No abstract

1. Chlorophyllase [EC 3.1.1.14] was extracted from the acetone-dried powder of the chloroplasts of greened rye seedlings with 1% cholate, and purified 870-fold with a yield of about 30%. The purification procedure was composed of fractionations with acetone and ammonium sulfate, and hydrophobic chromatography on a phenyl-Sepharose CL-4B column. 2. The purified enzyme was pure as analyzed by molecular-sieve chromatography and isoelectric electrophoresis. It had an isoelectric point of 4.5 and a molecular weight of 39,000. 3. The purified enzyme was stable at pH 6-9 and 4 degrees C. At pH 7.5, it was stable in the presence and absence of 30% acetone. However, at 30 degrees C, it was not stable above a 10% concentration of acetone. 4. The purified enzyme hydrolyzed chlorophylls a and b from spinach into chlorophyllides a and b and phytols, respectively; and bacteriochlorophyll a from Rhodospirillum rubrum into bacteriochlorophyllide a and a derivative of phytol, possibly all-trans-geranylgeraniol. The hydrolysis rates were stimulated to their maxima in the presence of 30% acetone; maximum stimulation was about 50% with bacteriochlorophyll a and about 400% with chlorophyll a. 5. At pH 7.5 and 30 degrees C in the presence of 30% acetone, the Km values and specific activities were 12 microM and 480 nmol . min-1 . mg-1 for chlorophylls a, and 4 microM and 170 nmol . min-1 . mg-1 for R. rubrum bacteriochlorophyll a, respectively.

The LH1 antenna complex and a native form of the LH2 complex were isolated from the carotenoidless R26 and R26.1 mutants of Rhodobacter sphaeroides by the use of a new detergent, sucrose monocholate. One-color, pump-and-probe transient Raman spectroscopy of these complexes using 351 nm, approximately 50 ps pulses showed the generation of the triplet state of bacteriochlorophyll a (BChl a), whereas measurements using 355 nm, approximately 12 ns pulses showed the generation of BChl a cation radical. Subpicosecond to nanosecond time-resolved absorption spectroscopy using 388 nm, 200 fs pulses for excitation showed rapid (<1 ps) generation of the triplet state and fast decay (<10 ps) of the singlet state of BChl a. Microsecond absorption spectroscopy confirmed the generation of BChl a cation radical. EPR spectroscopy using 532 nm, approximately 5 ns pulses for excitation established the generation of BChl a cation radical. The EPR line width suggested that the unpaired electron is shared by two BChl a molecules. In LH1, the yield of BChl a cation radical per complex was estimated to be about 80% of that in the reaction center, and in LH2 about 50%. Thus, rapid generation of the triplet state, and its subsequent transformation into the cation-radical state of BChl a have been shown to be intrinsic properties of B870 and B850 BChl a assembly in the carotenoidless LH1 and LH2 antenna complexes. In the case of the carotenoid-containing LH2 complex, the triplet states of BChl a and carotenoid (spheroidene) were generated immediately after excitation, but the triplet-state BChl a was quenched efficiently by the carotenoid so that no BChl a cation radical was generated. Thus, the photoprotective function of the carotenoid in this antenna complex is shown.

The frequency of the ring-breathing (C,-C, stretching) Raman line of bacteriochlorophyll a (BChl) in the T I state (abbreviated as ur") was determined in 16 different solvents. The u," frequencies were in the region of 1578-1581 cm-l in polar solvents forming the hexa-coordinated monomers, while they were in the region of 1585-1591 cm-1 in polar solvents forming the penta-coordinated monomers. The u," frequencies were 1597 and 1599 cm-i in methylene chloride and carbon tetrachloride forming penta-coordinated BChl aggregates. Possible mechanisms in which the Y," frequency reflects the states of intermolecular interactions are discussed.The Y," frequency of BChl that was bound to the light-harvesting complex (LHC) of Rhodobacter sphaeroides R26 was found to be 1598 cm-I , a result which suggests that a pair of BChl molecules form a dimer in the LHC in the T I state.