Analysis of the temperature-dependence of P(+)HA(-) charge recombination in the Rhodobacter sphaeroides reaction center suggests nanosecond temperature-independent protein relaxation.

The temperature dependence of charge recombination of the pair P(+)HA(-) in isolated reaction centers from the purple bacterium Rhodobacter sphaeroides with prereduced quinone QA was studied by sub-nanosecond to microsecond time-scale transient absorption. Overall, the kinetics slowed down substantially upon cooling from room temperature to ∼200 K, and then remained virtually unchanged down to 77 K, indicating the coexistence of two competitive pathways of charge recombination, a thermally-activated pathway appearing only above ~200 K and a temperature-independent pathway. In our modelling, the thermally activated pathway includes an uphill electron transfer from HA(-) to BA(-) leading to transient formation of the state P(+)BA(-), whereas the temperature-independent pathway is due to direct downhill electron transfer from HA(-) to P(+). At all temperatures studied, the kinetics could be approximated by a four-component decay. Detailed analysis of the lifetimes and amplitudes of particular phases over the range of temperatures suggests that the kinetically resolved phases reveal the consecutive appearance of three conformational states characterized by an increasing free energy gap between the states P(+)BA(-) and P(+)HA(-). The initial gap between these states was estimated to be only ~8 meV, the intermediate gap being ~92 meV, and the final gap ~135 meV, with no dependence on temperature. It was also calculated through a very straightforward approach that the relaxation process from the initial to the intermediate state occurs within 0.6 ± 0.1 ns, whereas the second step of relaxation from the intermediate to the final state takes 11 ± 2 ns. Both phases of the protein relaxation process are essentially temperature-independent. Possible alternative models to describe the experimental data that cannot be definitely excluded are also discussed.