Primary Reactions of Bacteriophytochrome Observed with Ultrafast

Dec 30, 2010 - Phytochromes are red-light photoreceptor proteins that regulate a variety of responses and cellular processes in plants, bacteria, and ...
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Primary Reactions of Bacteriophytochrome Observed with Ultrafast Mid-Infrared Spectroscopy )

K. C. Toh,†, Emina A. Stojkovic,‡,^ Alisa B. Rupenyan,†,# Ivo H. M. van Stokkum,† Marian Salumbides,† Marie-Louise Groot,† Keith Moffat,‡,§ and John T. M. Kennis*,† †

Biophysics Group, Department of Physics and Astronomy, Faculty of Sciences, VU University, De Boelelaan 1081, 1081HV Amsterdam, The Netherlands ‡ Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, United States § Institute for Biophysical Dynamics, The University of Chicago, Chicago, Illinois 60637, United States

bS Supporting Information ABSTRACT: Phytochromes are red-light photoreceptor proteins that regulate a variety of responses and cellular processes in plants, bacteria, and fungi. The phytochrome light activation mechanism involves isomerization around the C15dC16 double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important recent development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues. Bphs covalently bind a biliverdin (BV) chromophore, naturally abundant in mammalian cells. Here, we report an ultrafast time-resolved mid-infrared spectroscopic study on the Pr state of two highly related Bphs from Rps. palustris, RpBphP2 (P2) and RpBphP3 (P3) with distinct photoconversion and fluorescence properties. We observed that the BV excited state of P2 decays in 58 ps, while the BV excited state of P3 decays in 362 ps. By combining ultrafast mid-IR spectroscopy with FTIR spectroscopy on P2 and P3 wild type and mutant proteins, we demonstrate that the hydrogen bond strength at the ring D carbonyl of the BV chromophore is significantly stronger in P3 as compared to P2. This result is consistent with the X-ray structures of Bph, which indicate one hydrogen bond from a conserved histidine to the BV ring D carbonyl for classical bacteriophytochromes such as P2, and one or two additional hydrogen bonds from a serine and a lysine side chain to the BV ring D carbonyl for P3. We conclude that the hydrogen-bond strength at BV ring D is a key determinant of excitedstate lifetime and fluorescence quantum yield. Excited-state decay is followed by the formation of a primary intermediate that does not decay on the nanosecond time scale of the experiment, which shows a narrow absorption band at ∼1540 cm-1. Possible origins of this product band are discussed. This work may aid in rational structure- and mechanism-based conversion of BPh into an efficient near-IR fluorescent marker.

’ INTRODUCTION Phytochromes are red-light photoreceptors that play a critical role in regulating various cellular functions in plant, fungal, and bacterial kingdoms.1-6 Bacteriophytochromes (Bphs) RpBphP2 (P2) and RpBphP3 (P3) from Rhodoseudomonas palustris in tandem regulate the synthesis of light harvesting LH4 complexes.7 The photosensory core of these proteins contains the PAS, GAF, and PHY domains that are covalently attached to an output/effector domain histidine kinase (HK). Bphs undergo reversible photoconversion between two metastable photoexcited states denoted as Pr, absorbing near 700 nm, and Pfr, absorbing near 750 nm. These states are interphotoconvertible through an isomerization mechanism of the C15dC16 double bond of a linear tetrapyrrole cofactor, biliverdin (BV). Both P2 and P3 bind BV autocatalytically at ring A through a covalent linkage with a conserved cysteine in the PAS domain. P2 and P3 have a distinct light response manifested in their absorption spectra. P2 undergoes classical Pr-Pfr photoconversion, whereas P3 is the only Bph to date that forms a Pnr state with a blue-shifted absorption spectrum, peaking at 645 nm. Like their r 2010 American Chemical Society

counterparts in plant, the photochemistry of Bphs proceeds through several intermediate stages before attaining a conformation of 15Ea of BV in light state (e.g., Pfr state) upon light activation.8-11 In an important recent development, Bph has been engineered for use as a fluorescent marker in mammalian tissues.12 Bph fluoresces in the near-IR at ∼720 nm, a wavelength less prone to scattering that can penetrate more deeply into tissue than light emitted by GFP-derived fluorescent proteins. The BV cofactor is a naturally occurring cofactor in mammalian tissue that covalently binds to a conserved cysteine in the bacteriophytochrome, and hence Bph can readily be genetically encoded. It shares such properties with flavin-binding photoreceptors such as LOV and BLUF domains for use as photonic switch or sensor.13-17 Special Issue: Graham R. Fleming Festschrift Received: July 23, 2010 Revised: December 9, 2010 Published: December 30, 2010 3778

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Figure 1. (A) Biliverdin (BV) binding site in the X-ray structure of Rhodopseudomonas palustris P3 (Protein Data Bank code 2OOL.20 (B) BV binding site in the X-ray structure of Deinoccocus radiodurans BPh (1ZTU).18 (C) BV chromophore in a ZZZssa configuration with ring and atom numbering. (D) Partial protein sequence alignment of bacteriophytochromes P3 and P2 from Rhodopseudomonas palustris, DrBphP from Deinoccocus radiodurans, AtBphP1 from Agrobacterium tumafeciens, PaBphP from Pseudomonas aeruginosa, and cyanobacterial phytochrome Cph1 from Synechocystis sp. pcc 6803. Numerical values indicate positions of amino acids in P3 primary sequence.

Phytochrome photochemistry is thus of considerable significance for biomedical research and technology. The recent determination of crystal structures of various BPhs and the cyanobacterial phytochrome Cph1 has explored the light-activated function of phytochromes.8,18-21 The linear tetrapyrrole chromophore is bound to the PAS (BPh) or GAF (Cph1) domain at ring A through covalent linkage to a conserved cysteine. BV is largely engulfed by the GAF domain, which provides most of the hydrogen bonding networks and steric and hydrophobic interactions to secure the chromophore in position. The PHY domain forms an extension to the photosensory core of phytochromes that works in tandem with the GAF domain for tuning of spectral properties and implementing photochemical effectiveness. In the Pr state the chromophore assumes a ZZZssa configuration (Figure 1) and is positioned in its binding pocket through steric interactions and hydrogen bonds from protein residues to the pyrrole rings and propionate side chains. Recent

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studies have indicated that 15Za to 15Ea isomerization of the chromophore at the C15dC16 double bond, which causes a flip of pyrrole ring D, accompanies formation of the Pfr state.8 The primary photoproduct, denoted Lumi-R, is formed on the 10-100 ps time scale and adopts the 15Ea configuration.22-27 The X-ray crystal structure of a classical Bph from Deinococcus radiodurans DrBphP and cyanobacterial phytochrome Cph1 shows a single hydrogen bond between a conserved histidine residue and the bilin chromophore ring D carbonyl in the chromophore binding pocket (Figure 1B).18,28,29 In the P3 X-ray crystal structure (Figure 1A), besides the conserved histidine, Lys-183 and Ser-297 are within hydrogen bonding distance to the ring D carbonyl.20 Importantly, P3 is the only Bph with three potential H-bonding partners interacting with D-ring carbonyl group in the Pr state, as shown in the sequence alignment (Figure 1D). These different aspects of classical Bph and P3 present an opportunity to study the influence of the chromophore binding pocket on the phytochrome photochemistry. In our previous femtosecond time-resolved absorption studies on P2 and P3 PAS-GAF-PHY constructs, we have shown that the excited-state lifetimes and the spectra of P3 are very different from P2 and other classical Bphs. Strikingly, the BV excited state of P3 decayed with a monoexponential time constant of 330 ps, significantly longer than observed in P2 and other phytochromes, which we related to the hydrogen bond strength at ring D of the BV chromophore.27 We determined that the two additional polar residues, lysine and serine located at the immediate vicinity of BV ring D, are responsible for a lowering the Lumi-R quantum yield and increasing the BV excited-state lifetime. In addition, we identified excited-state proton transfer (ESPT) from the BV pyrrole rings to the protein backbone or a bound water as the process that deactivates the BV excited state to the Pr state.27 Taken together, the fluorescence quantum yield of P3 is significantly higher than that of classical Bph and with detailed knowledge about its excited-state dynamics, P3 forms an attractive starting material to generate a highly fluorescent deep-tissue fluorescent probe by means of rational structure- and mechanismbased engineering.27 The vibrational spectrum of a protein or a protein-bound chromophore contains a wealth of information about its structure, the interaction with the environment, and electronic properties. Time-resolved IR spectroscopy is a powerful tool that can reveal many of the dynamic structural and physical-chemical properties of chromophores involved in (photo)biological reactions.30,31 In addition, it can reveal the involvement of those parts of the protein that partake in the ongoing reactions. As the primary reactions in biological photoreceptors proceed on the ultrafast time scale, femtosecond mid-IR spectroscopy is a method of choice to identify reaction mechanisms of biological photoreceptors.32-42 The femtosecond time-resolved infrared absorption study on Cph1 had shown that methine bridges, ring A/D of the phycocyanobilin (PCB) chromophore, were involved in structural changes in its primary photochemistry.24 The application of femtosecond IR on another bacteriophytochrome, Agp1, had given a similar conclusion.42 Structural changes related to the methine bridges was also inferred by steady-state resonance Raman studies on the cryo-trapped intermediate states of various phytochromes.43 However, information about the structural evolution of BV in the early photochemistry of P2 and P3 is lacking. In this work, we extend our previous studies by comparing the early photochemistry of P2 and P3 in the Pr state using ultrafast mid-IR spectroscopy. We have studied the full photosensory 3779

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PAS-GAF-PHY core of P2 and P3, as well as their short PASGAF constructs. Our results support our earlier observation that excited-state decay is significantly slower in P3 as compared to P2. Comparison of time-resolved IR spectra and FTIR spectra of P2 and P3 and site-directed mutants where hydrogen bonding to ring D was modified provides direct spectroscopic evidence that hydrogen bonding to the ring D carbonyl is indeed significantly stronger in P3 than in P2.

’ MATERIAL AND METHODS Sample Preparation. The detailed preparation of wild type P2 (PAS-GAF), P2 (PAS-GAF-PHY), and the P2 (PAS-GAFPHY) M169K/A382S (P2KS) mutant and of wild type P3 (PASGAF), P3 (PAS-GAF-PHY), and the P3 (PAS-GAF-PHY) K183M/S297A (P3MA) mutant bacteriophytochrome proteins was described previously.20 For the ultrafast mid-IR experiments, the proteins were dissolved in D2O buffer (20 mM Tris 3 HCl, pD 8 at room temperature). For the FTIR experiments, the proteins were dissolved in H2O buffer (20 mM Tris 3 HCl, pH 8 at room temperature). Femtosecond Mid-IR Spectroscopy. The experimental setup is a home-built spectrometer based on a 1 kHz amplified Ti: sapphire laser system operating at 1 kHz (Spectra Physics Hurricane) that allows visible pump/mid infrared probe in a time window from 180 fs to 3 ns, as previously described.30,32 The red excitation pulse was generated by means of a noncollinear optical parametric amplifier and centered around 680 nm, at an excitation energy of 150-250 nJ. The infrared probe had a spectral width of 200 cm-1, was spectrally dispersed after the sample, and was detected with a 32-element array detector, leading to a spectral resolution of 6 cm-1. Vibrational spectra between 1780 and 1450 cm-1 were taken in two intervals and simultaneously analyzed. Spectra were recorded at 100 time delay points between -20 ps and þ2.8 ns. During the experiments, the sample cell was continuously translated with a Lissajous scanner, which ensured sample refreshment after each laser shot and a time interval of 1 min between successive exposures to the laser beams. Background illumination to photorevert the Bph sample to Pr was provided with a LED with a center wavelength at 750 nm (P2 PASGAF-PHY, P2 PAS-GAF and P3 PAS-GAF) or 650 nm (P3 PASGAF-PHY). Data Analysis. The time-resolved data can be described in terms of a parametric model in which some parameters, such as those descriptive of the instrument response function (IRF), are wavenumber-dependent, whereas others, such as the lifetime of a certain spectrally distinct component, underlay the data at all wavenumbers. The presence of parameters that underlay the data at all wavenumbers allow the application of global analysis techniques, which model wavenumber-invariant parameters as a function of all data.44 The femtosecond transient absorption data were globally analyzed using a kinetic model consisting of sequentially interconverting evolution-associated difference spectra (EADS), i.e., 1 f 2 f 3 f ... (Figures 2, 3 and 5A) in which the arrows indicate successive monoexponential decays of increasing time constant, which can be regarded as the lifetime of each EADS. The first EADS corresponds to the time-zero difference spectrum. This procedure enables us to clearly visualize the evolution of the (excited and intermediate) states of the system. It is important to note that a sequential analysis is mathematically equivalent to a parallel (sum-of-exponentials) analysis.44 The analysis program calculates both EADS and

Figure 2. Time-resolved spectroscopy of the Rps. palustris P2 PASGAF-PHY construct. (A) Evolution-associated difference spectra (EADS) and their corresponding lifetimes resulting from global analysis of ultrafast mid-IR experiments upon excitation at 680 nm. (B) kinetic traces at 1702, 1591, and 1540 cm-1.

decay-associated difference spectra (DADS), and the time constants that follow from the analysis apply to both. In general, the EADS may well reflect mixtures of molecular states such as may arise, for instance, from heterogeneous ground states or branching at any point in the molecular evolution or inverted kinetics.45-52 Throughout the manuscript, the EADS are shown in the main text and the corresponding DADS are shown in the Supporting Information. The advantage of showing EADS over DADS is that the former are intuitively more easily interpreted. A detailed account of the global analysis methodology is given in the Supporting Information. 3780

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with a nitrogen cooled photovoltaic MCT detector (20 MHz, KV 100, Kolmar Technologies, Inc.). Two LEDs, emitting at 680 and 750 nm, were used to convert P2 to its light or dark states, respectively. For P3, LEDs emitting at 680 and 650 nm were used instead. The light minus dark FTIR data were obtained, by subtracting an initially recorded protein dark-state spectrum as the background spectrum, from the light activated (using the 680 nm LED) protein spectrum. Background and sample interferogram data were averaged from 500-2000 interferogram scans, at 4 cm-1 spectra resolution. Measurements were repeated by illuminating the sample with a 750 nm (on P2) or a 650 nm (on P3) light to deactivate the light state of the protein and by taking a background and a light activated spectrum. The FTIR sample was prepared using a drop of 2 mL of sample at OD700 nm of ∼100 (in 20 mM Tris/HCl pH8 buffer) and spread between two tightly fixed CaF2 windows.

’ RESULTS AND DISCUSSION

Figure 3. Time-resolved spectroscopy of the Rps. palustris P2 PAS-GAF construct. (A) Evolution-associated difference spectra (EADS) and their corresponding lifetimes resulting from global analysis of ultrafast mid-IR experiments upon excitation at 680 nm. (B) Kinetic traces at 1708, 1588, and 1541 cm-1.

To account for unresolved fast relaxation processes within the instrument response that become apparent as a sharp peak around zero time delay, a pulse follower was included in the global analysis procedure. To avoid any effect of prezero signals arising from perturbed free induction decay (FID)53 and unresolved relaxation dynamics around zero delay on the outcome of global analysis procedure, the prezero to 0.5 ps spectra were given a low weight. Differential Fourier-Transform Infrared (FTIR) Spectroscopy. The differential FTIR data were recorded at room temperature using a FTIR spectrometer (IFS 66s Bruker) equipped

Ultrafast Mid-IR Spectroscopy of P2. The reaction dynamics of P2 (PAS-GAF-PHY) in the Pr state was investigated from a subpicosecond time scale up to 3 ns by means of ultrafast mid-IR spectroscopy. The sample was excited at 680 nm, and a spectral range of 1470-1780 cm-1 that covers the CdO and CdC vibration regions was monitored. The data were globally analyzed in terms of a kinetic scheme with sequentially interconverting species, where each species is characterized by an EADS that has a specific lifetime. One decay lifetime of 58 ps and a nondecaying component were required for an adequate fit of the data. The EADS are shown in Figure 2A, the corresponding DADS are shown in Figure S1 of Supporting Information Figure 2B shows kinetic traces at selected wavenumbers. Note that prezero signals in the kinetics, most apparent in the 1702 cm-1 trace in Figure 2B, arise from perturbed FID53 and do not relate the Bph photophysics. In some of the kinetics, an unresolved fast relaxation within the instrument response becomes apparent as a peak around zero delay. It is taken into account by including a pulse follower in the global analysis procedure and not further interpreted. The 58 ps component is assigned to the BV excited state. In our visible pump-probe experiments, we observed a major excitedstate decay component of 50 ps, consistent with the present data. In addition, 0.4, 4, and 250 ps components were observed with visible transient absorption spectroscopy.27 The absence of these components in our femtosecond mid-IR data is likely due to limited signalto-noise, or to a relative insensitivity of the IR spectra to the dynamics associated with these time constants. The 58 ps EADS shows major bleach bands in the range 1570-1640 cm-1, attributed to the BV chromophore CdC methine bridges vibrational bands.24,42 These bands are located at 1591, 1613, and 1635 cm-1. Their frequencies closely resemble those in the Pr state of plant phytochrome A (PhyA), DrBphP, and Agrobacterium bacteriophytochrome 1 (Agp1), as observed with resonant Raman spectroscopy.54,55 The largest bleach band at 1591 cm-1 in our femtosecond mid-IR data is assignable to the ring C-D methine bridge stretch band56 (see Figure 1C for BV ring and atom numbering). The bleach band at ∼1630 cm-1 is assigned to the ring A-B methine bridge (C4dC5) vibration band.56 Due to the reduction of bond orders in the excited states, these bleach bands are expected to be downshifted in the S1 state.24 The 58 ps EADS shows major bleach bands at 1728 and 1702 cm-1 (Figure 2A). In PhyA, the high-frequency carbonyl 3781

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Figure 4. Time-resolved spectroscopy of the Rps. palustris P3 PASGAF-PHY construct. (A) Evolution-associated difference spectra (EADS) and their corresponding lifetimes resulting from global analysis of ultrafast mid-IR experiments upon excitation at 680 nm. (B) Kinetic traces at 1678, 1588, and 1541 cm-1.

band at 1730 cm-1 was assigned to ring A C1dO stretching through 18O isotope labeling of PCB at this site.57,58 Thus, the 1728 cm-1 band can confidently be assigned to the BV ring A C1dO stretch vibration. It follows that the 1702 cm-1 band is associated with the BV ring D C19dO vibration, which is consistent with assignments made for Agp1 by Diller and coworkers26 and Bartl and co-workers.59 The CdO stretching bands observed here have a lower frequency than those found in Cph1 (located at 1738 and ∼1720 cm-1 respectively), probably due to a different environment of the chromophore. Also, the different conjugation at ring A between BV and PCB may play a

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role. The 58 ps EADS shows a bleach band at 1540 cm-1 that was observed previously in femtosecond mid-IR spectroscopy on phytochromes but not interpreted.24,42 The final nondecaying component is formed from the BV excited state in 58 ps and persists through our experimental time scale of 3 ns. It is assigned to the primary photoproduct Lumi-R. The nondecaying component has a low amplitude and most of its features do not rise above the noise level, in keeping with the low quantum yield of Lumi-R formation of 0.13.27 Surprisingly, however, is the occurrence of a very sharp absorption band at 1541 cm-1, at the same frequency as the bleach in the 58 ps EADS. Figure 2B shows the kinetic trace that demonstrates the rise of this positive-amplitude feature. Its possible origin will be discussed further on in the paper. We also performed ultrafast IR experiments on the P2 PASGAF construct that undergoes limited photoconversion20 under identical experimental conditions. Two time constants of 4 and 175 ps and a long-lived component were required for an adequate fit of the data. Figure 3A shows the resulting EADS, while kinetic traces at selected wavelengths are shown in Figure 3B. Figure S2 (Supporting Information) shows the DADS. The BV excited-state lifetime is significantly longer at 175 ps than in the P2 PAS-GAF-PHY construct, which agrees with our findings from ultrafast visible spectroscopy.60 The infrared signature of excited-state BV is essentially the same as that in P2 PAS-GAF-PHY, with CdO bleaches at 1734 and 1708 cm-1, methine bridge stretches at 1588, 1611, and 1635 cm-1, and a bleach at 1536 cm-1. As in the PAS-GAF-PHY construct, the long-lived photoproduct shows a pronounced induced absorption band at 1541 cm-1. In the P2 PAS-GAF construct, a 4 ps component was resolved. Inspection of the corresponding DADS (Figure S1, Supporting Information) reveals a pattern of alternating negative and positive bands at similar amplitudes, with a negative band at 1690 cm-1, a positive band at 1655 cm-1, and a broad negative band near 1590 cm-1. The pattern of the 4 ps DADS does not resemble BV excited-state decay, as the negative feature at 1690 cm-1 does not correspond to a BV CdO vibration in the Pr state, and the broad negative feature near 1590 cm-1 does not resemble the BV methine bridge stretches in Pr as observed for the 175 ps DADS. Hence, we conclude that the 4 ps component represents a relaxation process in the excited state.27 Ultrafast Mid-IR Spectroscopy of P3. We investigated the reaction dynamics of the P3 PAS-GAF-PHY construct in the Pr state by means of ultrafast mid-IR spectroscopy to uncover structural aspects of its photoreaction. The sample was excited at 680 nm and a spectral range of 1470-1780 cm-1 that covers the CdO and CdC vibration regions was monitored. Global analysis indicated that three lifetime components of 37 and 362 ps and a nondecaying component were required for an adequate fit of the data. The EADS are shown in Figure 4A, whereas kinetic traces at selected wavenumbers are shown in Figure 4B. Figure S3 (Supporting Information) shows the DADS. The first EADS (Figure 4A, circles) has a lifetime of 37 ps, similar to the 53 ps component observed with ultrafast visible spectroscopy. Its origin will be discussed below. It evolves into the EADS that has a lifetime of 362 ps (Figure 4A, solid diamonds). The 362 EADS corresponds to a relaxed form of the BV excited state similar to, although somewhat shorter than, that observed with ultrafast visible spectroscopy, which had a lifetime of 450 ps.27 It has an overall shape similar to that of the BV excited state of P2 (Figure 2A) but differs in some important 3782

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The Journal of Physical Chemistry A aspects. It shows strong bleach bands at 1588, 1603, and 1630 cm-1 that represent the BV chromophore CdC methine bridges vibrational bands. The strongest bleach band at 1588 cm-1 is assigned to the BV ring C-D methine bridge (C15dC16) stretching. The 1630 cm-1 bleach band is assigned to the ring A-B methine bridge (C4dC5) stretching. These bands are similar to those observed in P2 (Figure 2A), Cph1 and Agp1.24,42 Inspection of the carbonyl region (1680-1740 cm-1) reveals a pattern that is quite different from that of P2: The ring A CdO stretching band is located at 1736 cm-1 (Figure 4A, solid diamonds), a frequency comparable to those found in P2, Cph1 and Agp1.24,42 Strikingly, a pronounced bleach is observed at 1678 cm-1, a frequency where P2 and other phytochromes show no such signal. Given its frequency and its prompt rise within the instrument response, it must correspond to a CdO mode of the BV chromophore. As the ring A C1dO was already firmly assigned to the 1736 cm-1 band,58 the 1678 cm-1 band most likely corresponds to the BV ring D C19dO stretch mode. We will substantiate this assignment below by means of FTIR spectroscopy on P2, P3, and site-directed mutants. This observation implies that in P3, the ring D CdO stretch mode has a significantly lower frequency than in other phytochromes. We will demonstrate below that the downshift of the ring D CdO frequency results from the increased hydrogen bond strength to ring D in P3. As in P2, a strong and broad bleach band at 1541 cm-1 is observed in the BV excited state of P3. The BV excited state evolves in 362 ps to the nondecaying EADS (open diamonds), which is assigned to the primary photoproduct Lumi-R. This EADS has a very low amplitude, in keeping with the low Lumi-R quantum yield of P3 of 0.06.27 As in P2, a sharp induced absorption band at 1541 cm-1 is observed. Figure 4B shows the rise of this product band. Other bands in this EADS are mostly buried in the noise and will not be further considered. The spectral evolution shows a 37 ps component in addition to the 362 ps and nondecaying components. Inspection of the DADS (Figure S3, Supporting Information) shows negative bands at 1730, 1690, and 1655 cm-1, a broad negative feature between 1580 and 1630 cm-1, and positive bands at 1675 cm-1 and at frequencies below 1530 cm-1. The mostly negative pattern suggests that the 37 ps component mainly represents a BV excited-state decay process. However, the negative signals at 1690 at 1660 cm-1 do not match the carbonyl frequencies in Pr, the bands at 1580-1630 are shifted by ∼6 cm-1 with respect to those of Pr, and we conclude that the 37 ps does not represent decay of the Pr excited state. We conclude that this component remains difficult to interpret in specific molecular terms. We also performed ultrafast IR experiments on the P3 PASGAF construct under identical experimental conditions. P3 PASGAF does not form the Pnr state and undergoes photoconversion to a Meta-R like state.20 Figure 5A shows the resulting EADS, kinetic traces at selected wavelengths are shown in Figure 5B. Figure S4 (Supporting Information) shows the DADS. The BV excited-state lifetime is significantly longer at 435 ps than in the P3 PAS-GAF-PHY construct, which agrees with our findings from ultrafast visible spectroscopy.60 In addition, the 9.5 ps component observed in the P3 PAS-GAF-PHY construct does not appear in these data. The infrared signature of excited-state BV is essentially the same as that in P3 PAS-GAFPHY, with the ring A CdO bleaches at 1741 cm-1, the ring D CdO bleach at 1677 cm-1, methine bridge stretches at 1588, 1612, and 1635 cm-1, and a bleach at 1546 cm-1. As in

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Figure 5. Time-resolved spectroscopy of the Rps. palustris P3 PAS-GAF construct. (A) Evolution-associated difference spectra (EADS) and their corresponding lifetimes resulting from global analysis of ultrafast mid-IR experiments upon excitation at 680 nm. (B) Kinetic traces at 1677, 1588, and 1541 cm-1.

the PAS-GAF-PHY construct, the long-lived photoproduct shows a pronounced induced absorption band at 1541 cm-1. FTIR Spectroscopy. In the ultrafast IR experiments on P3, we observed a BV CdO band at a particularly low frequency of 1678 cm-1 (Figures 4A and 5A) as compared to P2 (Figures 2A and 3A) and other classical (bacterio)phytochromes24,42 and assigned it to the ring D carbonyl stretch mode. The lowering of the ring D carbonyl frequency is most likely due to the stronger hydrogen bonding at this site: the P3 X-ray structure shows 2-3 amino acids hydrogen bonding to the carbonyl of ring D in dark state, i.e., the conserved His-299, Lys-183, and Ser-297.20 In classical phytochromes, only a conserved His hydrogen bonds to ring D18,28,61 (Figure 1). To investigate this idea, we performed 3783

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The Journal of Physical Chemistry A light-minus-dark FTIR spectroscopy on wild type P3 and the P3MA mutant,27 where the two polar amino acids Lys-183 and Ser-297 are mutated to methionine and alanine, respectively, eliminating the two hydrogen bonds. Also, FTIR spectra were taken on the classical Bph P2 and its P2KS mutant, where Met169 and Ala-382 were replaced by polar residues Lys and Ser. All FTIR experiments were performed on PAS-GAF-PHY constructs. We note that the P2KS and P3MA mutants retain their respective wild-type photoconversion properties, i.e., P2KS converts to Pfr and P3MA converts to Pnr.20 Figure 6A shows the light-minus-dark FTIR spectra of P3 wild type (solid line) and the P3MA mutant (crossed symbol line). In P3 wild type, carbonyl bleaches are observed at 1734 and 1685 cm-1 (Figure 6A, solid line), assigned to ring A C1dO and ring D C19dO, respectively. This result is consistent with those of ultrafast IR spectroscopy, which indicated ring A and D carbonyl frequencies at 1736 and 1678 cm-1 (Figure 4A) (note that the FTIR spectra were taken in H2O and ultrafast IR spectra in D2O, giving rise to slightly different frequencies). In the P3MA mutant, the bleach at 1685 cm-1 has disappeared and a new bleach at 1711 cm-1 has appeared (Figure 6A, crossed symbol line), indicating that the BV ring D carbonyl shifts up by 26 cm-1 upon replacement of the hydrogen bonding amino acids Ser and Lys by nonpolar amino acids Met and Ala. Figure 6B shows the light-minus-dark FTIR spectra of P2 wild type (solid line) and the P2KS mutant (crossed symbol line). In P2 wild type, which forms only a single hydrogen bond from a conserved His to ring D, bleaches are observed at 1732 and 1703 cm-1 (Figure 6B, solid line), assigned to the ring A C1dO and ring D C19dO, respectively. With ultrafast IR, similar frequencies are observed at 1728 and 1702 cm-1 (Figure 2A). In the P2KS mutant, where M169 was replaced by Lys and A283 by Ser (the equivalent amino acids in P3), it is expected that two additional hydrogen bonds are formed to the ring D carbonyl. Indeed, the FTIR spectrum of the P2KS mutant (Figure 6B, crossed symbol line) shows a downshifting of ring D C19dO stretching frequency from 1703 (in WT) to 1676 cm-1 (in P2KS) in the dark state. We conclude that in P3 wild type in the dark, hydrogen bonding to the ring D carbonyl is significantly stronger than in P2, consistent with the X-ray structures of P3 and classical (B)ph (Figure 1).20,28,61 This result further corroborates our earlier work,27 where we identified the factors that determine the BV excited-state lifetimes and isomerization quantum yields of wildtype and mutants of P2 and P3. We concluded that the hydrogenbond strength to ring D is rate-limiting for isomerization and the excited-state lifetime and that the quantum yields of fluorescence and isomerization are determined by excited-state deprotonation of biliverdin at the pyrrole rings, in competition with hydrogenbond rupture between the D-ring and the apoprotein.27 Origin of 1540 cm-1 Band. An interesting observation of the present work is a band near 1540 cm-1 that appears as a broad bleach in the BV excited state (thus corresponding to Pr) and a narrow induced absorption at essentially the same frequency in the primary photoproduct in all Bph variants studied here, i.e., P2 PAS-GAF-PHY, P2 PAS-GAF, P3 PAS-GAF-PHY, and P3 PASGAF. Because the 1540 cm-1 band in the primary photoproduct has an amplitude that exceeds all other product bands, it might correspond to a molecular state that plays an integral role in the primary photochemistry of bacteriophytochrome. The question arises what specific vibrational mode(s) of BV these bands belong to. We first note that the negative (Pr) and positive

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Figure 6. (A) Light-minus-dark FTIR spectroscopy of wild type P3 PAS-GAF-PHY (solid line) and the P3MA PAS-GAF-PHY mutant (crossed line). (B) Light-minus-dark FTIR spectroscopy of wild type P2 PAS-GAF-PHY (solid line) and the P2KS PAS-GAF-PHY mutant (crossed line).

(photoproduct) bands do not necessarily relate to the exact same vibrational mode. In previous ultrafast mid-IR experiments on Cph1 and Agp1, a similar but smaller bleach band at ∼1540 cm-1 was observed.24,42 With resonant Raman spectroscopy on plant PhyA in D2O, Pr shows a band at 1547 cm-1, and cryotrapped Lumi-R exhibited a sharp band at 1541 cm-1.43 In DrBphP, a band at 1546 cm-1 was observed in D2O with resonant Raman spectroscopy.55 In neither of these studies were the bands near 1540 cm-1 interpreted. It does not belong to the N-H in-plane bending vibrational band (at ∼1570 cm-1 in PhyA, Agp1 and DrBphP in H2O10,54,55) because this band downshifts to below 1100 cm-1 upon deuteration.43,55 As a first possible origin of the 1540 cm-1 band, calculations and IR absorption experiments on the model compound biliverdin dimetyl ester have indicated a mode at 1543 cm-1 in D2O that corresponded to the CdC ring stretch and C-vinyl stretch band of ring D.57 As a second possibility for the origin of the ∼1540 cm-1 band, recent DFT calculations have indicated that in a pyrrole-N deuterated PCB chromophore in a ZZZssa configuration, a band near 1540 cm-1 arises that includes mainly stretching coordinates from ring B and, to a minor content, from the B-C and A-B methine bridges. In the ZZEssa configuration, this band slightly shifts to 1542 cm-1.62 It is difficult to 3784

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The Journal of Physical Chemistry A understand how such a band assignment would relate to Lumi-R formation because no significant changes are thought to occur at ring B upon isomerization about the C15dC16 double bond. We note, however, that the long-lived 1540 cm-1 absorption does not necessarily relate to Lumi-R and may correspond to a ground-state intermediate on the pathway of Pr reformation. Such ground-state intermediates were recently observed for various phytochromes.26,63 At this stage the origin of the 1540 cm-1 bands remain unclear and will be the subject of further studies.

’ CONCLUSIONS Here, we have reported an ultrafast mid-IR study of two related bacteriophytochromes: P2, which shows classical Pr-Pfr photochemistry and P3, which shows an unusual Pr - Pnr photochemistry. In P2, BV excited-state decay occurs with a time constant of 58 ps, largely consistent with our results from visible transient absorption spectroscopy which indicated a biexponential decay with a main decay component of 60 ps. Excited-state decay in P3 is significantly slower with a time constant of 362 ps, which is also consistent with visible transient absorption results.27 In our previous work, we proposed that the slower excited-state decay of P3 is related to an increased hydrogen bond strength at ring D, with three amino acid side chains (His, Lys, and Ser) competing for hydrogen bonding to the ring D carbonyl in P3.20,27 In P2, only one such hydrogen bond can form from conserved His to ring D.18,61 Here, we obtained direct spectroscopic evidence for increased hydrogen bond strength at ring D in P3: ultrafast IR spectroscopy on P2 and P3, and FTIR spectroscopy on the P2 and P3 wild types and P3MA and P2KS mutants indicated that in P3, the ring D C19dO stretch mode has an unusually low vibrational frequency at 1685-1678 cm-1. In contrast, in P2 the ring D C19dO stretch mode is located at 1703 cm-1, which demonstrates that P3 has one or two additional hydrogen bonds to ring D. ’ ASSOCIATED CONTENT

bS

Supporting Information. DADS spectra. Discussion of model based data analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Phone þ31205987212. )

Present Addresses

Imperial College, London, United Kingdom. Department of Biology, Northeastern Illinois University, Chicago. # Chemistry Department, University of Amsterdam, Amsterdam, The Netherlands. ^

’ ACKNOWLEDGMENT We are grateful to Peter Hildebrandt of Technical University Berlin for sharing unpublished results. We thank Jos Thieme for technical support. K.C.T. and J.T.M.K. were supported by the Earth and Life Sciences Council of The Netherlands Foundation for Scientific Research (NWO-ALW) through a VIDI grant to J. T.M.K. E.A.S and K.M. were supported by an NIH grant GM036452 to K. M.

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Bph, bacteriophytochrome; BV, biliverdin; PCB, phycocyanobilin; EADS, evolution-associated difference spectrum; DADS, decay-associated difference spectrum; ESPT, excited-state proton transfer

’ REFERENCES (1) Borthwick, H. A.; Hendricks, S. B.; Parker, M. W.; Toole, E. H.; Toole, V. K. Proc. Natl. Acad. Sci. U.S.A. 1952, 38, 662. (2) Butler, W. L.; Norris, K. H.; Siegelman, H. W.; Hendricks, S. B. Proc. Natl. Acad. Sci. U.S.A. 1959, 45, 1703. (3) Davis, S. J.; Vener, A. V.; Vierstra, R. D. Science 1999, 286, 2517. (4) Hughes, J.; Lamparter, T.; Mittmann, F.; Hartmann, E.; Gartner, W.; Wilde, A.; Borner, T. Nature 1997, 386, 663. (5) Lamparter, T.; Michael, N.; Mittmann, F.; Esteban, B. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11628. (6) Blumenstein, A.; Vienken, K.; Tasler, R.; Purschwitz, J.; Veith, D.; Frankenberg-Dinkel, N.; Fischer, R. Curr. Biol. 2005, 15, 1833. (7) Giraud, E.; Zappa, S.; Vuillet, L.; Adriano, J. M.; Hannibal, L.; Fardoux, J.; Berthomieu, C.; Bouyer, P.; Pignol, D.; Vermeglio, A. J. Biol. Chem. 2005, 280, 32389. (8) Yang, X.; Kuk, J.; Moffat, K. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 14715. (9) Inomata, K.; Hammam, M. A.; Kinoshita, H.; Murata, Y.; Khawn, H.; Noack, S.; Michael, N.; Lamparter, T. J. Biol. Chem. 2005, 280, 24491. (10) Borucki, B.; von Stetten, D.; Seibeck, S.; Lamparter, T.; Michael, N.; Mroginski, M. A.; Otto, H.; Murgida, D. H.; Heyn, M. P.; Hildebrandt, P. J. Biol. Chem. 2005, 280, 34358. (11) von Stetten, D.; Seibeck, S.; Michael, N.; Scheerer, P.; Mroginski, M. A.; Murgida, D. H.; Krauss, N.; Heyn, M. P.; Hildebrandt, P.; Borucki, B.; Lamparter, T. J. Biol. Chem. 2007, 282, 2116. (12) Shu, X.; Royant, A.; Lin, M. Z.; Aguilera, T. A.; Lev-Ram, V.; Steinbach, P. A.; Tsien, R. Y. Science 2009, 324, 804. (13) Drepper, T.; Eggert, T.; Circolone, F.; Heck, A.; Krauss, U.; Guterl, J. K.; Wendorff, M.; Losi, A.; Gartner, W.; Jaeger, K. E. Nat. Biotechnol. 2007, 25, 443. (14) Schroder-Lang, S.; Schwarzel, M.; Seifert, R.; Strunker, T.; Kateriya, S.; Looser, J.; Watanabe, M.; Kaupp, U. B.; Hegemann, P.; Nagel, G. Nat. Methods 2007, 4, 39. (15) Strickland, D.; Moffat, K.; Sosnick, T. R. Proc. Natl. Acad. Sci. U. S.A. 2008, 105, 10709. (16) Wu, Y. I.; Frey, D.; Lungu, O. I.; Jaehrig, A.; Schlichting, I.; Kuhlman, B.; Hahn, K. M. Nature 2009, 461, 104. (17) Chapman, S.; Faulkner, C.; Kaiserli, E.; Garcia-Mata, C.; Savenkov, E. I.; Roberts, A. G.; Oparka, K. J.; Christie, J. M. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 20038. (18) Wagner, J. R.; Brunzelle, J. S.; Forest, K. T.; Vierstra, R. D. Nature 2005, 438, 325. (19) Wagner, J. R.; Zhang, J.; Brunzelle, J. S.; Vierstra, R. D.; Forest, K. T. J. Biol. Chem. 2007, 282, 12298. (20) Yang, X.; Stojkovic, E. A.; Kuk, J.; Moffat, K. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 12571. (21) Rohmer, T.; Lang, C.; Hughes, J.; Essen, L. O.; Gartner, W.; Matysik, J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 15229. (22) Andel, F., III; Hansson, K. C.; Gai, F.; Anfinrud, P. A.; Mathies, R. A. Biospectroscopy 1997, 3, 421. (23) Heyne, K.; Herbst, J.; Stehlik, D.; Esteban, B.; Lamparter, T.; Hughes, J.; Diller, R. Biophys. J. 2002, 82, 1004. (24) van Thor, J. J.; Ronayne, K. L.; Towrie, M. J. Am. Chem. Soc. 2007, 129, 126. (25) Dasgupta, J.; Frontiera, R. R.; Taylor, K. C.; Lagarias, J. C.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 1784. (26) Schumann, C.; Gross, R.; Michael, N.; Lamparter, T.; Diller, R. ChemPhysChem 2007, 8, 1657. (27) Toh, K. C.; Stojkovic, E. A.; van Stokkum, I. H. M.; Moffat, K.; Kennis, J. T. M. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 9170. (28) Essen, L. O.; Mailliet, J.; Hughes, J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 14709. 3785

dx.doi.org/10.1021/jp106891x |J. Phys. Chem. A 2011, 115, 3778–3786

The Journal of Physical Chemistry A (29) Cornilescu, G.; Ulijasz, A. T.; Cornilescu, C. C.; Markley, J. L.; Vierstra, R. D. J. Mol. Biol. 2008, 383, 403-413. (30) Groot, M. L.; van Wilderen, L. J. G. W.; Di Donato, M. Photochem. Photobiol. Sci. 2007, 6, 501. (31) Kotting, C.; Gerwert, K. ChemPhysChem 2005, 6, 881. (32) Groot, M. L.; van Wilderen, L. J. G. W.; Larsen, D. S.; van der Horst, M. A.; van Stokkum, I. H. M.; Hellingwerf, K. J.; van Grondelle, R. Biochemistry 2003, 42, 10054. (33) Herbst, J.; Heyne, K.; Diller, R. Science 2002, 297, 822. (34) Heyne, K.; Mohammed, O. F.; Usman, A.; Dreyer, J.; Nibbering, E. T. J.; Cusanovich, M. A. J. Am. Chem. Soc. 2005, 127, 18100. (35) Schumann, C.; Gross, R.; Wolf, M. M. N.; Diller, R.; Michael, N.; Lamparter, T. Biophys. J. 2008, 94, 3189. (36) Stoner-Ma, D.; Jaye, A. A.; Matousek, P.; Towrie, M.; Meech, S. R.; Tonge, P. J. J. Am. Chem. Soc. 2005, 127, 2864. (37) van Thor, J. J.; Ronayne, K. L.; Towrie, M. J. Am. Chem. Soc. 2007, 129, 126. (38) van Wilderen, L. J. G. W.; van der Horst, M. A.; van Stokkum, I. H. M.; Hellingwerf, K. J.; van Grondelle, R.; Groot, M. L. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15050. (39) Bonetti, C.; Mathes, T.; van Stokkum, I. H. M.; Mullen, K. M.; Groot, M. L.; van Grondelle, R.; Hegemann, P.; Kennis, J. T. M. Biophys. J. 2008, 95, 4790. (40) Alexandre, M. T. A.; Domratcheva, T.; Bonetti, C.; van Wilderen, L.; van Grondelle, R.; Groot, M. L.; Hellingwerf, K. J.; Kennis, J. T. M. Biophys. J. 2009, 97, 227. (41) Kennis, J. T. M.; Groot, M. L. Curr. Opin. Struct. Biol. 2007, 17, 623. (42) Schumann, C.; Gross, R.; Michael, N.; Lamparter, T.; Diller, R. ChemPhysChem 2007, 8, 1657. (43) Kneip, C.; Hildebrandt, P.; Schlamann, W.; Braslavsky, S. E.; Mark, F.; Schaffner, K. Biochemistry 1999, 38, 15185. (44) van Stokkum, I. H. M.; Larsen, D. S.; van Grondelle, R. Biochimica Et Biophysica Acta-Bioenergetics 2004, 1657, 82. (45) Gradinaru, C. C.; Kennis, J. T. M.; Papagiannakis, E.; van Stokkum, I. H. M.; Cogdell, R. J.; Fleming, G. R.; Niederman, R. A.; van Grondelle, R. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 2364. (46) Papagiannakis, E.; Kennis, J. T. M.; van Stokkum, I. H. M.; Cogdell, R. J.; van Grondelle, R. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 6017. (47) Gauden, M.; Yeremenko, S.; Laan, W.; van Stokkum, I. H. M.; Ihalainen, J. A.; van Grondelle, R.; Hellingwerf, K. J.; Kennis, J. T. M. Biochemistry 2005, 44, 3653. (48) Gauden, M.; Grinstead, J. S.; Laan, W.; van Stokkum, H. M.; Avila-Perez, M.; Toh, K. C.; Boelens, R.; Kaptein, R.; van Grondelle, R.; Hellingwerf, K. J.; Kennis, J. T. M. Biochemistry 2007, 46, 7405. (49) Berera, R.; Herrero, C.; van Stokkum, L. H. M.; Vengris, M.; Kodis, G.; Palacios, R. E.; van Amerongen, H.; van Grondelle, R.; Gust, D.; Moore, T. A.; Moore, A. L.; Kennis, J. T. M. Proc. Natl. Acad. Sci. U.S. A. 2006, 103, 5343. (50) Bonetti, C.; Alexandre, M. T. A.; van Stokkum, I. H. M.; Hiller, R. G.; Groot, M. L.; van Grondelle, R.; Kennis, J. T. M. Phys. Chem. Chem. Phys. 2010, 12, 9256. (51) Berera, R.; van Stokkum, I. H. M.; Kodis, G.; Keirstead, A. E.; Pillai, S.; Herrero, C.; Palacios, R. E.; Vengris, M.; van Grondelle, R.; Gust, D.; Moore, T. A.; Moore, A. L.; Kennis, J. T. M. J. Phys. Chem. B 2007, 111, 6868. (52) Berera, R.; van Grondelle, R.; Kennis, J. T. M. Photosynth. Res. 2009, 101, 105. (53) Hamm, P. Chem. Phys. 1995, 200, 415. (54) Andel, F., 3rd; Lagarias, J. C.; Mathies, R. A. Biochemistry 1996, 35, 15997. (55) Wagner, J. R.; Zhang, J.; von Stetten, D.; Gunther, M.; Murgida, D. H.; Mroginski, M. A.; Walker, J. M.; Forest, K. T.; Hildebrandt, P.; Vierstra, R. D. J. Biol. Chem. 2008, 283, 12212. (56) Margulies, L.; Toporowics, M. J. Am. Chem. Soc. 1984, 106, 7331. (57) Smit, K.; Matysik, J.; Hildebrandt, P.; Mark, F. J. Phys. Chem. 1993, 97, 11887.

ARTICLE

(58) Foerstendorf, H.; Benda, C.; Gartner, W.; Storf, M.; Scheer, H.; Siebert, F. Biochemistry 2001, 40, 14952. (59) Piwowarski, P.; Ritter, E.; Hofmann, K. P.; Hildebrandt, P.; von Stetten, D.; Scheerer, P.; Michael, N.; Lamparter, T.; Bartl, F. ChemPhysChem 2010, 11, 1207. (60) Toh, K. C.; Stojkovic, E. A.; van Stokkum, I. H. M.; Moffat, K.; Kennis, J. T. M. Manuscript submitted 2010. (61) Wagner, J. R.; Zhang, J. R.; Brunzelle, J. S.; Vierstra, R. D.; Forest, K. T. J. Biol. Chem. 2007, 282, 12298. (62) Borucki, B.; von Stetten, D.; Seibeck, S.; Lamparter, T.; Michael, N.; Mroginski, M. A.; Otto, H.; Murgida, D. H.; Heyn, M. P.; Hildebrandt, P. J. Biol. Chem. 2005, 280, 34358. (63) van Wilderen, L.; Clark, I. P.; Towrie, M.; van Thor, J. J. J. Phys. Chem. B 2009, 113, 16354.

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