Dewey Holten

Primary photochemistry of reaction centers from the photosynthetic purple bacteria

Photosynthetic organisms transform the energy of sunlight into chemical potential in a specialized membrane-bound pigment-protein complex called the reaction center. Following light activation, the reaction center produces a charge-separated state consisting of an oxidized electron donor molecule and a reduced electron acceptor molecule. This primary photochemical process, which occurs via a series of rapid electron transfer steps, is complete within a nanosecond of photon absorption. Recent structural data on reaction centers of photosynthetic bacteria, combined with results from a large variety of photochemical measurements have expanded our understanding of how efficient charge separation occurs in the reaction center, and have changed many of the outstanding questions.

Temperature and detection-wavelength dependence of the picosecond electron-transfer kinetics measured in Rhodopseudomonas sphaeroides reaction centers. Resolution of new spectral and kinetic components in the primary charge-separation process

Abstract We have examined the temperature dependence of the rate of electron transfer to ubiquinone from the bacteriopheophytin (BPh) that serves as an initial electron acceptor (I) in reaction centers of Rhodopseudomonas sphaeroides . The kinetics were measured from the decay of the 665-nm absorption band of the reduced BPh (BPh − or I − ) and from the recovery of the BPh band at 545 nm, following excitation of reaction centers in polyvinyl alcohol films with 30-ps flashes. The measured time constant decreases from 229 ± 25 ps at 295 K to 97 ± 8 ps near 100 K and then remains constant down to 5 K. The temperature dependence of the kinetics can be rationalized on the assumption that the reaction results in changes in the frequencies of numerous low-energy nuclear (vibrational) modes of the electron carriers and/or the protein. The kinetics measured in the absorption bands near 765 and 795 nm show essentially the same temperature dependence as those measured at 545 or 665 nm, but the time constants vary with detection wavelength. The time constant measured in the 795-nm region (70 ± 10 ps at 5 and 76 K) is shorter than that seen in the absorption bands of the BPh; the time constant measured at 758 nm is longer. Time constants measured with reaction centers in solution at 288 K also vary with the detection wavelength. These results can be explained on the assumption that the absorption changes measured at some wavelengths reflect nuclear relaxations rather than electron transfer. The absorption changes at 795 nm probably reflect a relaxation of the bacteriochlorophyll molecules that are near neighbors of the BPh and the primary electron donor (P). Those near 530 and 755 nm probably are due to the second BPh molecule, which does not appear to undergo oxidation or reduction.

Primary photochemical processes in isolated reaction centers of Rhodopseudomonas viridis

Picosecond and nanosecond spectroscopic techniques have been used to study the primary electron transfer processes in reaction centers isolated from the photosynthetic bacterium Rhodopseudomonas viridis. Following flash excitation, the first excited singlet state (P*) of the bacteriochlorophyll complex (P) transfers an electron to an intermediate acceptor (I) in less than 20 ps. The radical pair state P+I-) subsequently transfers an electron to another acceptor (X) in about 230 ps. There is an additional step of unknown significance exhibiting 35 ps kinetics. P+ subsequently extracts an electron from a cytochrome, with a time constant of about 270 ns. At low redox potential (X reduced before the flash), the state P+I- (or PF) lives approx. 15 ns. It decays, in part, into a longer lived state (PR), which appears to be a triplet state. State PR decays with an exponential time of approx. 55 microseconds. After continuous illumination at low redox potential (I and X both reduced), excitation with an 8-ps flash produces absorption changes reflecting the formation of the first excited singlet state, P*. Most of P* then decays with a time constant of 20 ps. The spectra of the absorbance changes associated with the conversion of P to P* or P+ support the view that P involves two or more interacting bacteriochlorophylls. The absorbance changes associated with the reduction of I to I- suggest that I is a bacteriopheophytin interacting strongly with one or more bacteriochlorophylls in the reaction center.

Charge separation in a reaction center incorporating bacteriochlorophyll for photoactive bacteriopheophytin

Site-directed mutagenic replacement of M subunit Leu214 by His in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides results in incorporation of a bacteriochlorophyll molecule (BChl) in place of the native bacteriopheophytin (BPh) electron acceptor. Evidence supporting this conclusion includes the ground-state absorption spectrum of the (M)L214H mutant, pigment and metal analyses, and time-resolved optical experiments. The genetically modified RC supports transmembrane charge separation from the photoexcited BChl dimer to the primary quinone through the new BChl molecule, but with a reduced quantum yield of 60 percent (compared to 100 percent in wild-type RCs). These results have important implications for the mechanism of charge separation in the RC, and rationalize the choice of (bacterio)pheophytins as electron acceptors in a variety of photosynthetic systems.

Picosecond-photodichroism studies of the transient states in Rhodopseudomonas sphaeroides reaction centers at 5 K. Effects of electron transfer on the six bacteriochlorin pigments

Abstract We have examined the dichroism of the visible and near-infrared absorption changes due to the early transient states in Rhodopseudomonas sphaeroides reaction centers imbedded in polyvinyl alcohol films at 5 K. The transient-state, ground-state and derivative spectra acquired under these conditions are highly resolved. Spectral features have been assigned to the bacteriochlorophyll (BChl) dimer (P) that serves as the primary electron donor, to each of the two additional BChls, and to the two bacteriopheophytin (BPh) molecules. The dichroism of the absorption changes, taken together with earlier results including our observation of a detection-wavelength dependence of the kinetics, argues that only one of the BPhs is a clearly resolved electron carrier prior to ubiquinone. The second BPh and the two BChls not constituting P display electrochromic effects and/or nuclear relaxations, possibly involving the protein, in response to the charge-separation process.

Subpicosecond and picosecond studies of electron transfer intermediates in Rhodopseudomonas sphaeroides reaction centers

The primary electron transfer processes in isolated reaction centers of Rhodopseudomonas sphaeroides have been investigated with subpicosecond and picosecond spectroscopic techniques. Spectra and kinetics of the absorbance changes following excitation with 0.7-ps 610-nm pulses, absorbed predominantly by bacteriochlorophyll (BChl), indicate that the radical pair state P+BPh-, in which an electron has been transferred from the BChl dimer (P) to a bacteriopheophytin (BPh), is formed with a time constant no greater than 4 ps. The initial absorbance changes also reveal an earlier state, which could be an excited singlet state, or a P+BChl- radical pair. The bleaching at 870 nm produced by 7 ps excitation at 530 nm (absorbed by BPh) or at 600 nm (absorbed predominantly by BChl) shows no resolvable delay with respect to standard compounds in solution, suggesting that the time for energy transfer from BPh to P is less than 7 ps. However, the bleaching in the BPh band at 545 nm following 7-ps 600-nm excitation, exhibits an 8- to 10-ps lag with respect to standard compounds. This finding is qualitatively similar to the 35-ps delay previously observed at 760 nm by Shuvalov at al. (Shuvalov, V.A., Klevanik, A.V., Sharkov, A.V., Matveetz, Y.A. and Kryukov, P.G. (1978) FEBS Lett. 91, 135-139) when 25-ps 880-nm excitation flashes were used. A delay in the bleaching approximately equal to the width of the excitation flash can be explained in terms of the opposing effects of bleaching due to the reduction of BPh, and absorbance increases due to short-lived excited states (probably of BChl) that turn over rapidly during the flash. The decay of the initial bleaching at 800 nm produced by 7-ps 530- or 600-nm excitation flashes shows a fast component with a 30-ps time constant, in addition to a slower component having the 200-ps kinetics expected for the decay of P+BPh-. the dependence on excitation intensity of the absorbance changes due to the 30-p]s component indicate that the quantum yield of the state responsible for this step is lower than that observed for the primary electron transfer reactions. This suggests that at least part of the transient bleaching at 800 nm is due to a secondary process, possibly caused by excitation with an excessive number of photons. If the 800-nm absorbing BChl (B) acts as an intermediate electron carrier in the primary photochemical reaction, electron transfer between B and the BPh must have a time constant no greater than 4 ps.

Control of electron transfer between the L- and M-sides of photosynthetic reaction centers

An aspartic acid residue has been introduced near ring V of the L-side accessory bacteriochlorophyll (BCHlL) or the photosynthetic reaction center in a rhodobacter capsulatus mutant in which a His also replaces Leu 212 on the M-polypeptide. The initial stage of charge separation in the G(M201)D/L(M212)H double mutant yields approximately 70 percent electron transfer to the L-side cofactors, approximately 15 percent rapid deactivation to the ground state, and approximately 15 percent electron transfer to the so-called inactive M-side bacteriopheophytin (BPhM). It is suggested here that the Asp introduced at M201 modulates the reduction potential of BCHlL, thereby changing the energetics of charge separation. The results demonstrate that an individual amino acid residue can, through its influence on the free energies of the charge-separated states, effectively dictate the balance between the forward electron transfer reactions on the L-side of the RC, the charge-recombination processes, and electron transfer to the M-side chromophores.

Porphyrins XXXV . Exciton coupling in μ-oxo Scandum dimers

Abstract The absorption and emission spectra at room temperature and at 77 K are reported for the monomers and μ-oxo dimers of (OEP)Sc(III) and (TPP)Sc(III). [Here (OEP) is octaethylporphin and (TPP) is tetraphenylporphin.] Exciton coupling effects are strongest in the B(Soret) band of [(OEP)Sc]2O dimer: (i) The peak is blue shifted by 11 nm; (ii) the Soret band has a long red tail out to 480 mn; (iii) the fluorescence polarization shows a broad negativ band ≈ 440 nm. A vibronic exciton coupling model can roughly interpret the data if there is substantial and variable tilting of the ring planes. Exciton effects are weaker in the B(Soret) band of [(TPP)SC]2O, presumably because there is less tilting. The effect of dimer formation on the Q band of [(OEP)Scl2O is to red shift the band ≈ 420 cm−1 and to nearly double the Q(0,0) halfwidth; there is no change in fluorescence yield with dimerization. Presumably for Q bands exciton coupling is weaker than inhomogeneous broadening. Both the phosphorescence yield and triplet lifetime at 77 K drop by case2 3 in the dimer, showing faster radiationless decay.

Nickel porphyrin photophysics and photochemistry. A picosecond investigation of ligand binding and release in the excited state

Abstract The photophysical behavior and some photochemical processes for nickel (II) porphyrins have been examined with picosecond transient absorption techniques. Detailed results are reported for Ni-octaethylporphyrin (NiOEP) and Ni-protoporphyrin IX dimethylester (NiPPDME) in toluene, pyridine and piperidine. Excitation flashes at six wavelengths between 355 and 532 nm have been employed. In toluene, rapid (⩽ 15 ps) radiationless decay occurs via several pathways to the low-lying 3B1g excited state. In the basic solvents pyridine and piperidine, excited states with nickel 1A1g (dz2) character have a tendency to release ligands bound to the metal. Excited states with nickel 1B1g or 3B1g (dz2, dx2-y2) character on the other hand, have an affinity for basic ligands, which rapidly bind to the metal. The competition between radiationless decay, ligand binding, and ligand release depends on the nickel porphyrin, solvent, and excitation wavelength. A set of “rules” has been developed that gives a consistent view of all of our results and those of previous investigators. These results may be helpful in understanding photoprocesses in other transition-metal porphyrins, including hemes, which have particular biological significance.

ELECTRON TRANSFER FROM PHOTOEXCITED SINGLET AND TRIPLET BACTERIOPHEOPHYTIN

Abstract— Nanosecond and picosecond. kinetic techniques have been used to study electron transfer from the first excited singlet state (Bph*) and the first excited triplet state (BphT) of bacteriopheophytin to p‐benzoquinone. Quenching of the first excited singlet state by 40 mMp‐benzoquinone results in a decrease in the lifetime of Bph* but does not lead directly to the formation of the π‐cation radical (Bph†). In the presence of 8 M methyl iodide and 40 mMp‐benzoquinone together, the singlet lifetime is reduced further; however, the quantum yield of BphT is enhanced due to the increased rate of intersystem crossing between Bph* and BphT. Electron transfer from BphT to p‐benzoquinone leads to the formation and detection of Bph†. The results are discussed in terms of the spin‐selectivity of the reverse electron transfer process within the intermediate charge transfer complexes.