Transient absorption spectroscopy has been used to study sub-picosecond energy transfer processes in isolated photosystem II (PS II) reaction centers. As reported previously [Durrant, J. R.; et al. Proc. Natl. Acad. Sci. U.S.A. 1992 , 89, 11632−11636], using long wavelength (694 nm) excitation, spectral evolution of the isotropic Q y band bleach/stimulated emission is dominated by energy transfer processes with a 100 ± 50 fs time constant. In contrast, depolarization of this signal occurs with a time constant of 400 ± 30 fs, from an initial anisotropy of ∼0.4 to a value of ∼0.15 at 1.5 ps. This decay of the anisotropy is attributed to energy transfer between at least two degenerate states contributing to reaction center absorption circa 680 nm, with these states having approximately orthogonal transition dipoles. The transient anisotropy barely changes between 1.5 and 60 ps, indicating that under these excitation conditions equilibration of the excitation energy between reaction center excited states occurs on a sub-picosecond time scale. Transient data collected for pheophytin Q x absorption bands indicate that pheophytin molecules are included in the 100 fs equilibration process. These results are discussed in the context of the PS II multimer model and are shown to be in good agreement with this model.

Pigment-protein interactions play a significant role in determining the properties of photosynthetic complexes. Site-directed mutants of Synechocystis PCC 6803 have been prepared which modify the redox potential of the primary radical pair anion and cation. In one set of mutants, the environment of P680, the primary electron donor of Photosystem II, has been modified by altering the residue at D1-His198. It has been proposed that this residue is an axial ligand to the magnesium cation. In the other set, the D1-Gln130 residue, which is thought to interact with the C9-keto group of the pheophytin electron acceptor, has been changed. The effect of these mutations is to alter the free energy of the primary radical pair state, which causes a change in the equilibrium between excited singlet states and radical pair states. We show that the free energy of the primary radical pair can be increased or decreased by modifications at either the D1-His198 or the D1-Gln130 sites. This is demonstrated by using three independent measures of quantum yield and equilibrium constant, which exhibit a quantitative correlation. These data also indicate the presence of a fast nonradiative decay pathway that competes with primary charge separation. These results emphasize the sensitivity of the primary processes of PS II to small changes in the free energy of the primary radical pair.

We compare primary charge separation in a photosystem II reaction center preparation isolated from a wildtype (WT) control strain of the cyanobacterium Synechocystis sp. PCC 6803 and from two site-directed mutants of Synechocystis in which residue 130 of the D1 polypeptide has been changed from a glutamine to either a glutamate (mutant D1-Gln130Glu), as in higher plant sequences, or a leucine residue (mutant D1-Gln130Leu). The D1-130 residue is thought to be close to the pheophytin electron acceptor. We show that, when P680 is photoselectively excited, the primary radical pair state P680 ؉ Ph؊ is formed with a time constant of 20 -30 ps in the WT and both mutants; this time constant is very similar to that observed in Pisum sativum (a higher plant). We also show that a change in the residue at position D1-130 causes a shift in the peak of the pheophytin Q x -band. Nanosecond and picosecond transient absorption measurements indicate that the quantum yield of radical pair formation ( RP ), associated with the 20 -30-ps component, is affected by the identity of the D1-130 residue. We find that, for the isolated photosystem II reaction center particle, RP higher plant > RP D1-Gln130Glu mutant > RP WT > RP D1-Gln130Leu mutant . Furthermore, the spectroscopic and quantum yield differences we observe between the WT Synechocystis and higher plant photosystem II, seem to be reversed by mutating the D1-130 ligand so that it is the same as in higher plants. This result is consistent with the previously observed natural regulation of quantum yield in Synechococcus PS II by particular changes in the D1 polypeptide amino acid sequence (Clark, A. K., Hurry, V. M., Gustafsson, P. and Oquist, G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11985-11989). Photosystem II (PS II)1 is unique in that it is the only complex of photosynthetic organisms that is able to catalyze the oxidation of water. Upon light absorption, primary charge separation results in the oxidation of P680, the primary electron donor of PS II, and the reduction of pheophytin (Ph). It is the high oxidizing potential of P680 ϩ that drives the secondary electron donor-side reactions of tyrosine and manganese oxidation and that leads ultimately to the splitting of water and release of molecular oxygen.All oxygenic photosynthetic organisms contain PS II; these include higher plants, algae, and cyanobacteria. The most commonly isolated reaction center complex from PS II (the D1/D2 cytochrome b 559 complex) binds six chlorophylls, two pheophytins, two ␤-carotenes, and one cytochrome b 559 (1, 2). Since this PS II reaction center lacks the secondary acceptors, Q A and Q B , and has little effective tyrosine Z activity (3), its photochemistry is limited to the formation of the primary radical pair state P680 ϩ Ph Ϫ and charge recombination pathways from this state (for review, see Ref. 4). The isolated reaction center is, however, an ideal system for spectroscopic studies of PS II primary photochemistry since the complications usually associated with energy transfer from a...

1. The mechanism of rabbit muscle phosphofructokinase was investigated by measurement of fluxes, isotope trapping and steady-state velocities at pH8 in triethanolamine/HCl buffer with 4mM free Mg2+. Most observations were made at I0.2. 2. The ratio Flux of fructose 1,6-bisphosphate--fructose 6-phosphate/Flux of fructose 1,6-bisphosphate-+ATP at zero ATP concentration increased hyperbolically from unity to about 3.2 as the concentration of fructose 6-phosphate was increased.Similarly, the ratio Flux of fructose 1,6-bisphosphate-*ATP/Flux of fructose 1,6-bisphosphate-fructose 6-phosphate at zero fructose 6-phosphate concentration increased from unity to about 1.4 as the concentration of ATP was increased. The addition of substrates must therefore be random, whatever the other aspects of the reaction. Further, from the plateau values of the ratios, it follows that the substrates dissociate very infrequently from the ternary complex and that at a low substrate concentration 72% of the reaction follows the pathway in which ATP adds first to the enzyme. 3. Isotope-trapping studies with [32P]ATP confirmed that ATP can bind first to the enzyme in rate-limiting step and that dissociation of ATP from the ternary complex is slow in relation to the forward reaction. No isotope trapping of [U-14C]-fructose 6-phosphate could be demonstrated. 4. The ratios Flux of ATP-+fructose 1,6-bisphosphate/Flux of ATP-ADP measured at zero ADP concentration and the reciprocal of the ratio measured at zero fructose 1,6-bisphosphate concentration did not differ significantly from unity. Calculated values for these ratios based on the kinetics of the reverse reaction and assuming ordered dissociations of products or a ping-pong mechanism gave values very significantly greater than unity. These findings exclude an ordered dissociation or a substantial contribution from a pingpong mechanism, and it is concluded that the reaction is sequential and that dissociation of products is random. 5. Rate constants were calculated for the steps in the enzyme reaction. The results indicate a considerable degree of co-operativity in the binding between the two substrates. 6. The observations on phosphofructokinase are discussed in relation to methods of measurement and interpretation of flux ratios and in relation to the mechansim of other kinase enzymes.Phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) catalyses the transfer of the terminal phosphate of ATP to the C-1 hydroxy group of Fru6P to produce Fru(1,6)P2 and ADP. It is an important glycolytic enzyme, particularly because it is subject to metabolic control and its velocity is modified by a Abbreviations used: Fru6P, fructose 6-phosphate; Fru(1,6)P2, fructose 1,6-bisphosphate.

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