Initial electron-transfer in the reaction center from Rhodobacter sphaeroides.

Abstract

The initial electron transfer steps in the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides have been investigated by femtosecond timeresolved spectroscopy. The experimental data taken at various wavelengths demonstrate the existence of at least four intermediate states within the first nanosecond. The difference spectra of the intermediates and transient photodichroism data are fully consistent with a sequential four-step model of the primary electron transfer: Light absorption by the special pair P leads to the state P*. From the excited primary donor P*, the electron is transferred within 3.5 ± 0.4 ps to the accessory bacteriochlorophyll B. State P+B-decays with a time constant of 0.9 ± 0.3 ps passing the electron to the bacteriopheophytin H. Finally, the electron is transferred from H-to the quinone QA within 220 ± 40 ps. According to present knowledge, an electron is transferred upon light absorption from the P along branch A to the quinone QA (8)(9)(10)(11)(12)(13) Materials and Experimental TechniquesRCs from two strains ofRb. sphaeroides, the wild-type strain (ATCC 17023) and the carotenoid-free strain (R 26.1), were isolated as described (39). The measurements were performed at room temperature (297 K) in cuvettes with a 1-mm path length with stirring. The concentration of the samples was adjusted to OD8w values between 0.5 and 1.0 mm' A synchronously pumped unidirectional ring dye laser (42) generated pulses with a duration of 60 fs at a wavelength of 860 nm. The energy of single pulses was increased to 20 lJ by a three-stage dye amplifier (repetition rate, 10 Hz). Each of these pulses was split into two parts. (i) The excitation pulse was focused to a 0.5-mm spot providing an energy density of not more than 100 uJ/cm2 in the sample. In the probed volume, -7% of the RCs were excited per pulse. This low excitation density prevents double excitation of the RC and related nonlinear processes. The exciting pulse, centered at 860 nm, had a bandwidth of <15 nm (full width at half-maximum). No spectral components of the exciting pulse were detected at wavelengths of <840 nm. Thus only the lowest electronic level ofP at 860 nm was excited. (ii) The probing pulse passed an adjustable delay line and was focused onto a 1-mm-thick jet of ethylene glycol to generate a femtosecond white-light pulse. A 10-to 20-nm-wide portion of this continuum was selected by means of a special dispersion compensating monochromator (43). The energy of the probing pulses was <7 p.J/cm2.

Spectroscopic characterization of reaction centers of the (M)Y210W mutant of the photosynthetic bacterium Rhodobacter sphaeroides

“…The late relative absorbance decrease can be fitted with a 220 ps time constant. This time constant is due to the electron transfer from (I) A t o QA (Holzapfel et al 1990). The decay of P* in the (M)Y210W mutant was best fitted by a sum of two exponentials with time constants of 5.1 -+ 0.8 ps (20%) and 36.0 + 1.8ps (70%).…”

Primary Electron Transfer Kinetics in Membrane-Bound Rhodobacter sphaeroides Reaction Centers: A Global and Target Analysis

“…The species-associated difference spectra (SADS) ( Figure 6) were consistent with the expected spectral properties of the intermediate states specified in the scheme. In particular, Figure 6C shows the 810 and 860 nm bleach, together with the positive band at 1020 nm, that would be expected for the radical pair state P + B L - (5,8,9,12,18). Note the difference of the 1020 nm band with the SADS of Figure 6D,E in the overlay Figure 6F.…”

“…The most favourable fit to the full set of experimental data from 690-1060 nm required five lifetimes. A combination of the model for RC kinetics from (5,6,8,9,12,18) and the idea of radical pair relaxation (5,18,(38)(39)(40) gave a five-component kinetic scheme ( Figure 5) that offered a satisfactory description of the data. The SADS of the two excited states and three radical pair states involved in this scheme were consistent with what is known about the spectra of these states.…”

“…The first concerns the much debated role of the accessory BChl B L , and specifically the proposal that the P + B L -radical pair state is formed as a true intermediate during electron transfer from P* to P + H L - (5,6,8,9,12,18). The analysis presented in this paper is entirely consistent with the view that the P + B L -state is formed as a detectable intermediate, with in our case a lifetime of 1.9 ps in Q A -reduced, membrane-bound wild type Rb.…”

“…First, a wavelength dependence of the kinetics of the bandshift of the B absorption band has been observed (11). An additional time component has been assigned in this spectral region, and also around 1020 nm (12), that was attributed to the formation of B L -as an intermediate in the electron transfer process (8,9). Furthermore, fluorescence emission from P* has been found to decay with multiple time constants that extend from a few to hundreds of picoseconds (7,(12)(13)(14)(15)(16)(17)(18)(19)(20).…”