Photophysical properties of structurally and electronically modified flavin derivatives determined by spectroscopy and theoretical calculations.

Four different riboflavin (RF) derivatives, two electronically modified compounds (1- and 5-deazariboflavin, 1DRF and 5DRF) and two sterically modified compounds (7,8-didemethyl- and 8-isopropylriboflavin, DMRF and iprRF), were subjected to a combination of time-resolved measurements (absorption and fluorescence) and high-level quantum chemical investigations. Both alkyl-modified flavins showed similar fluorescence properties as the parent compound, yet 5DRF had a larger quantum yield of fluorescence (PhiF = 0.52) than RF (PhiF = 0.27). Interestingly, 1DRF did not show fluorescence at all under these steady state conditions. The triplet quantum yield was different for the modified flavins such that no triplet formation was found for 1DRF, whereas the other compounds all formed triplet states (PhiTR for 5DRF of 0.64 and 0.50 and 0.23 for iprRF and DMRF, respectively). The triplet states of the two alkyl-modified flavins decayed with similar time constants as the parent compound, whereas a shorter lifetime was measured for 5DRF (tauTR = 15 micros, compared to tauTR = 29 micros for RF). In the calculations, the flavin derivatives were modeled as lumiflavins, that is, without the ribityl chain. We conclude that for aqueous solutions of DMRF, iprRF, and 5DRF intersystem crossing (ISC) takes place from the S1 1(pipi*) to the T2 3(pipi*) state by a vibronic spin-orbit coupling mechanism, a process common to most flavins, whereas ISC is slow in excited 1DRF due to the absence of a close-by triplet state.

Flavodoxin relaxes in microseconds upon excitation of the flavin chromophore: detection of a UV-visible silent intermediate by laser photocalorimetry.

A small contraction concomitant with the relaxation of the protein in ca. 3 mus is observed upon ns-laser excitation at 455 nm of the Cys69Ala (C69A) mutant of flavodoxin II from Azotobacter vinelandii. This constitutes another example of detection of a UV-vis silent transient species through a photocalorimetric technique. The contraction is attributed to reorganization of protein dihedral bonds and of water molecules at relatively long distances from the flavin chromophore, after the protein has received the heat shock from the relaxing photoproduced charge transfer state. This study constitutes a preliminary step towards the understanding of the origin of protein movements taking place upon electron transfer reactions, e.g. between a photoinduced electron donor (or acceptor) and an accepting (or donating) cofactor in a protein.

A photoprotection mechanism involving the D(2) branch in photosystem II cores with closed reaction centers.

Nanosecond transient absorption spectroscopy has been used to study reaction centre (RC) chlorophyll triplet quenching by carotenoid in intact photosystem II cores from T. elongatus with closed RCs. We found a triplet beta-carotene ((3)Car) signal (absorption difference maximum at 530 nm) that is sensitized by the RC chlorophyll (Chl) triplet with a formation time of ca. 190 ns, has a decay time of 7 micros and is formed with a quantum yield between 10 and 20%. The (3)Car signal is assigned to the beta-carotene on the D(2) branch of the RC. We thus propose a new photoprotection mechanism operative in closed RCs where-as a consequence of the negative charge on the quinone Q(A)-the triplet chlorophyll ((3)Chl) is formed by the radical pair (RP) mechanism on the normally inactive D(2) branch where it can be subsequently quenched by the D(2) beta-carotene. We suggest that the D(2) branch becomes active when the RCs are closed under high light fluence conditions. Under these conditions the D(2) branch plays a photoprotective role. This interpretation allows combining many seemingly inconsistent observations in the literature and reveals the so far missing RC triplet quenching mechanism in photosystem II. The newly proposed mechanism also explains the reason why this RC triplet quenching is not observed in isolated D(1)-D(2)-cyt b(559) RCs. If Q(A) is either not present at all (as in the isolated RC) or is not charged (as in open RCs or with doubly reduced Q(A)) then the RC (3)Chl is formed on the D(1) branch. The D(1) branch (3)Chl can not be quenched due to the large distance to the beta-carotene. This interpretation is actually in line with the well-known (3)RC quenching mechanism in bacterial RCs, where also the carotenoid in the (analogous to the D(2) branch) B-branch of the RC becomes the quencher.

A large entropic term due to water rearrangement is concomitant with the photoproduction of anionic free-base porphyrin triplet states in aqueous solutions.

The enthalpy change, DeltaTH, and volume change, DeltaTV, associated with triplet state formation upon excitation of free-base meso-tetra-(4-sulfonatophenyl)porphyrin, TSPP4-, its Zn derivative, ZnTSPP4-, and meso-tetra-(4-carboxyphenyl)porphyrin, TCPP4-, were obtained in aqueous solutions by the application of laser-induced optoacoustics spectroscopy in the presence of phosphate salts of various monovalent cations (Li+, Na+, K+, NH4+ and Cs+). A linear correlation was found between DeltaTH and DeltaTV at different phosphate concentrations for the free-base porphyrins. The intercepts (132 +/- 8 kJ mol(-1) for TSPP4- and 164 +/- 23 kJ mol(-1) for TCPP4-) of these plots correspond to the respective value of the triplet energy content obtained from phosphorescence at 77 K (140 and 149 kJ mol(-1)). This suggests that DeltaTG for the triplet state formation is independent of the medium and an enthalpy-entropy compensation is responsible for the much smaller and salt-dependent DeltaTH values obtained at room temperature. The Gibbs energy for triplet state formation of the free-base porphyrins at room temperature is thus mainly determined by the entropic term due to solvent rearrangement. The DeltaTH values for 3ZnTSPP4- at different buffer concentrations and different cations are all between 130 and 150 kJ mol(-1), close to the triplet energy obtained from phosphorescence (E(T) = 155 kJ mol(-1)). The solvent structure and the nature of the counterion have a negligible influence on the 3ZnTSPP4-formation due to the blockage of the electron pairs on the central N atoms. Thus, the small DeltaTV value should be due to intrinsic bond changes upon 3ZnTSPP4- formation and no correlation between DeltaTH and DeltaTV should be expected in this case. The enthalpy change determines the Gibbs energy for 3ZnTSPP4-formation at room temperature.

Photoinduced electron transfer from tetrasulfonated porphyrin to benzoquinone revisited. The structural volume-normalized entropy change correlates with marcus reorganization energy.

Time-resolved laser-induced optoacoustic spectroscopy was used for the determination of the enthalpy, DeltaTH, and structural volume changes, DeltaTV, concomitant with triplet state formation upon excitation of meso-tetrakis(4-sulfonatophenyl)porphyrin, TSPP(4-), as well as with the triplet state electron-transfer (ET) quenching by benzoquinone, BQ (DeltaRH and DeltaRV). The values of DeltaTH and DeltaTV for (3)TSPP(4-) formation in the presence of different cations (Li+, Na+, K+, NH4+, and Cs+) correlated with each other and afforded a value of DeltaTG = 140 +/- 20 kJ mol(-1), equal to the value of E+ at 77 K, but much larger than the DeltaTH values in solution at room temperature, due to the large entropic factor in solution. The influence of the cations on DeltaTH and DeltaTV (a contraction ranging from 5.4 to 3.8 cm3 mol(-1)) is attributed to changing chromophore-water interactions in the ground and triplet states. Upon quenching of 3TSPP(4-) by BQ, the quantum yield of free radical formation, PhiR = 0.66 +/- 0.04, is the same in the solutions of the five cations. The values of DeltaRH and DeltaRV are small and have a large error. The energy level of the free radicals formed is thus very similar to that of 3TSPP(4-). TDeltaRS and X = TDeltaRS/DeltaRV, i.e., the structural volume change-normalized entropy change for free radicals formation, were derived using average values of DeltaRH and DeltaRV together with the calculated DeltaRG degrees . The measured Marcus reorganization energy, lambda, and X fall into the lambda vs X linear dependence we previously found for the radical formation upon ET quenching of triplet flavins (3FMN and 3FAD) by amines and amino acids. Thus, X = TDeltaRS/DeltaRV in aqueous solutions is a property of the particular donor-acceptor pair linearly correlated to the corresponding Marcus reorganization energy. The value of X is much larger than the predicted value applying the electrostriction concepts in view of the noncontinuum nature of the aqueous solutions.

Entropy changes drive the electron transfer reaction of triplet flavin mononucleotide from aromatic amino acids in cation-organized aqueous media. A laser-induced optoacoustic study.

The thermodynamic parameters for the formation of the free radicals upon electron transfer quenching of the flavin triplet state (3FMN) by tryptophan and tyrosine, Delta(FR)H and Delta(FR)V, were obtained in aqueous solution by the application of laser-induced optoacoustic spectroscopy at various temperatures. The Delta(FR)H and Delta(FR)V values include the electron transfer and charge separation steps plus the protonation of the FMN anion radical and the deprotonation of the amino-acid cation radical. A linear correlation was found between the Delta(FR)H and Delta(FR)V values for each of the amino acids in phosphate buffers of [CH3(CH2)3]4N+, Li+, NH4+, K+ and Cs+. The compensation between Delta(FR)H and Delta(FR)V within the salt series, and the independent evaluation of the Gibbs energy for electron transfer Delta(ET)G(o) afforded the entropy change, Delta(FR)S, for the reaction, different for the two amino acids. The values of Delta(FR)H, Delta(FR)V and Delta(FR)S in each buffer are mainly determined by the changes in strength and probably number of hydrogen bonds between the reacting partners and water produced along all steps leading to the radicals FMNH* and A*. The Delta(FR)V values linearly correlate with the tabulated entropy of organization of the water structure for the five cations, DeltaS(o)(cat). The entropy change upon formation of the free radicals, Delta(FR)S, quantitatively correlated to the Delta(FR)V value, drives the separation of the ion pair after the electron transfer reaction in the case of highly organizing cations. The ratio X = T Delta(FR)S/Delta(FR)V = (55 +/- 9) kJ cm(-3) for Trp as 3FMN quencher is smaller than X = (83 +/- 9) kJ cm(-3) for Tyr as quencher. These values are discussed in conjunction with the Marcus reorganization energy, as calculated from the Gibbs activation energy of the electron transfer process, which is independent of the salt present but different for each of the two quenchers.

Conformational and photophysical studies on porphyrin-containing donor-bridge-acceptor compounds. Charge separation in micellar nanoreactors.

Photophysical studies with semi-rigid, 1, and flexible, 2, donor-bridge-acceptor (D-b-A2+) molecules with D a porphyrin and A2+ a methyl viologen moiety, were performed in neat polar solvents as well as included in surfactant (DTAB) aqueous and in reverse AOT/n-alkane micelles. The micelles acted as nanoreactors for the photoinduced electron transfer reaction upon laser excitation. In spite of the longer lifetime of the charge separated (CS) state in the semi-rigid tetrad 1(ca. 200 ns vs. ca. 100 ns for the flexible dyad 2), the CS formation quantum yield, for example in acetonitrile, was lower for the former (phi(CS) = 0.13) than for the latter (0.58). Comparison of the time-resolved fluorescence data in neat solvent and in the micelles yielded the phi(CS) values in the dilute micellar solutions. Application of laser-induced optoacoustic spectroscopy at various temperatures to 1 dissolved in a polar organic solvent (benzonitrile, BZN) included in aqueous DTAB nanoreactors afforded structural volume changes for the production in hundreds of ps of the CS state upon excitation of a polar molecule. The contraction during CS formation upon excitation of the collapsed conformer in BZN is attributed to the entering of solvent into the open molecular cavity. The opening upon formation of the CS state due to photoinduced electron transfer in the 1 collapsed conformation arises from the repulsion of the two positively charged ends in this state, as previously calculated. Inclusion of 1 in reverse AOT micelles in various n-alkanes also led to a contraction upon excitation, but the data had much more error due to the limited range of variability of the ratio of thermoelastic parameters. The data obtained with the more flexible "supermolecule" 2 showed the predicted large conformation flexibility of these molecules.