Triplet formation on a monomeric chlorophyll in the photosystem II reaction center as studied by time-resolved infrared spectroscopy.

The process of formation of the triplet state of chlorophyll in the photosystem II (PS II) reaction center complex was studied by means of time-resolved infrared (IR) spectroscopy. Using a dispersive-type IR spectrometer with a time resolution of approximately 55 ns, transient spectra in the C=O stretching region (1760--1600 cm(-1)) were measured at 77 K. The data were analyzed by singular-value decomposition and subsequent least-squares fitting. Two distinct spectral components having different kinetic behaviors were resolved. One had spectral features characterized by negative peaks at 1740 and 1680 cm(-1) and an overall positive background and was assigned to the P(680)(+)Phe(-)/P(680)Phe radical pair by static FTIR measurements of the P(680)(+)/P(680) and Phe(-)/Phe differences. The other had prominent negative and positive peaks at 1668 and 1628 cm(-1), respectively, which were previously assigned to the keto C==O change upon triplet formation of the monomeric chlorophyll denoted as Chl(T) [Noguchi, T., Tomo, T., and Inoue, Y. (1998) Biochemistry 37, 13614-13625]. The former component of P(680)(+)Phe(-)/P(680)Phe exhibited a multiphasic decay with time constants of 77 ns (75%), 640 ns (18%), 8.3 micros (4%), and 0.3 ms (3%), while the latter component of (3)Chl(T)/Chl(T) was formed with a single-exponential rise with a time constant of 57 ns and had a lifetime of 1.5 ms. From the observations that only the two spectral components were resolved without any other triplet intermediates and the time constant of (3)Chl(T) formation roughly agreed with or seemed even faster than that of the major phase of the P(680)(+)Phe(-) decay, two alternative mechanisms of triplet formation are proposed. (i) (3)Chl(T) is directly formed from P(680)(+)Phe(-) by charge recombination at Chl(T), and (ii) (3)P(680) is formed, and then the triplet is transferred to Chl(T) with a time constant of much less than 50 ns. The location of Chl(T) in the D1 subunit as the monomer chlorophyll corresponding to the accessory bacteriochlorophyll in the L subunit of purple bacteria is favored to explain the former mechanism as well as the triplet properties reported in the literature. The physiological role of the triplet formation on Chl(T) is also discussed.