The orange carotenoid protein (OCP) is a two-domain photoactive protein that noncovalently binds an echinenone (ECN) carotenoid and mediates photoprotection in cyanobacteria. In the dark, OCP assumes an orange, inactive state known as OCPO; blue light illumination results in the red active state, known as OCPR. The OCPR state is characterized by large-scale structural changes that involve dissociation and separation of C-terminal and N-terminal domains accompanied by carotenoid translocation into the N-terminal domain. The mechanistic and dynamic-structural relations between photon absorption and formation of the OCPR state have remained largely unknown. Here, we employ a combination of time-resolved UV–visible and (polarized) mid-infrared spectroscopy to assess the electronic and structural dynamics of the carotenoid and the protein secondary structure, from femtoseconds to 0.5 ms. We identify a hereto unidentified carotenoid excited state in OCP, the so-called S* state, which we propose to play a key role in breaking conserved hydrogen-bond interactions between carotenoid and aromatic amino acids in the binding pocket. We arrive at a comprehensive reaction model where the hydrogen-bond rupture with conserved aromatic side chains at the carotenoid β1-ring in picoseconds occurs at a low yield of <1%, whereby the β1-ring retains a trans configuration with respect to the conjugated π-electron chain. This event initiates structural changes at the N-terminal domain in 1 μs, which allow the carotenoid to translocate into the N-terminal domain in 10 μs. We identified infrared signatures of helical elements that dock on the C-terminal domain β-sheet in the dark and unfold in the light to allow domain separation. These helical elements do not move within the experimental range of 0.5 ms, indicating that domain separation occurs on longer time scales, lagging carotenoid translocation by at least 2 decades of time.

Despite the apparent similarity between the plant Photosystem II reaction center (RC) and its purple bacterial counterpart, we show in this work that the mechanism of charge separation is very different for the two photosynthetic RCs. By using femtosecond visible-pump-mid-infrared probe spectroscopy in the region of the chlorophyll ester and keto modes, between 1,775 and 1,585 cm ؊1 , with 150-fs time resolution, we show that the reduction of pheophytin occurs on a 0.6-to 0.8-ps time scale, whereas P ؉ , the precursor state for water oxidation, is formed after Ϸ6 ps. We conclude therefore that in the Photosystem II RC the primary charge separation occurs between the ''accessory chlorophyll'' ChlD1 and the pheophytin on the so-called active branch.electron transfer ͉ photosynthesis ͉ pump-probe T he primary steps of energy and electron transfer in green plants' photosynthesis occur in two large protein complexes called Photosystem I and Photosystem II (PSII). PSII is an aggregate of many individual pigment-protein complexes. The core of PSII consists of the chlorophyll (Chl)-binding antennaproteins CP43 and CP47, which feed excitation energy into the D 1 D 2 cytb559 reaction center (RC). Crystal structures of PSII cores from cyanobacteria have been resolved with increasingly high resolution (1-3); currently, the resolution is 3.2 Å (4). The structure of the PSII RC shows four Chls and two pheophytins (H) arranged in two branches very similar to the bacterial RC. In the heart of the PSII RC, there is a dimer of Chls, and in each branch there is one monomeric Chl and one H. Furthermore, there are two distant Chls bound to the periphery of the PSII RC. Although the structure suggests there may be a ''special pair'' of strongly electronically coupled pigments in the PSII RC, the visible absorption spectrum does not show a distinct band. This finding is in contrast to the bacterial RC, where the lowest energy absorption band fully originates from one of the exciton transitions of a special pair of bacteriochlorophylls.Since the first purification of the PSII RC in 1987 (5), it has been speculated that its way of operation would be similar to that of the bacterial RC, with a special pair that upon excitation drives a charge separation in Ϸ3 ps. This idea was based on the strong homology between the bacterial RC and the PSII RC, the strong similarity in the pigment composition, even details in the way the pigments interacted with the protein, and the near-identity of the electron transfer events at the acceptor side. Conversely, it was clear that major differences between the two RCs had to exist at the electron donor side where in the PSII RC charge separation eventually leads to the oxidation of water and the production of molecular oxygen, requiring a very large oxidation potential of the primary electron donor (Ͼ1.2 V vs. 0.45 V in the bacterial RC).In the mid-1990s, it was recognized that energy transfer and charge separation in the PSII RC most likely proceeded in a manner that is very different from that in the b...

The role of conformational changes in explaining the huge catalytic power of enzymes is currently one of the most challenging questions in biology. Although it is now widely regarded that enzymes modulate reaction rates by means of short- and long-range protein motions, it is almost impossible to distinguish between conformational changes and catalysis. We have solved this problem using the chlorophyll biosynthetic enzyme NADPH:protochlorophyllide (Pchlide) oxidoreductase, which catalyses a unique light-driven reaction involving hydride and proton transfers. Here we report that prior excitation of the enzyme-substrate complex with a laser pulse induces a more favourable conformation of the active site, enabling the coupled hydride and proton transfer reactions to occur. This effect, which is triggered during the Pchlide excited-state lifetime and persists on a long timescale, switches the enzyme into an active state characterized by a high rate and quantum yield of formation of a catalytic intermediate. The corresponding spectral changes in the mid-infrared following the absorption of one photon reveal significant conformational changes in the enzyme, illustrating the importance of flexibility and dynamics in the structure of enzymes for their function.

CP43 is a chlorophyll-protein complex that funnels excitation energy from the main light-harvesting system of photosystem II to the photochemical reaction center. We purified CP43 from spinach photosystem II membranes in the presence of the nonionic detergent n-dodecyl-beta,D-maltoside and recorded its spectroscopic properties at various temperatures between 4 and 293 K by a number of polarized absorption and fluorescence techniques, fluorescence line narrowing, and Stark spectroscopy. The results indicate two "red" states in the Q(y) absorption region of the chlorophylls. The first peaks at 682.5 nm at 4 K, has an extremely narrow bandwidth with a full width at half-maximum of approximately 2.7 nm (58 cm(-1)) at 4 K, and has the oscillator strength of a single chlorophyll. The second peaks at approximately 679 nm, has a much broader bandshape, is caused by several excitonically interacting chlorophylls, and is responsible for all 4 K absorption at wavelengths longer than 685 nm. The Stark spectrum of CP43 resembles the first derivative of the absorption spectrum and has an exceptionally small overall size, which we attribute to opposing orientations of the monomer dipole moments of the excitonically coupled pigments.

BLUF domains constitute a recently discovered class of photoreceptor proteins found in bacteria and eukaryotic algae. BLUF domains are blue-light sensitive through a FAD cofactor that is involved in an extensive hydrogen-bond network with nearby amino acid side chains, including a highly conserved tyrosine and glutamine. The participation of particular amino acid side chains in the ultrafast hydrogen-bond switching reaction with FAD that underlies photoactivation of BLUF domains is assessed by means of ultrafast infrared spectroscopy. Blue-light absorption by FAD results in formation of FAD(*-) and a bleach of the tyrosine ring vibrational mode on a picosecond timescale, showing that electron transfer from tyrosine to FAD constitutes the primary photochemistry. This interpretation is supported by the absence of a kinetic isotope effect on the fluorescence decay on H/D exchange. Subsequent protonation of FAD(*-) to result in FADH(*) on a picosecond timescale is evidenced by the appearance of a N-H bending mode at the FAD N5 protonation site and of a FADH(*) C=N stretch marker mode, with tyrosine as the likely proton donor. FADH(*) is reoxidized in 67 ps (180 ps in D(2)O) to result in a long-lived hydrogen-bond switched network around FAD. This hydrogen-bond switch shows infrared signatures from the C-OH stretch of tyrosine and the FAD C4=O and C=N stretches, which indicate increased hydrogen-bond strength at all these sites. The results support a previously hypothesized rotation of glutamine by approximately 180 degrees through a light-driven radical-pair mechanism as the determinant of the hydrogen-bond switch.

Photoactive yellow protein (PYP) is a bacterial blue light sensor that induces Halorhodospira halophila to swim away from intense blue light. Light absorption by PYP's intrinsic chromophore, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. Here we describe the initial structural changes of the chromophore and its nearby amino acids, using visible pump/mid-infrared probe spectroscopy. Upon photoexcitation, the trans bands of the chromophore are bleached, and shifts of the phenol ring bands occur. The latter are ascribed to charge translocation, which probably plays an essential role in driving the trans to cis isomerization process. We conclude that breaking of the hydrogen bond of the chromophore's C=O group with amino acid Cys69 and formation of a stable cis ground state occur in approximately 2 ps. Dynamic changes also include rearrangements of the hydrogen-bonding network of the amino acids around the chromophore. Relaxation of the coumaryl tail of the chromophore occurs in 0.9-1 ns, which event we identify with the I(0) to I(1) transition observed in visible spectroscopy.

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.

Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira halophila. The formation of PYP's signaling state is initiated by trans-cis isomerization of the p-coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is Ϸ0.3. Using femtosecond visible pump͞mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I0, both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I0-to-I1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.ground state intermediate ͉ hydrogen bond ͉ quantum yield ͉ picosecond ͉ vibrational P YP (photoactive yellow protein) belongs to the Xanthopsins, a family of blue-light photoreceptors that contain 4-hydroxycinnamic acid as their photoactive chromophore (see refs. 1-3 for a review). PYP is a small protein and therefore an attractive model system for exploring how a chromophore and protein interact to sense light and send a biological signal. Its photocycle has been characterized by various experimental techniques, such as fluorescence (4, 5), (time-resolved) FTIR (6-8), (timeresolved) x-ray crystallography (9-12), NMR (13), Stark spectroscopy (14), and pump(-dump)-probe spectroscopy (15, 16). X-ray diffraction on PYP crystals has demonstrated that the PYP chromophore is covalently linked (see Fig. 1) to the protein backbone by means of . It is further embedded in a hydrogen-bonding network consisting of . In the ground state, the chromophore is in a deprotonated trans form, negatively charged, and possibly stabilized by the positive Arg-52 residue (12). After photoexcitation, the chromophore forms a red-shifted intermediate, referred to as I 0 , within a few picoseconds. This intermediate has a shifted absorption maximum from 446 to 500 nm. The second intermediate, I 1 (or pR or PYP L ), absorbs maximally at 480 nm and is formed in 1-3 ns (15-20). This intermediate is followed by protonation of the chromophore and a large structural change of the protein on a mil...