solid lines in Fig. 30c). At this wavelength, bacteriopheophytin has its absorption peak, therefore, bleach at this wavelength (negative DA) signals about the electron landing on this pigment. The bleaching disappears when the electron moves on to the quinon. Initial positive signal is evidently due to the excited state absorption of P*, because it appears at the very early times, when the special pair P is excited.
In summary, we have three experimental facts
- P* excited state disappears within 3.5 ps.
- Electron lands on bacteriopheophytin H in ~3.5 ps and moves on within ~220 ps after the excitation.
- Both bacteriopheophytin bleaching and accessory chlorophyll bleaching (785 nm trace) show an additional process with characteristic time of 0.9 ps, i.e. to get an adequate description of the data at least 4 kinetic components are required.
These experimental facts led W.Zinth with coworkers to the following scheme of primary charge separation in the reaction center:
i.e. the excited special pair P* transfers the electron to the accessory bacteriochlorophyll B within 3.5 ps, then the electron quickly shifts to bacteriopheophytin H , from which it reaches the primary quinon QA in 220 ps. The electron ‘escapes’ from the accessory bacteriochlorophyll much faster (0.9 ps) than it arrives (3.5 ps), therefore the population of P+B- state is very small at all times. This is why this state remained undetected for a long time, and the received wisdom was that the accessory bacteriochlorophyll does not participate in the electron transfer chain. The quality of data and sophisticated modeling performed by W. Zinth and coworkers showed for the first time that this pigment is an integral part of charge separation chain. Later, this was confirmed by other experiments .