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

The phosphorescence of singlet oxygen ((1)X( *)) photosensitized by the carotenoidless reaction center (RC) of Rhodobacter sphaeroides R26.1 has been investigated, using H(2)O and D(2)O 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/tau)-exp(-t/zeta)], regardless of whether (1)X( *) is sensitized by P(dagger) or M(dagger) (where the dagger denotes triplet excitation and M is a water-soluble molecule). The time constant zeta was identified with the decay time of (1)X( *); when P(dagger) is the sensitizer, one finds zeta(P)((1))=3.3+/-0.3 micros, and zeta(P)((2))=34+/-3 micros, where the superscripts 1 and 2 refer to H(2)O and D(2)O, respectively; the corresponding values for sensitization by M(dagger) (in the absence of RC) are zeta(M)((1))=3.7+/-0.4 micros, and zeta(M)((2))=75+/-5 micros. The addition of RC's to the solution of M in D(2)O reveals that the RC is a quencher of (1)X( *); however, for equal concentrations of the RC, zeta(P)((2))<zeta(M)((2)), showing that (1)X( *) 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 tau(P) are similar in both solvents, ca. 2 micros, but this time constant does not figure in the disappearance of P(dagger), which follows a bi-exponential course, alpha(1)exp(-t/tau(1))+alpha(2)exp(-t/tau(2)). The time constants tau(1) and tau(2) (72+/-5 micros and 12+/-1 micros, respectively) as well as the factor alpha(2) are insensitive to the oxygen content, and quenching of P(dagger) is manifested only through a threefold reduction in the magnitude of alpha(1); these data imply the absence of dynamic quenching and heterogeneity of the RC. The mean lifetime of (1)X( *) inside the protein matrix is identified with tau(P), and the absence of a prompt component in the phosphorescence signal rationalized by proposing that the radiative decay of (1)X( *) within the RC is much slower than that in an aqueous environment.