ARTICLE pubs.acs.org/JPCA
Protein-Induced Excited-State Dynamics of Protochlorophyllide Robert Hanf,† Sonja Fey,‡ Benjamin Dietzek,†,§ Michael Schmitt,† Christiane Reinbothe,^ Steffen Reinbothe,^ Gudrun Hermann,‡ and J€urgen Popp*,†,§ †
Institute for Physical Chemistry and Abbe Centre of Photonics, Friedrich-Schiller-University Jena, Helmholtzweg 4, D-07743 Jena, Germany ‡ Institute of Biochemistry and Biophysics, Friedrich-Schiller-University Jena, Philosophenweg 12, D-07743 Jena, Germany § Institute of Photonic Technology (IPHT) Jena, Albert-Einstein-Strasse 9, D-07745 Jena, Germany ^ Centre dEtudes et de Recherches sur les Macromolecules Organiques, Universite Joseph Fourier, Centre National de la Recherche Scientifique, Federation de Recherche en Evolution, 3017, B.P.53, F-38041 Grenoble Cedex 9, France
bS Supporting Information ABSTRACT: The light-driven NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme of chlorophyll biosynthesis in angiosperms. POR’s unique requirement for light to become catalytically active makes the enzyme an attractive model to study the dynamics of enzymatic reactions in real time. Here, we use picosecond time-resolved fluorescence and femtosecond pump probe spectroscopy to examine the influence of the protein environment on the excited-state dynamics of the substrate, protochlorophyllide (PChlide), in the enzyme/substrate (PChlide/POR) and pseudoternary complex including the nucleotide cofactor NADP+ (PChlide/NADP+/ POR). In comparison with the excited-state processes of unbound PChlide, the lifetime of the thermally equilibrated S1 excited state is lengthened from 3.4 to 4.4 and 5.4 ns in the PChlide/POR and PChlide/NADP+/POR complex, whereas the nonradiative rates are decreased by ∼30 and 40%, respectively. This effect is most likely due to the reduced probability of nonradiative decay into the triplet excited state, thus keeping the risk of photosensitized side reactions in the enzyme low. Further, the initial reaction path involves the formation of an intramolecular charge-transfer state (SICT) as an intermediate product. From a strong blue shift in the excited-state absorption, it is concluded that the SICT state is stabilized by local interactions with specific protein sites in the catalytic pocket. The possible relevance of this result for the catalytic reaction in the enzyme POR is discussed.
’ INTRODUCTION Chlorophyll (Chl) plays a dominant role in oxygenic photosynthesis and, thus, in the conversion of solar energy into chemical energy. Depending on the protein environment, it is involved in light-harvesting as well as electron-transfer reactions, which are coupled to proton translocation and generation of the proton electrochemical gradient driving ATP synthesis.1 4 To allow high efficiency of photosynthesis and to avoid photodamage due to the phototoxity of Chl, efficient scavenging mechanisms operate in the thylakoid membranes harboring the photosystems. Moreover, the biosynthetic pathway leading to Chl is tightly regulated and adapted to the light environment.5 10 One of the key regulatory steps in Chl biosynthesis of all oxygenproducing photosynthetic organisms is the light-driven reduction of protochlorophyllide (PChlide) into chlorophyllide (Chlide) by the enzyme NADPH:protochlorophyllide oxidoreductase (POR, EC 1.3.1.33).11 16 POR catalyzes the proton and hydride transfer to the C17 C18 double bond in ring D of PChlide to produce Chlide, the direct precursor of Chl r 2011 American Chemical Society
(Figure 1). The proton and hydride ions are derived from a highly conserved tyrosine residue of the apoenzyme and the nicotinamid ring of the coenzyme NADPH, respectively.11 16 On the basis of latest studies, the catalytic mechanism consists of a light-activated conformational change at the active site of the enzyme/substrate complex, which is initiated by the absorption of a first photon. The absorption of a second photon triggers the actual photochemical reaction, which results in the formation of a reaction intermediate (I675*) with rate constants of ∼300 and ∼3.7 ns 1.17 20 The molecular nature of this intermediate remains unclear, but most likely, it represents an essential precursor for the subsequent hydride and proton transfer reactions occurring on the microsecond time scale.16,21 In addition to the regulatory function in chlorophyll biosynthesis, the POR enzyme also plays a significant photoprotective role. Due to the efficient binding and transformation of PChlide into Chlide in a Received: September 30, 2010 Published: June 16, 2011 7873
dx.doi.org/10.1021/jp2035899 | J. Phys. Chem. A 2011, 115, 7873–7881
The Journal of Physical Chemistry A
Figure 1. Scheme for the photoreduction of protochlorophyllide (PChlide) to chlorophyllide (Chlide) by the enzyme POR (A) and scheme for the ultrafast dynamics of free PChlide in solution (B). FC refers to the Franck Condon region; S1 denotes the thermally equilibrated S1 excited state; Sx, the secondary excited state; and SICT, the intermediate charge-transfer state. The time constants, τi, refer to the following values: τ1 = 3 ps, τ2 = 27 ps, τ3 = 200 ps, and τ4 = 3.4 ns, respectively.27,29
fast reaction with high quantum yield, it prevents the accumulation of free PChlide in the light and thus limits PChlide photodestruction and photooxidative damage caused by formation of singlet oxygen and reactive oxygen species.13,22 24 Current research work is being focused on an analysis of the very early steps in POR-driven enzyme catalysis. Hereby, POR is especially suited for performing studies on the role of the protein environment on the dynamics of the reaction. POR establishes stable substrate/cosubstrate ternary complexes in the dark, and catalysis can be initiated by a short light pulse to produce the respective POR product complex. This unique behavior enables the observation of the initial photophysical and photochemical processes at ultrafast time scales. This approach requires, however, detailed knowledge of the excited-state processes of the free non-enzyme-bound substrate, PChlide, in solution outside the protein cavity. For this reason, we have previously investigated the excited-state dynamics of PChlide in solvents of different physical properties, assuming that the solvent conditions can mimic specific environmental conditions in the enzyme/substrate complex.25 31 On the basis of picosecond time-resolved fluorescence and femtosecond pump probe experiments, a model has been proposed that explains the early light-induced dynamic events in the PChlide molecule.25 31 Accordingly, the initially excited Franck Condon state population undergoes decay into two different relaxation channels, a reactive and a nonreactive one (Figure 1). Along the nonreactive path, vibrational relaxations and cooling lead within ∼3 ps (τ1) to the population of the thermally relaxed S1 excited state, which decays by intersystem crossing on a nanosecond time scale (τ4) into a long-lived triplet state. In parallel, ultrafast excited-state motions out of the Franck Condon region open the reactive path and lead within 22 27 ps (τ2) to the formation of an intermediate photoproduct (SICT). This photoproduct is nonfluorescent and decays into the initial ground state with a characteristic time constant of 200 ps
ARTICLE
(τ3). From the available experimental evidence, it appears that the photoproduct represents an intramolecular charge-transfer state with the charge-transfer character being due to the electronwithdrawing effect of the isocyclic fifth ring directly attached to the conjugated π-electron system of the porphyrin macrocycle (Figure 1).32 It is conceivable that the charge separation within the SICT state could facilitate the photoreduction of PChlide and therefore might also play an important role for the initial catalytic processes in the POR enzyme. To vindicate our initial model, the excited-state dynamics of PChlide upon binding to the POR enzyme were analyzed. The results indicate that these dynamics are controlled by the specific protein environment at the substrate binding site of the POR enzyme molecule. As will be shown, the local active-site environment affects the formation of the intramolecular charge-transfer state (SICT) and quenches the formation of the triplet state in the de-excitation path of PChlide. The corresponding studies were performed with recombinant POR A from barley heterologously expressed in Escherichia coli. Unlike former studies, which have been focused on bacterial enzymes, this work includes POR from a higher plant.17 20,33,34
’ EXPERIMENTAL SECTION Expression and Purification of POR A from Barley (Hordeum vulgare). His-tagged POR from barley (H. vulgare)
was overexpressed and purified in a manner similar to the procedure described in Buhr et al.24 In brief, the initial cDNA encoding the POR A protein was subcloned into the expression vector pQE30 (Quiagen) at the BamHI and HindIII restriction sites. The vector DNA (pQE30-His-POR) was then transformed into competent E. coli M15 host cells. For overexpression of the recombinant POR protein, the E. coli cells were grown in Luria broth (LB) containing 25 μg/mL kanamycin and 100 μg/mL ampicillin. Exponentially growing cells were treated with 1 mM isopropyl β-D- thiogalactopyranoside (IPTG) to induce overexpression and grown for an additional 3 h at 37 C. After harvesting by centrifugation, the cell pellet was resuspended in binding buffer (50 mM Tris/HCl, 0.5 M NaCl, 20 mM imidazole, 20% glycerol, and 0.1% Triton X-100 at pH 7.6) and disrupted by cell lyses with lysozyme (1 mg/mL) and subsequent sonication on ice. For purification, the cell lysate was subjected to an affinity chromatography on Ni agarose using a HisTrap FF column (GE Healthcare). The column was washed with 10 volumes of binding buffer, and the POR protein was eluted with washing buffer containing 300 mM imidazole. The enzyme fractions obtained from this elution step were desalted on a HiPrep desalting column (GE Healthcare) with a buffer containing 50 mM Tris/HCl, 300 mM NaCl, 20% glycerol and 0.1% Triton X-100 at pH 7.6. PChlide Preparation. PChlide a was isolated from 5-day-old, dark-grown oat seedlings (Avena sativa) as reported recently.27 Briefly, the tips of the oat coleoptiles were treated with 15 mM 5-aminolevulinic acid in 35 mM potassium phosphate buffer for 48 h. Subsequent to the disruption of the oat coleoptiles by homogenization, PChlide was extracted into an ice-cold 10 mM Tricin buffer (pH 7.5) containing 75% (v/v) acetone. After centrifugation, the PChlide was transferred into diethyl ether and, in a following step, into a 4:1 mixture of methanol and 0.01 M ammonia. Finally, the PChlide was again extracted with a water/methanol mixture and fractionated by HPLC on a reversed-phase RP-18 column in a linear 20 80% acetonitrile 7874
dx.doi.org/10.1021/jp2035899 |J. Phys. Chem. A 2011, 115, 7873–7881
The Journal of Physical Chemistry A gradient. The fractions containing highly pure PChlide were lyophilized and stored at 250 K in a refrigerator until use. Reconstitution of the POR/PChlide Complexes. For the measurement of the POR/PChlide complexes in the timeresolved experiments, 10 4 M POR in activity buffer (50 mM Tris/HCl, 300 mM NaCl, 20% glycerol, 0.1% Triton X-100, 7 mM DTT, pH 7.6) was mixed with PChlide to yield a final concentration of 4 10 5 M and allowed to equilibrate in the dark for 2 h. In the case of the ternary POR/PChlide/NADP+ complex, NADP+ was added to the above POR/PChlide assay to a final concentration of 1 mM. To avoid inner filter effects, the concentration ratio of the samples for the stationary fluorescence measurements was adjusted such that the absorbance of PChlide was below 0.05 at the excitation wavelength. Triton-X-100 (0.1%) and 20% glycerol are the usual additives in the activity buffer because they increase the solubility of the POR enzyme, which is associated with etioplast membranes in nature.17,18,21,33,34 They affect neither the enzymatic activity nor the absorption spectrum of PChlide in the enzyme/substrate complexes. Extent of PChlide Binding. The apparent binding constant (KD) for PChlide binding to the POR enzyme from cyanobacteria is similar in the presence of either NADPH or NADP+ (7.7 and 2.8 μM, respectively).33,34 Using the apparent binding constant determined in our experiments for the POR enzyme from barley with NADPH as cofactor (KD = 3 μM) results in ∼95% PChlide binding to POR. Steady-State Fluorescence Spectroscopy. Fluorescence emission and excitation spectra were recorded with a Spex Fluorolog-2 spectrofluorometer in the spectral region between 400 and 800 nm using different wavelengths in the Soret and Q-band for excitation. The quantum yields were determined from corrected emission spectra using quinine sulfate in ethanol as standard and calculated according to: ΦF = ΦFR(I/I)R(ODR/OD)(n2/n2R), where ΦF is the quantum yield, I is the integrated fluorescence intensity, OD is the optical density at the excitation wavelength, n is the refractive index, and the subscript R refers to the fluorescence standard. In the low temperature experiments, the concentration of glycerol was increased to 60% in the activity buffer. Time-Resolved Spectroscopy. The ultrafast transient absorption setup has been described elsewhere in detail.35 Briefly, the output of an amplified Ti:sapphire laser (Libra, Coherent Inc.) was split and used to pump two noncollinear optical-parametric amplifiers (TOPASwhite, LightConversion Ltd.) independently. The TOPAS output (10 μJ,
