Article pubs.acs.org/JPCB
Excited-State Properties of Protochlorophyllide Analogues and Implications for Light-Driven Synthesis of Chlorophyll Derren J. Heyes,*,† Samantha J. O. Hardman,† David Mansell,† Aisling Ní Cheallaigh,†,§ John M. Gardiner,† Linus O. Johannissen,† Gregory M. Greetham,‡ Michael Towrie,‡ and Nigel S. Scrutton*,† †
Manchester Institute of Biotechnology and School of Chemistry, The University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K. ‡ Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Harwell Oxford, Didcot OX11 0QX, U.K. S Supporting Information *
ABSTRACT: Protochlorophyllide (Pchlide), an intermediate in the biosynthesis of chlorophyll, is the substrate for the lightdriven enzyme protochlorophyllide oxidoreductase. Pchlide has excited-state properties that allow it to initiate photochemistry in the enzyme active site, which involves reduction of Pchlide by sequential hydride and proton transfer. The basis of this photochemical behavior has been investigated here using a combination of time-resolved spectroscopies and density functional theory calculations of a number of Pchlide analogues with modifications to various substituent groups. A keto group on ring E is essential for excited-state charge separation in the molecule, which is the driving force for the photoreactivity of the pigment. Vibrational “fingerprints” of specific regions of the Pchlide chromophore have been assigned, allowing identification of the modes that are crucial for excitedstate chemistry in the enzyme. This work provides an understanding of the structural determinants of Pchlide that are important for harnessing light energy.
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INTRODUCTION The chlorophyll precursor protochlorophyllide (Pchlide) is the major pigment found in seedlings and etiolated plants and acts as an essential light-activated trigger for subsequent formation of chlorophyll and development of the plant.1−3 Pchlide is the substrate and chromophore for the light-driven enzyme protochlorophyllide oxidoreductase (POR), which catalyzes the reduction of the C17−C18 double bond to form chlorophyllide (Chlide) and is an important regulatory step in chlorophyll biosynthesis.1−3 The reaction catalyzed by POR involves a light-driven hydride transfer from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to the C17 position of the Pchlide molecule,4,5 followed by a thermally activated proton transfer from a conserved Tyr residue to the C18 position, both reactions involving quantum mechanical tunneling.6 Although the chemical steps in the POR reaction cycle proceed on the microsecond time scale,5 catalysis is dependent on excited-state processes in the Pchlide molecule. Timeresolved studies on the isolated Pchlide pigment have demonstrated that Pchlide is an intrinsically reactive molecule with multiexponential dynamics7−12 that depends strongly on solvent polarity.7,8,12 The identification of a number of Pchlide © XXXX American Chemical Society
excited-state species suggests multiphasic quenching of Pchlide excited-state emission via solvation of an intramolecular charge transfer (ICT) state7−12 and subsequent formation of a triplet state on the nanosecond time scale.11,12 Moreover, timedependent density functional theory (DFT) calculations have confirmed the ICT character of the Pchlide excited state in methanol and, in turn, this is thought to induce site-specific solvation of the photoexcited Pchlide molecule via strengthening of H-bonding interactions.13 A detailed understanding of the exact mechanism of photochemistry in the ternary enzyme−substrate complex has proven more challenging, although it was recently proposed that excited-state interactions between active site residues and the carboxyl group at the C17 position result in the formation of a “reactive” ICT state.14 This creates a highly polarized C17−C18 double bond, thereby facilitating subsequent nucleophilic attack of the negatively charged hydride from NADPH to the C17 position of Pchlide.14 Therefore, it is likely that the dipolar character of the ICT state in Pchlide is crucial for harnessing the light energy to drive Received: January 17, 2017 Published: January 24, 2017 A
DOI: 10.1021/acs.jpcb.7b00528 J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B
Figure 1. Light-driven reduction of the C17−C18 double bond of Pchlide to form Chlide is catalyzed by POR and requires NADPH as a cofactor. The dashed box indicates the double bond that is reduced during the reaction and the red circles show the regions of the Pchlide molecule that have previously been shown to be important for activity (central Mg, ring E, and the side chain at the C17 position). The structures of all of the Pchlide derivatives described in the present study are shown underneath, with modifications indicated by dashed red circles.
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POR catalysis and, ultimately, the development of the plant. The highly reactive nature of the excited state is thought to be caused by the presence of a number of substituent groups on the Pchlide molecule, such as the electron-withdrawing carbonyl group on ring E,12,15,16 the central Mg ion, and the carboxylic acid side chain at the C17 position, all of which have been shown to be important for enzymatic photoreduction (Figure 1).17 We have now synthesized a number of Pchlide analogues that contain alterations to all of these positions (compounds A−F in Figure 1) and used a combination of spectroscopic techniques and computational approaches to understand how the structural changes affect the photochemical and excited-state properties of the Pchlide molecule that are so crucial for POR catalysis.
EXPERIMENTAL METHODS
Synthesis of Pchlide Analogues. All chemicals and solvents were purchased from Sigma Aldrich, except where specified, and were of analytical grade or higher. All pigments were synthesized from commercially available pheophorbide a to yield target compounds A−F (Figure S1) and were verified by NMR and mass spectrometry techniques. Chemical syntheses were monitored by thin-layer chromatography (TLC) using aluminum foil-coated TLC plates carrying silica gel 60 F254 (0.2 mm thickness; Merck). Ultraviolet light and/ or phosphomolybdic acid (10 g of phosphomolybdic acid in 100 mL of absolute ethanol) were used to detect the compounds. Purification of the compounds was carried out
B
DOI: 10.1021/acs.jpcb.7b00528 J. Phys. Chem. B XXXX, XXX, XXX−XXX
Article
The Journal of Physical Chemistry B
D′, E′, and F′ were then subjected to the above Mg insertion (D″, E″, F″) and DDQ oxidation (D, E, F), as described for the synthesis of A. Time-Resolved Spectroscopy. Ground state absorbance spectra were recorded using a Cary 50 UV/visible spectrophotometer (Agilent Technologies). The samples contained each analogue (A−F) at an optical density (OD) of ∼0.8 in methanol for time-resolved visible measurements or an OD of ∼0.07 at 430 nm in 2H-methanol for time-resolved infrared (IR) spectroscopy measurements. Visible transient absorption spectroscopy was carried out using a Ti:sapphire amplifier (hybrid Coherent Legend Elite-F-HE, 1 kHz repetition rate, 800 nm, ∼120 fs pulse duration); a noncollinear optical parametric amplifier (light conversion TOPAS White) was used to generate the pump beam. Data on 17,18-didehydropheophorbide a (B) were collected using a different Ti:sapphire amplifier system (Spectra Physics Solstice Ace; 6 mJ of 800 nm pulses at 1 kHz with a 100 fs pulse duration), a Topas Prime OPA with an associated NirUVis unit, which was used to generate the pump beam. In both cases, the pump beam was centered at 430 nm, with 1 μJ pulse power and a beam diameter of ∼150 μm, which was depolarized before the sample. A broad band, ultrafast, pump−probe transient absorbance spectrometer, “Helios” (Ultrafast systems LLC), was used to collect “fast” data, with a time resolution of around 0.2 ps. Data were collected for approximately 30 min per dataset at randomly arranged time delays ranging from 300 fs to 3 ns. The probe beam consisted of a white-light continuum generated in a sapphire crystal. A broad band, subnanosecond, pump−probe transient absorbance spectrometer, “Eos” (Ultrafast systems LLC), was used to collect “slow” data. Data were collected for approximately 30 min per dataset at randomly arranged time delays ranging from 0.5 ns to 17.2 μs. A 2 kHz white-light continuum fiber laser was used to generate the probe pulses. The delay between the pump and probe was managed electronically. Samples were magnetically stirred in a 2 mm path length quartz cuvette. After correcting the fast data for spectral chirp, the fast and slow datasets were combined by scaling the full slow dataset by a fixed factor to match the intensity of the ground state bleach feature in the fast dataset at similar time points (datasets overlap between ∼0.5 and 3 ns). Infrared transient absorption spectroscopy was carried out at the Ultra facility (Central Laser Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, UK), using the time-resolved multiple probe spectroscopy technique.23 The samples were contained between two CaF2 windows separated by a Teflon spacer to give a path length of approximately 100 μm. An excitation wavelength of 430 nm, with a 0.5 μJ pulse power and a beam diameter of ∼150 μm, set at the magic angle with respect to the probe beam, was used. The sample was flowed through the cell and the sample holder, rastered to avoid sample damage. Difference spectra were generated relative to those from the ground state in the spectral window of 1500−1800 cm−1, with a spectral resolution of ∼3 cm−1. Data were collected for approximately 30 min per dataset, at randomly arranged time delays, ranging from 300 fs to 2 ns. Fluorescence experiments were carried out using a Ti:sapphire amplifier system (Spectra Physics Solstice Ace), producing 6 mJ of 800 nm pulses at 1 kHz, with a 100 fs pulse duration. A portion of the output of the amplifier was used to pump a Topas Prime OPA with an associated NirUVis unit, which was used to generate the pump beam (∼0.4 μJ) centered
using column chromatography (Fluka Analytical high-puritygrade silica gel, 60 Å pore size, 220−440 mesh particle size, 35−75 μm particle size). NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer and referenced to the solvent. Coupling constants (J values) are expressed in Hz and were calculated using iNMR processing software. Proton peaks are indicated according to their appearance (e.g., singlet, doublet, triplet) or as a multiplet, where no defined multiplicity is observed and/or overlap of multiple signals occurs. Lowresolution mass spectrometry was performed on Waters SQD2 (Q-MS with ES+, ES−, and APCI source). High-resolution mass spectrometry was performed on a mixture of Waters QTOF micro (Q-TOF reflection with an ES+ ion source, accurate mass determination
