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.

The primary light-induced charge separation in reaction Centers of Rkfobacter sphueroides was investigated with femtosecond time resolution. The absorption changes in the time range 100 fs to 1 ns observed after direct excitation of the primary donor P at 860 mn could only be explained by a kinetic model which uses three time constants. This fmding supports the following reaction scheme: (i) the electronically excited primary donor P* decays with a time constant of 3.5 ps and populates a very short-lived intermediate involving a reduced accessory bacteriochlorophyll molecule; (ii) with a time constant of 0.9 ps the electron is transferred to the neighboring bacteriopheophytin molecule; and (iii) from there within 200 ps to the quinone.

ABSTRACT:Femtosecond spectroscopy was used in combination with site-directed mutagenesis to study the influence of tyrosine M210 (YM210) on the primary electron transfer in the reaction center of Rhodobacter sphaeroides. The exchange of YM210 to phenylalanine caused the time constant of primary electron transfer to increase from 3.5 f 0.4 ps to 16 f 6 ps while the exchange to leucine increased the time constant even more to 22 f 8 ps. The results suggest that tyrosine M210 is important for the fast rate of the primary electron transfer.x e primary photochemical event during photosynthesis of bacteriochlorophyll-(Bchl-) containing organisms is a lightinduced charge separation within a transmembrane protein complex called the reaction center (RC). The crystal structures of RC's from Rhodopseudomonas (Rps.) viridis and Rhodobacter (Rb.) sphaeroides have been solved to high resolution [reviewed in Deisenhofer and Michel (1989), Chang et al. (1986), Tiede et al. (1988), andRees et al. (1989)l. The RC from Rb. sphaeroides contains three protein subunits referred to as L, M, and H, according to their respective mobilities in SDS-polyacrylamide gels. Associated with the L and M subunits are the cofactors, consisting of four Bchl a, two bacteriopheophytin (Bph) a, one atom of non-heme ferrous iron, two quinones (QA and Qe), and in some species one carotenoid [reviewed in Parson (1987) and Feher et al.' Financial support was from the Deutsche Forschungsgemeinschaft,

The resonance-enhanced multiphoton ionization (REMPI) of the OH radical in the gas phase was studied using an isothermal discharge-flow reactor for the production of OH radicals (H+NO,+OH+NO), tunable laser light from an excimer-pumpeddye laser, and a time-of-flight (TOF) mass spectrometer. A mass-resolved REMPI spectrum was found in the wavelength region of 290-3 10 nm, which is assigned to a (3 + I ) ionization process. An ab initio quantum-chemical calculation predicts the Rydberg state, verified by the experiment.

The primary photosynthetic reactions in whole membranes of the antenna-deficient mutant strain U43 (pTXA6-10) of Rhodobacter capsulatus are studied by transient absorption and emission spectroscopy with subpicosecond time resolution. Extensive similarities between the transient absorption data on whole membranes and on isolated reaction centers support the idea that the primary processes in isolated reaction centers are not modified by the isolation procedure.