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.

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.

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.

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.

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.

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.

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.