Conformational changes in an ultrafast light-driven enzyme determine catalytic activity.

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.

Spectroscopic characterization of the first ultrafast catalytic intermediate in protochlorophyllide oxidoreductase.

The enzyme NADPH:protochlorophyllide oxidoreductase (POR) catalyses the reduction of protochlorophyllide into chlorophyllide, a precursor of chlorophyll a in photosynthetic organisms. The enzyme binds the substrate and the cofactor in the dark and catalysis is initiated by the absorption of light by the substrate. We have carried out spectroscopic measurements with ultrafast time resolution under single pulse conditions, which reveal a biphasic formation of the first catalytic intermediate, I675* with average rates of (3.7 ± 0.7 ps)(-1) and (177 ± 78 ps)(-1), as obtained from a systematic analysis of 15 datasets. Measurements in the mid-IR absorption spectral region show that I675* is associated with a decrease of the PChlide C[double bond, length as m-dash]O keto oscillator strength. The spectroscopic changes in the visible and mid-IR regions are specific for the enzyme reaction as they do not occur in the photoexcited substrate alone. In deuterated samples, the rates of I675* formation are reduced by a factor of 1.5-2 compared to protonated samples, suggesting the involvement of a proton movement in this reaction step. The quantum yield of this step is determined to be 0.64 ± 0.11, and the quantum yield of the final reaction product formed on a later time scale, chlorophyllide, is 0.26 ± 0.06. Several possible interpretations of these data are discussed.

Enzyme activation and catalysis: characterisation of the vibrational modes of substrate and product in protochlorophyllide oxidoreductase.

The light-dependent reduction of protochlorophyllide, a key step in the synthesis of chlorophyll, is catalyzed by the enzyme protochlorophyllide oxidoreductase (POR) and requires two photons (O. A. Sytina et al., Nature, 2008, 456, 1001-1008). The first photon activates the enzyme-substrate complex, a subsequent second photon initiates the photochemistry by triggering the formation of a catalytic intermediate. These two events are characterized by different spectral changes in the infra-red spectral region. Here, we investigate the vibrational frequencies of the POR-bound and unbound substrate, and product, and thus provide a detailed assignment of the spectral changes in the 1800-1250 cm(-1) region associated with the catalytic conversion of PChlide:NADPH:TyrOH into Chlide:NADP(+):TyrO(-). Fluorescence line narrowed spectra of the POR-bound Pchlide reveal a C=O keto group downshifted by more than 20 cm(-1) to a relatively low vibrational frequency of 1653 cm(-1), as compared to the unbound Pchlide, indicating that binding of the chromophore to the protein occurs via strong hydrogen bond(s). The frequencies of the C=C vibrational modes are consistent with a six-coordinated state of the POR-bound Pchlide, suggesting that there are two coordination interactions between the central Mg atom of the chromophore and protein residues, and/or a water molecule. The frequencies of the C=C vibrational modes of Chlide are consistent with a five-coordinated state, indicating a single interaction between the central Mg atom of the chromophore and a water molecule. Rapid-scan FTIR measurements on the Pchlide:POR:NADPH complex at 4 cm(-1) spectral resolution reveal a new band in the 1670 cm(-1) region. The FTIR spectra of the enzyme activation phase indicate involvement of a nucleotide-binding structural motif, and an increased exposure of the protein to solvent after activation.

Modeling of multi-exciton transient absorption spectra of protochlorophyllide aggregates in aqueous solution.

Protochlorophyllide (Pchlide) is a natural porphyrin, a precursor of chlorophyll, synthesized by plants for its photosynthetic apparatus. The pigment spontaneously forms aggregates when dissolved in neat water solution. We present here calculations of the transient absorption spectra and its comprising components (ground-state bleach, stimulated emission, and excited-state absorption) for a strongly excitonically coupled linear chain of four Pchlide chromophores, using exciton theory with phenomenological Gaussian line shapes and without energetic disorder. A refined multiexciton model that includes static disorder is applied to fit the experimental power-dependent transient absorption spectra of aqueous protochlorophyllide and the kinetics for delay times up to 20 ps after photoexcitation. We show that population up to the 4-exciton manifold is sufficient to explain the pronounced saturation of the bleaching and the shape changes in the instantaneous, t = 0.2 ps transient spectra when the pulse energy is increased from 10 to 430 nJ per pulse. The decay of the multiexciton manifold is relatively slow and is preceded by a spectroscopically distinct process. We suggest that the exciton states in the Pchlide aggregates are mixed with charge-transfer states (CTS) and that the population and repopulation of the CTS coupled to the exciton states explains the relatively slow decay of the multiexciton manifold. The relevance of our results to the optical properties and dynamics of natural photosynthetic complexes and the possible physical origin of CTS formation are discussed.

Single and multi-exciton dynamics in aqueous protochlorophyllide aggregates.

In plants, the oxidoreductase enzyme POR reduces protochlorophyllide (Pchlide) into chlorophyllide (Chlide), using NADPH as a cofactor. The reduction involves the transfer of two electrons and two protons to the C17═C18 double bond of Pchlide, and the reaction is initiated by the absorption of light by Pchlide itself. In this work we have studied the excited state dynamics of Pchlide dissolved in water, where it forms excitonically coupled aggregates, by ultrafast time-resolved transient absorption and fluorescence experiments performed in the 480-720 nm visible region and in the 1780-1590 cm(-1) mid-IR region. The ground state visible absorption spectrum of aqueous Pchlide red shifts and broadens in comparison to the spectrum of monomeric Pchlide in organic solvents. The population of the one-exciton state occurs at low excitation densities, of <1 photon per aggregate. We characterized the multiexciton manifolds spectra by measuring the absorption difference spectra at increasingly higher photon densities. The multiexciton states are characterized by blue-shifted stimulated emission and red-shifted excited state absorption in comparison to those of the one-exciton manifold. The relaxation dynamics of the multiexciton manifolds into the one-exciton manifold is found to occur in ∼10 ps. This surprisingly slow rate we suggest is due to the intrinsic charge transfer character of the PChlide excited state that leads to solvation, stabilizing the CT state, and subsequent charge recombination, which limits the exciton relaxation.

Ultrafast catalytic processes and conformational changes in the light-driven enzyme protochlorophyllide oxidoreductase (POR).

The enzyme POR (protochlorophyllide oxidoreductase), from the family of alcohol dehydrogenases, reduces protochlorophyllide into chlorophyllide on the absorption of light. The reduction involves the transfer of two protons and two electrons and is an important regulatory step in the biosynthesis of chlorophyll. In recent years, due to the availability of large quantities of the pure enzyme, much of the catalytic reaction has been unravelled by using a variety of spectroscopic methods, including ultrafast initial events in catalysis. In addition, it has been demonstrated that a light-activated conformational change of the protein is necessary to activate catalysis. This makes POR a very important model system to study the relationship between structural changes of enzymes and functionality.

Protochlorophyllide excited-state dynamics in organic solvents studied by time-resolved visible and mid-infrared spectroscopy.

Protochlorophyllide (PChlide) is a precursor in the biosynthesis of chlorophyll. Complexed with NADPH to the enzyme protochlorophyllide oxidoreductase (POR), it is reduced to chlorophyllide, a process that occurs via a set of spectroscopically distinct intermediate states and is initiated from the excited state of PChlide. To obtain a better understanding of these catalytic events, we characterized the excited state dynamics of PChlide in the solvents tetrahydrofuran (THF), methanol, and Tris/Triton buffer using ultrafast transient absorption in the visible and mid-infrared spectral regions and time-resolved fluorescence emission experiments. For comparison, we present time-resolved transient absorption measurements of chlorophyll a in THF. From the combined analysis of these experiments, we derive that during the 2-3 ns excited state lifetime an extensive multiphasic quenching of the emission occurs due to solvation of the excited state, which is in agreement with the previously proposed internal charge transfer (ICT) character of the S1 state ( Zhao , G. J. ; Han , K. L. Biophys. J. 2008 , 94 , 38 ). The solvation process in methanol occurs in conjunction with a strengthening of a hydrogen bond to the Pchlide keto carbonyl group. We demonstrate that the internal conversion from the S2 to S1 excited states is remarkably slow and stretches out on to the 700 fs time scale, causing a rise of blue-shifted signals in the transient absorption and a gain of emission in the time-resolved fluorescence. A triplet state is populated on the nanosecond time scale with a maximal yield of approximately 23%. The consequences of these observations for the catalytic pathway and the role of the triplet and ICT state in activation of the enzyme are discussed.