Nanosecond fluorescence from isolated photosynthetic reaction centers of Rhodopseudomonas sphaeroides

The time-course of fluorescence from reaction centers isolated from Rhodopseudomonas sphaeroides was measured using single-photon counting techniques. When electron transfer is blocked by the reduction of the electron-accepting quinones, reaction centers exhibit a relatively long-lived (delayed) fluorescence due to back reactions that regenerate the excited state (P*) from the transient radical-pair state, P^F. The delayed fluorescence can be resolved into three components, with lifetimes of 0.7, 3.2 and 11 ns at 295 K. The slowest component decays with the same time-constant as the absorbance changes due to P^F, and it depends on both temperature and magnetic fields in the same way that the absorbance changes do. The time-constants for the two faster components of delayed fluorescence are essentially independent of temperature and magnetic fields. The fluorescence also includes a very fast (prompt) component that is similar in amplitude to that obtained from unreduced reaction centers. The prompt fluorescence presumably is emitted mainly during the period before the initial charge-transfer reaction creates P^F from P*. From the amplitudes of the prompt and delayed fluorescence, we calculate an initial standard free-energy difference between P* and P^F of about 0.16 eV at 295 K, and 0.05 eV at 80 K, depending somewhat on the properties of the solvent. The multiphasic decay of the delayed fluorescence is interpreted in terms of relaxations in the free energy of P^F with time, totalling about 0.05 eV at 295 K, possibly resulting from nuclear movements in the electron-carriers or the protein.