pheophytin-a and bacteriopheophytin

The bacteriopheophytin a molecules at the H(A) and H(B) binding sites of reaction centers (RCs) of the Y(M210)W mutant of Rhodobacter sphaeroides were chemically exchanged with plant pheophytin a. The Y(M210)W mutation slows down the formation of H(A)(-), presumably by raising the free energy level of the P(+)B(A)(-) state above that of P* due to increasing the oxidation potential of the primary electron donor P and lowering the reduction potential of the accessory bacteriochlorophyll B(A). Exchange of the bacteriopheophytins with pheophytin a on the contrary lowers the redox potential of H(A), inhibiting its reduction. A combination of the mutation and pigment exchange was therefore expected to make the A-side of the RC incapable of electron transfer and cause the excited state P* to deactivate directly to the ground state or through the B-side, or both. Time-resolved absorption difference spectroscopy at 10 K on the RCs that were modified in this way showed a lifetime of P* lengthened to about 500 ps as compared to about 200 ps measured in the original Y(M210)W RCs. We show that the decay of P* in the pheophytin-exchanged preparations is accompanied by both return to the ground state and formation of a new charge-separated state, the absorption difference spectrum of which is characterized by bleachings at 811 and 890 nm. This latter state was formed with a time constant of ca. 1.7 ns and a yield of about 30%, and lasted a few nanoseconds. On the basis of spectroscopic observations these bands at 811 and 890 nm are tentatively attributed to the presence of the P(+)B(B)(-) state, where B(B) is the accessory bacteriochlorophyll in the "inactive" B-branch of the cofactors. The B(B) molecules in Y(M210)W RCs are suggested to be spectrally heterogeneous, absorbing in the Q(y) region at 813 or 806 nm. The results are discussed in terms of perturbation of the free energy level of the P(+)B(B)(-) state and absorption properties of the B(B) bacteriochlorophyll in the mutant RCs due to a long-range effect of the Y(M210)W mutation on the protein environment of the B(B) binding pocket.

Topics: Electron Transport; Light-Harvesting Protein Complexes; Mutation; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Plants; Rhodobacter sphaeroides; Spectrophotometry; Temperature; Time Factors

Recent studies of reaction centres from Rhodobacter sphaeroides (R-26), in which bacteriopheophytins a were replaced by plant pheophytins a, have shown that at low temperature the excited state of primary electron donor P* is converted to the state P+B-(A) (where B(A) is a bacteriochlorophyll a monomer in branch A) which has a long lifetime (about 600 ps [8]). This allows the direct measurement of the free energy difference between P* and P+B-(A) using the temperature dependence of the recombination fluorescence from P+B-(A). The data show that P+B-(A) is located below P* by 550+/-30 mV. Thus, the primary conversion of P* leads to the formation of P+B-(A) which is below P* in energy and is a real intermediate in electron transfer.

Topics: Kinetics; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Spectrometry, Fluorescence; Thermodynamics

Low temperature optical and photochemical properties of Rhodobacter sphaeroides (R-26) reaction centers, in which bacteriopheophytin a has been replaced by plant pheophytin a, are reported. Modified reaction centers preserve the ability for photoinduced electron transfer from the primary electron donor P to the primary quinone acceptor QA at 80K. The triplet state ESR signal of modified reaction centers with prereduced QA at 10K shows an electron spin polarization pattern and ZFS parameters analogous to those for the triplet state 3P in non-treated reaction centers. It was found that at low temperature both P+QA- and 3P states are formed via a precursor radical pair P+I- in which I is the introduced plant pheophytin molecule. This shows that acceptor systems of bacterial and plant (photosystem II) reaction centers are mutually replacable in structural and functional aspects.

Topics: Circular Dichroism; Electron Spin Resonance Spectroscopy; Electron Transport; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Plants; Rhodobacter sphaeroides; Spectrophotometry

The major part (> 90%) of bacteriopheophytin a in reaction centers (RCs) of Rhodobacter sphaeroides was substituted by plant pheophytin a. In modified RCs the photochemical formation of P+Qa- occurs with with a quantum efficiency of 79%. The intermediary state P+I- displayed a recombination time constant of 1.5 ns, and the electron transfer from I- to Qa was characterized by a time constant of 540 ps. On the basis of spectral properties of P+I- for native and modified RCs, it was suggested that bacteriopheophytin, as well as bacteriochlorophyll monomers located in L protein branch, have a transition at 545 nm with approx. equal extinction coefficients. Accordingly, the state P+I- in modified RCs is proposed to consist of a thermodynamic mixture of P+BL- (approximately 80%) and P+Phe- (approximately 20%).

Topics: Bacteriochlorophylls; Electron Transport; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Plant Proteins; Rhodobacter sphaeroides; Spectrophotometry; Time Factors