ENERGETICS AND KINETICS OF RADICAL PAIRS IN REACTION CENTERS FROM RHODOBACTER-SPHAEROIDES - A FEMTOSECOND TRANSIENT ABSORPTION STUDY

Femtosecond transient absorption spectra on reaction centers from Rhod obacter sphaeroides wild type have been recorded with high time and wa velength resolution and a very high S/N ratio in the 500-940 nm range with a diode array system. The data have been analyzed by global analy sis. Five lifetime components of 1.5, 3.1, 10.8, and 148 ps and long-l ived (several nanoseconds) were required to fit the entire three-dimen sional data surface adequately with a single set of lifetimes and deca y-associated difference spectra(DADS). Up to 30 ps. there is little di spersion in the lifetimes, but in the longer time range (50-250 ps), a substantial variation in lifetime was observed? depending on detectio n wavelength. The data from the global analysis have been subjected to kinetic modeling comparing sequential kinetic schemes either includin g (reversible model) or excluding (forward model) back-reactions in th e early electron transfer process(es). Thus, the molecular rate consta nts for the model(s) and the difference spectra of the pure intermedia tes [species-associated difference spectra (SADS)]were obtained. The d ata unequivocally confirm the necessity of an electron transfer interm ediate with spectral characteristics of P+B-H prior to the formation o f the P+BH- state (P is special pair, B is accessory chlorophyll, and H is pheophytin), irrespective of the model chosen. Besides being in m uch better agreement with the observation of long-lived fluorescence k inetics components, the reversible model results in SADS, in particula r for the P+BH- state, that are in somewhat better agreement with expe ctations than for the pure forward model. For these and other reasons, the reversible model is preferred over the pure forward model. The el ectrochromic shifts of the H bands in the P+B- state and of the B band s in the P+H- state are revealed clearly in the spectra, thus supporti ng the assignments. Within the reversible model, the rate constant for the forward reaction in the first step P double dagger double right a rrow P+B-H is slightly larger [k(12) approximate to (2.48 ps)(-1)] tha n for the second step P+B-H double right arrow P+BH- [k(23) approximat e to (2.53 ps)(-1)], in contrast to the pure forward model. From the r ate constants for the respective back-reactions, the free energy diffe rences Delta G relative to P far the states P+B-H and P+BH- have been determined to be -41 and -91 meV, respectively. Thus, the free energy difference for the P+BH- state at early times after electron transfer is by a factor of 2-3 smaller than assumed so far. This has the impor tant consequence that a quasi-equilibrium exists from about 10 ps unti l further electron transfer on the 200 ps time scale with a substantia l percentage (approximate to 16%) of the P+B-H state present. These re sults present the first direct evidence from transient absorption data , where the nature of the intermediate can be assigned, for the validi ty of the slow radical pair relaxation concept. The results have vario us consequences for understanding the mechanism of the overall electro n transfer reaction and imply a much more active role of the protein i n the early charge separation processes of the reaction center than as sumed so far. The data art discussed in terms of current electron tran sfer theory. It is suggested that the two first-electron steps operate at a rate very close to the maximal possible rate.