The photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila was examined by time-resolved difference absorption spectroscopy in the wavelength range of 300-600 nm. Both time-gated spectra and single wavelength traces were measured. Global analysis of the data established that in the time domain between 5 ns and 2 s only two intermediates are involved in the room temperature photocycle of PYP, as has been proposed before (Meyer T.E., E. Yakali, M. A. Cusanovich, and G. Tollin. 1987. Biochemistry. 26:418-423; Meyer, T. E., G. Tollin, T. P. Causgrove, P. Cheng, and R. E. Blankenship. 1991. Biophys. J. 59:988-991). The first, red-shifted intermediate decays biexponentially (60% with tau = 0.25 ms and 40% with tau = 1.2 ms) to a blue-shifted intermediate. The last step of the photocycle is the biexponential (93% with tau = 0.15 s and 7% with tau = 2.0 s) recovery to the ground state of the protein. Reconstruction of the absolute spectra of these photointermediates yielded absorbance maxima of about 465 and 355 nm for the red- and blue-shifted intermediate with an epsilon max at about 50% and 40% relative to the epsilon max of the ground state. The quantitative analysis of the photocycle in PYP described here paves the way to a detailed biophysical analysis of the processes occurring in this photoreceptor molecule.

Two complementary aspects of the thermodynamics of the photoactive yellow protein (PYP), a new type of photoreceptor that has been isolated from Ectothiorhodospira halophila, have been investigated. First, the thermal denaturation of PYP at pH 3.4 has been examined by global analysis of the temperature-induced changes in the UV-VIS absorbance spectrum of this chromophoric protein. Subsequently, a thermodynamic model for protein (un)folding processes, incorporating heat capacity changes, has been applied to these data. The second aspect of PYP that has been studied is the temperature dependence of its photocycle kinetics, which have been reported to display an unexplained deviation from normal Arrhenius behavior. We have extended these measurements in two solvents with different hydrophobicities and have analyzed the number of rate constants needed to describe these data. Here we show that the resulting temperature dependence of the rate constants can be quantitatively explained by the application of a thermodynamic model which assumes that heat capacity changes are associated with the two transitions in the photocycle of PYP. This result is the first example of an enzyme catalytic cycle being described by a thermodynamic model including heat capacity changes. It is proposed that a strong link exists between the processes occurring during the photocycle of PYP and protein (un)folding processes. This permits a thermodynamic analysis of the light-induced, physiologically relevant, conformational changes occurring in this photoreceptor protein.

It is generally accepted that electron transfer in bacterial photosynthesis is driven by the first singlet excited state of a special pair of bacteriochlorophylls (P*). We have examined the first steps of electron transfer in a mutant of the Rhodobacter sphaeroides reaction center in which charge separation from P* is dramatically slowed down. The results provide for the first time clear evidence that excitation of the monomeric bacteriochlorophyll in the active branch of the reaction center (B(A)) drives ultrafast transmembrane electron transfer without the involvement of P*, demonstrating a new and efficient mechanism for solar energy transduction in photosynthesis. The most abundant charge-separated intermediate state probably is P+B(A)-, which is formed within 200 fs from B(A)* and decays with a lifetime of 6.5 ps into P+H(A)-. We also see evidence for the involvement of a B(A)+H(A)- state in the alternative pathway.

A pathway of electron transfer is described that operates in the wild-type reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides. The pathway does not involve the excited state of the special pair dimer of bacteriochlorophylls (P*), but instead is driven by the excited state of the monomeric bacteriochlorophyll (B A *) present in the active branch of pigments along which electron transfer occurs. Pump-probe experiments were performed at 77 K on membrane-bound RCs by using different excitation wavelengths, to investigate the formation of the charge separated state P ؉ H A ؊ . In experiments in which P or B A was selectively excited at 880 nm or 796 nm, respectively, the formation of P ؉ H A ؊ was associated with similar time constants of 1.5 ps and 1.7 ps. However, the spectral changes associated with the two time constants are very different. Global analysis of the transient spectra shows that a mixture of P ؉ B A ؊ and P* is formed in parallel from B A * on a subpicosecond time scale. In contrast, excitation of the inactive branch monomeric bacteriochlorophyll (B B ) and the high exciton component of P (P ؉ ) resulted in electron transfer only after relaxation to P*. The multiple pathways for primary electron transfer in the bacterial RC are discussed with regard to the mechanism of charge separation in the RC of photosystem II from higher plants.

The photocycle of the photoactive yellow protein (PYP) isolated from Ectothiorhodospira halophila was analyzed by flash photolysis with absorption detection at low excitation photon densities and by temperature-dependent laser-induced optoacoustic spectroscopy (LIOAS). The quantum yield for the bleaching recovery of PYP, assumed to be identical to that for the phototransformation of PYP (pG), to the red-shifted intermediate, pR, was phi R = 0.35 +/- 0.05, much lower than the value of 0.64 reported in the literature. With this value and the LIOAS data, an energy content for pR of 120 kJ/mol was obtained, approximately 50% lower than for excited pG. Concomitant with the photochemical process, a volume contraction of 14 ml/photoconverted mol was observed, comparable with the contraction (11 ml/mol) determined for the bacteriorhodopsin monomer. The contraction in both cases is interpreted to arise from a protein reorganization around a phototransformed chromophore with a dipole moment different from that of the initial state. The deviations from linearity of the LIOAS data at photon densities > 0.3 photons per molecule are explained by absorption by pG and pR during the laser pulse duration (i.e., a four-level system, pG, pR, and their respective excited states). The data can be fitted either by a simple saturation process or by a photochromic equilibrium between pG and pR, similar to that established between the parent chromoprotein and the first intermediate(s) in other biological photoreceptors. This nonlinearity has important consequences for the interpretation of the data obtained from in vitro studies with powerful lasers.

Absorbance difference kinetics were measured on quinone-reduced membrane-bound wild type Rhodobacter sphaeroides reaction centers in the wavelength region from 690 to 1060 nm using 800 nm excitation. Global analysis of the data revealed five lifetimes of 0.18, 1.9, 5.1, and 22 ps and a long-lived component for the processes that underlie the spectral evolution of the system. The 0.18 ps component was ascribed to energy transfer from the excited state of the accessory bacteriochlorophyll (B*) to the primary donor (P*). The 1.9 ps component was associated with a state involving a BChl anion absorbing in the 1020 nm region. This led to the conclusion that primary electron transfer is best described by a model in which the electron is passed from P* to the acceptor bacteriopheophytin (H L ) via the monomeric bacteriochlorophyll (B L ), with the formation of the radical pair state P + B L-. An analysis assuming partial direct charge separation from B* [Van Brederode, M. E., Jones, M. R., and Chem. Phys. Lett. 268,[143][144][145][146][147][148][149] was also consistent with the data. Within the framework of a five component model, the 5.1 and 22 ps lifetimes were associated with charge separation and relaxation of the P + H L -radical pair state respectively, providing a description which adequately accounted for the complex kinetics of decay of P*. Alternatively, by assuming that the 5.1 and 22 ps components originate from a single component with a multi-exponential decay, a simpler analysis with only four components could be employed, resulting in only a small increase (7%) in the weighted root mean square error of the fit. In both descriptions part of the decay of P* proceeds with a lifetime of about 2 ps. The relative merits of these alternative descriptions of the primary events in light-driven electron transfer are discussed. Similar measurements on YM210H mutant reaction centers revealed four lifetimes of 0.2, 3.1, and 12 ps and a long-lived component. The 3.1 and 12 ps lifetimes are ascribed to multi-exponential decay of the P* state. The differences with the WT data are discussed.The reaction center (RC) of purple bacteria is responsible for the conversion of light energy into a transmembrane electrical potential. The structures of the RC from two species of purple bacteria, Rhodopseudomonas (Rps.) Viridis and Rhodobacter (Rb.) sphaeroides, have been solved to high resolution (1, 2). They reveal an assembly of protein subunits and redox cofactors that are arranged around an axis of approximate 2-fold symmetry. The primary donor of electrons (P) is a pair of excitonically-coupled bacteriochloropyll a (BChl) molecules positioned close to the periplasmic face of the protein. The remaining redox cofactors, two monomeric BChls (B), two molecules of bacteriopheophytin a (H), and two ubiquinones (Q), are arranged around the symmetry axis in two branches that span the membrane dielectric. Excitation of P initiates a sequence of electron transfer reactions that result in the separation of charge across the ...

In photosynthetic reaction centers, the excitation with light leads to the formation of a charge separated state across the photosynthetic membrane. For the reaction center of purple nonsulphur bacteria, it was previously generally assumed that this primary charge separation could only start with the excitation of the so-called special pair of bacteriochlorophyll molecules located in the heart of the RC. However, recently new and ultrafast pathways of charge separation have been discovered in the bacterial RC that are driven directly by the excited state of the accessory monomeric bacteriochlorophyll present in the active branch of cofactors. These results demonstrate that the route for energy conversion in photosynthesis can be much more flexible than previously thought. We suggest that the existence of multiple charge separation routes is particularly relevant for the mechanism of charge separation in the photosystem II reaction center of higher plants.z 1999 Federation of European Biochemical Societies.

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