essential that in vitro esr observations be interpreted in terms of the chlorophyll species actually present. Chlorophyll is able to act both as electron donor and electron acceptor in chargetransfer complexes. The central Mg atom of chlorophyll is coordinatively unsaturated when it has the coordination number 4, and at least one of the Mg axial positions must always be occupied by an electron donor group. In the absence of other nucleophiles, the ketone C-O function in Ring V of one chlorophyll molecule serves as donor to the Mg atom of another, forming chlorophyll dimers, (Chl2), or oligomers, (ChlW)3. Extraneous nucleophiles (bases) can compete for the coordination site at Mg, with disruption of the chlorophyllchlorophyll interactions, to form chlorophyll-ligand adducts, (Chl-L). The nature of the nucleophile determines whether the chlorophyll-nucleophile adduct is monomeric or polymeric. Bifunctional ligands, such as dioxane, pyrazine, 1, diazobicyclo(2.2.2.)octane, and, in particular, water can cross-link chlorophyll molecules or chlorophyll dimers by coordination to Mg to form large (chlorophyll-nucleophile) micelles (to be published). The chlorophyll species present in a particular experiment are very sensitive to temperature, adventitious nucleophiles such as water, and solvent, factors not always taken into account in previous work. EXPERIMENTAL METHODSChlorophyll samples were dried, prior to solution preparation, by codistillation with CCL (36). The solvents used for in vitro measurements were first dried over Linde 3A molecular sieve and degassed under reduced pressure. Solutions were prepared and oxidant was added in a nitrogen-filled dry box or on the vacuum line.Irradiations were performed with a 150-W Varian Eimac lamp. Infrared components were removed by 2 cm of water and 2 dichroic infrared-rejecting filters. All irradiations were by red light with a Corning 2404 sharp cut-off filter. All in vitro measurements were made in 4-mm quartz esr tubes at -170'C; in vivo measurements were made at room temperature on concentrated slurries of cells held in a Varian water cell (V4548).625

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In vitro and in vivo triplet state electron paramapnetic resonance (epr) spectra of bacteriochlorophylls (Bchls) show important differences in (a) electron spin polarization (esp), and (b) zero field splitting (ZFS) parameters. The unusual esp and ZFS properties of the observed in vivo triplet state are best interpreted as arising from a short-lived radical pair precursor.(not directly observable by epr) formed in or with the special pair of bacteriochlorophyll molecules involved in the primary photo-act.The successful observations of electron paramagnetic resonance (epr) triplet signals in photosynthetic bacteria by Dutton, Leigh, and coworkers (1-4) have revived interest in speculations advanced many years ago [e.g., Franck et al. (5)] that chlorophyll (Chl) triplet states may be involved in the primary events of photosynthesis. The magnetic properties of the lowest excited triplet state of all of the important chlorophylls have now been characterized in vitro (6-12), which makes it possible to compare in vivo and in vitro triplet spectra. In this communication we show that the bacteriochlorophyll (Bchl) special pair (BB)t previously postulated to participate in the primary light conversion step (13,14) provides an eminently suitable framework for the interpretation of the in vivo triplet state electron spin polarization (esp) and zero field splitting (ZFS). We propose that if normal chemistry of photosynthesis is blocked, the radical pair state formed in the special pair in the primary photo-act decays to a triplet state (observable by epr) whose esp reflects the unusual spin population of the radical pair intermediate. METHODSThe methods we used for observing the triplets (11,12) and can be related to those at zero field by mixing coefficients that depend on the strength and direction of the magnetic field. The selective population and depopulation of the triplet spin sublevels results in a non-Boltzmann distribution of spin populations in the triplet manifold. This manifests itself in triplet spectra that differ from the normal intensity pattern in that some of the transitions show enhanced absorption (a) while the others are in emission (e), i.e., show esp.

The visible absorption spectra of chlorophyll a monomer, dimer, and oligomers in solution have been analyzed with respect to peak positions, extinction coefficients, oscillator strengths, and dipole strengths. Exciton theory has been used to relate features in the dimer and oligomer spectra to features in the monomer spectrum. Conversion from penta-to hexacoordination at Mg strongly red shifts the Qx transition so that Qx(O,O) appears between Qy(O,O) and QJ(O,l). The relative bulk solvent environmental shifts of the Qy transition in carbon tetrachloride and n-octane have been computed. Because chlorophyll a oligomers have absorption spectra strikingly similar to antenna chlorophyll in green plants, oligomeric chlorophyll a is proposed as a model for antenna chlorophyll.

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