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The photophysical behavior of several porphyrin compounds were studied to determine the sequential charge transfer processes of molecules with simple organic building blocks. Excitation at 308 nm produced an acceptor moiety with a characteristic charge-transfer emission instead of fluorescence. The porphyrin compounds exhibited variations in their fluorescence with different quantum yield values and decay times. Estimations of the energetics of charge transfer are presented.

On the basis of sequence comparison with the M subunit of the reaction center of purple bacteria, no residues in photosystem II can be clearly identified that may be predicted to correspond to the His residue that binds one of the accessory bacteriochlorophylls in the purple bacterial reaction center. However, the Arg180 residue of the D2 protein is close to where this residue is predicted to be and could conceivably serve as a chlorophyll ligand. To analyze the function of Arg180, it was changed to nine different amino acids in the cyanobacterium Synechocystis sp. PCC 6803. Except for the Arg180-->Gln (R180Q) mutant, the resulting strains were no longer photoautotrophic. The properties of photosystem II upon mutation of Arg180 were probed in strains from which photosystem I had been deleted genetically. Mutations at the Arg180 residue affected oxygen evolution capacity and the amount of photosystem II that was present in thylakoids. Surprisingly, in the Arg180 mutants, EPR signals that may originate from the oxidized redoxactive Tyr160 of the D2 protein (Y(D)ox) were small and generally did not resemble the usual signal IIs, signifying an effect of the Arg180 mutations on the environment surrounding Tyr160. In addition, in most mutants, the charge recombination kinetics between the primary electron-accepting quinone in photosystem II (Q(A)-) and oxidized species on the donor side were faster upon introducing mutations at Arg180 suggesting an increased steady-state concentration of P680+ in the mutants. However, Arg180 mutations also affected Q(A)- oxidation by the secondary electron-accepting quinone (Q(B)). HPLC analysis showed that, in the Arg180 mutants that were assayed, the pheophytin/chlorophyll ratio of photosystem II had not changed, indicating that the mutations did not lead to a pheophytinization of one of the chlorophyll molecules. Even though the results presented do not provide positive evidence that Arg180 of the D2 protein corresponds in function to the ligand to the central Mg in an accessory bacteriochlorophyll in reaction centers of purple bacteria, it is clear that changes in Arg180 greatly affect Tyr160 and P680. Various scenarios are discussed that are compatible with the data presented, and include an apparently close interaction between Arg180, His189, and Tyr160, and the possibility of the involvement of multiple chlorophylls to together form P680.

In photosystem II of green plants the key photosynthetic reaction consists of the transfer of an electron from the primary donor called P680 to a nearby pheophytin molecule. We analyzed the temperature dependence of this reaction by subpicosecond transient absorption spectroscopy over the temperature range 20-240 K using isolated photosystem II reaction centers from spinach. After excitation in the red edge of the Qy absorption band, the decay of the excited state can conveniently be described by two kinetic components that both accelerate with temperature. This temperature behavior differs remarkably from that observed in purple bacterial reaction centers. We attribute the first component, which accelerates from 2.6 ps at 20 K to 0.4 ps at 240 K, to charge separation after direct excitation of P680, and explain its temperature dependence by an intermediate that lies in energy above the singlet-excited P680 and that possibly has charge-transfer character. The second component accelerates from 120 ps at 20 K to 18 ps at 240 K and is attributed to charge separation after direct excitation of the "trap" state near-degenerate with P680 and subsequent slow energy transfer from this trap state to P680. We suggest that the slow energy transfer from the trap state to P680 plays an important role in the kinetics of radical pair formation at room temperature.

The pigment composition of the isolated photosystem II reaction center complex in its most stable and pure form currently is a matter of considerable debate. In this contribution, we present a new method based on a combination of gel filtration chromatography and diode array detection to analyze the composition of photosystem II reaction center preparations. We show that the method is very sensitive for the detection of contaminants such as the core antenna protein CP47, pigment-free and denatured reaction center proteins, and unbound chlorophyll and pheophytin molecules. We also present a method by which the photosystem II reaction center complex is highly purified without using Triton X-100, and we show that in this preparation the contamination with CP47 is less than 0.1%. The results strongly indicate that the photosystem II reaction center complex in its most stable and pure form binds six chlorophyll a, two pheophytin a, and two beta-carotene molecules and that the main effect of Triton X-100 is the extraction of beta-carotene from the complex. Analysis of 4 K absorption and emission spectra indicates that the spectroscopic properties of this preparation are similar to those obtained by a short Triton X-100 treatment. In contrast, preparations obtained by long Triton X-100 treatment show decreased absorption of the shoulder at 684 nm in the 4 K absorption spectrum and an increased number of pigments that trap excitation energy at very low temperatures. We conclude that the 684 nm shoulder in the 4 K absorption spectrum should at least in part be attributed to the primary electron donor of photosystem II.