Excited-State Dynamics of Protochlorophyllide Revealed by Subpicosecond Infrared Spectroscopy

Abstract

To gain a better understanding of the light-induced reduction of protochlorophyllide (PChlide) to chlorophyllide as a key regulatory step in chlorophyll synthesis, we performed transient infrared absorption measurements on PChlide in d4-methanol. Excitation in the Q-band at 630 nm initiates dynamics characterized by three time constants: τ₁ = 3.6 ± 0.2, τ₂ = 38 ± 2, and τ₃ = 215 ± 8 ps. As indicated by the C13'=O carbonyl stretching mode in the electronic ground state at 1686 cm⁻¹, showing partial ground-state recovery, and in the excited electronic state at 1625 cm⁻¹, showing excited-state decay, τ₂ describes the formation of a state with a strong change in electronic structure, and τ₃ represents the partial recovery of the PChlide electronic ground state. Furthermore, τ₁ corresponds with vibrational energy relaxation. The observed kinetics strongly suggest a branched reaction scheme with a branching ratio of 0.5 for the path leading to the PChlide ground state on the 200 ps timescale and the path leading to a long-lived state (>>700 ps). The results clearly support a branched reaction scheme, as proposed previously, featuring the formation of an intramolecular charge transfer state with ∼25 ps, its decay into the PChlide ground state with 200 ps, and a parallel reaction path to the long-lived PChlide triplet state.

Excited‐State Charge Separation in the Photochemical Mechanism of the Light‐Driven Enzyme Protochlorophyllide Oxidoreductase

“…[21] Global analysis was used to model the time-resolved absorption difference spectra from these measurements by fitting the data to yield species associated difference spectra (SADS). [22] Thee volution of the spectral features allows the mechanism of intermediate formation to be determined, whilst the kinetic parameters are indicative of the overall rate of loss of each species,i ncluding both conversion to the proceeding state and return to the ground state (see the Supporting Information for further detail). Small differences in fitted lifetimes between the visible and IR datasets can be attributed to differences in solvation (H 2 O in visible or D 2 Oi nI R) and lower signal-to-noise ratios at longer IR time delays.For clarity only the SADS are shown in the main manuscript, whereas ground-state absorption spectra ( Figure S1), raw time-resolved data ( Figures S2-8) and an analysis of the global fitting ( Figures S9-11) can be found in the Supporting Information.…”

“…[15,22,23] In the Pchlide only sample the data can be modeled with four component lifetimes.T he short-lived S1 component is resolved in the IR data, but not in the visible data, due to the smaller initial step sizes.T he lifetimes of the slower components are similar in both the visible and IR measurements.T he SADS,w hich have been fitted with ac ombination of Gaussian components (Figure S9), show that the proposed solvation of the ICT state (SADS3) is accompanied by changes in the 1550 cm À1 region ( Figure 2C and Figure S9). This is in agreement with previous ultrafast time-resolved IR measurements,w hich suggested that this step is associated with structural changes to the C13 carbonyl and delocalized C=Cand C=Nmodes.…”

Response to the Comments by L. O. Björn on our Paper “Catalytic Efficiency of a Photoenzyme—An Adaptation to Natural Light Conditions”

“…All these experiments were also performed on recombinant enzymes expressed in Escherichia coli or on a pigment-free, monomeric enzyme isolated from etiolated oat (Avena sativa) seedlings. [24] The absorbance spectra of the reconstituted POR-PChlide-NADPH complexes are strictly reproducible and, as can be seen from Figure S1 of our manuscript L. O. Bjçrn is referring to, not disturbed by scattered light. In the article of L. O. Bjçrn the results obtained from those enzymes are subjected to criticisms in so far as they would differ from those published more than 40 years ago.…”

“…[24] The absorbance spectra of the reconstituted POR-PChlide-NADPH complexes are strictly reproducible and, as can be seen from Figure S1 (0,2)]. The enzymes from barley do not include the transit peptide, which is only necessary to import the precursor proteins from the cytoplasm into plastids.…”

Plant Protochlorophyllide Oxidoreductases A and B

“…Preparation of PChlide-PChlide a was isolated from 5-dayold, dark grown oat seedlings (Avena sativa L.) as reported recently (28). Briefly, the coleoptiles were treated with 15 mM 5-aminolevulinic acid in 35 mM potassium phosphate buffer for 48 h. Following disruption of the coleoptiles by homogenization, PChlide was extracted into an ice-cold 10 mM Tricine buffer, pH 7.5, 75% acetone (v/v) mixture.…”