Formation and geminate quenching of singlet oxygen in purple bacterial reaction center

Abstract

The phosphorescence of singlet oxygen ( 1X ∗) photosensitized by the carotenoidless reaction center (RC) of Rhodobacter sphaeroides R26.1 has been investigated, using H 2O and D 2O as the suspending media. To enhance (under neutral conditions) the triplet quantum yield of the special pair P 870 (P) by the radical pair mechanism, the quinone acceptor Q A was removed by means of a chemical treatment. The phosphorescence signal fits the functional form P 0[exp (− t/ τ) − exp(− t/ ζ)], regardless of whether 1X ∗ is sensitized by P † or M † (where the dagger denotes triplet excitation and M is a water-soluble molecule). The time constant ζ was identified with the decay time of 1X ∗; when P † is the sensitizer, one finds ζ P ( 1 ) = 3.3 ± 0.3 μ s , and ζ P ( 2 ) = 34 ± 3 μ s , where the superscripts 1 and 2 refer to H 2O and D 2O, respectively; the corresponding values for sensitization by M † (in the absence of RC) are ζ M ( 1 ) = 3.7 ± 0.4 μ s , and ζ M ( 2 ) = 75 ± 5 μ s . The addition of RC’s to the solution of M in D 2O reveals that the RC is a quencher of 1X ∗; however, for equal concentrations of the RC, ζ P ( 2 ) < ζ M ( 2 ) , showing that 1X ∗ is deactivated, after its entry into the suspending medium, mainly by the solvent or the same RC which acts as the sensitizer. The values of τ P are similar in both solvents, ca. 2 μs, but this time constant does not figure in the disappearance of P †, which follows a bi-exponential course, α 1exp(− t/ τ 1) + α 2exp(− t/ τ 2). The time constants τ 1 and τ 2 (72 ± 5 μs and 12 ± 1 μs, respectively) as well as the factor α 2 are insensitive to the oxygen content, and quenching of P † is manifested only through a threefold reduction in the magnitude of α 1; these data imply the absence of dynamic quenching and heterogeneity of the RC. The mean lifetime of 1X ∗ inside the protein matrix is identified with τ P, and the absence of a prompt component in the phosphorescence signal rationalized by proposing that the radiative decay of 1X ∗ within the RC is much slower than that in an aqueous environment.