Primary Reactions of Bacteriophytochrome Observed …ivo/pub/2011/Toh_JPCA115_3778.pdfPrimary Reactions of Bacteriophytochrome Observed with Ultrafast ... ﬂuorescesinthenear-IRat∼720nm

Published: December 30, 2010

r 2010 American Chemical Society 3778 dx.doi.org/10.1021/jp106891x | J. Phys. Chem. A 2011, 115, 3778–3786

ARTICLE

pubs.acs.org/JPCA

Primary Reactions of Bacteriophytochrome Observed with UltrafastMid-Infrared SpectroscopyK. C. Toh,†, ) Emina A. Stojkovi�c,‡,^ 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 regulatea variety of responses and cellular processes in plants, bacteria, and fungi. Thephytochrome light activation mechanism involves isomerization around theC15dC16 double bond of an open-chain tetrapyrrole chromophore, resulting ina flip of its D-ring. In an important recent development, bacteriophytochrome(Bph) has been engineered for use as a fluorescent marker in mammaliantissues. Bphs covalently bind a biliverdin (BV) chromophore, naturallyabundant in mammalian cells. Here, we report an ultrafast time-resolved mid-infrared spectroscopic study on the Pr state of twohighly related Bphs from Rps. palustris, RpBphP2 (P2) and RpBphP3 (P3) with distinct photoconversion and fluorescenceproperties. We observed that the BV excited state of P2 decays in 58 ps, while the BV excited state of P3 decays in 362 ps. Bycombining ultrafast mid-IR spectroscopy with FTIR spectroscopy on P2 and P3 wild type andmutant proteins, we demonstrate thatthe hydrogen bond strength at the ring D carbonyl of the BV chromophore is significantly stronger in P3 as compared to P2. Thisresult is consistent with the X-ray structures of Bph, which indicate one hydrogen bond from a conserved histidine to the BV ring Dcarbonyl for classical bacteriophytochromes such as P2, and one or two additional hydrogen bonds from a serine and a lysine sidechain to the BV ring D carbonyl for P3. We conclude that the hydrogen-bond strength at BV ring D is a key determinant of excited-state lifetime and fluorescence quantum yield. Excited-state decay is followed by the formation of a primary intermediate that doesnot decay on the nanosecond time scale of the experiment, which shows a narrow absorption band at∼1540 cm-1. Possible originsof this product band are discussed. This workmay aid in rational structure- andmechanism-based conversion of BPh into an efficientnear-IR fluorescent marker.

’ INTRODUCTION

Phytochromes are red-light photoreceptors that play a criticalrole in regulating various cellular functions in plant, fungal, andbacterial kingdoms.1-6 Bacteriophytochromes (Bphs) RpBphP2(P2) and RpBphP3 (P3) from Rhodoseudomonas palustris intandem regulate the synthesis of light harvesting LH4complexes.7 The photosensory core of these proteins containsthe PAS, GAF, and PHY domains that are covalently attached toan output/effector domain histidine kinase (HK). Bphs undergoreversible photoconversion between two metastable photoex-cited states denoted as Pr, absorbing near 700 nm, and Pfr,absorbing near 750 nm. These states are interphotoconvertiblethrough an isomerization mechanism of the C15dC16 doublebond of a linear tetrapyrrole cofactor, biliverdin (BV). Both P2and P3 bind BV autocatalytically at ring A through a covalentlinkage with a conserved cysteine in the PAS domain. P2 and P3have a distinct light response manifested in their absorptionspectra. P2 undergoes classical Pr-Pfr photoconversion,whereas P3 is the only Bph to date that forms a Pnr state witha blue-shifted absorption spectrum, peaking at 645 nm. Like their

counterparts in plant, the photochemistry of Bphs proceedsthrough several intermediate stages before attaining a conforma-tion of 15Ea of BV in light state (e.g., Pfr state) upon lightactivation.8-11

In an important recent development, Bph has been engineeredfor use as a fluorescent marker in mammalian tissues.12 Bphfluoresces in the near-IR at∼720 nm, a wavelength less prone toscattering that can penetrate more deeply into tissue than lightemitted by GFP-derived fluorescent proteins. The BV cofactor isa naturally occurring cofactor in mammalian tissue that cova-lently binds to a conserved cysteine in the bacteriophytochrome,and hence Bph can readily be genetically encoded. It shares suchproperties with flavin-binding photoreceptors such as LOV andBLUF domains for use as photonic switch or sensor.13-17

Special Issue: Graham R. Fleming Festschrift

Received: July 23, 2010Revised: December 9, 2010

3779 dx.doi.org/10.1021/jp106891x |J. Phys. Chem. A 2011, 115, 3778–3786

The Journal of Physical Chemistry A ARTICLE

Phytochrome photochemistry is thus of considerable significancefor biomedical research and technology.

The recent determination of crystal structures of various BPhsand the cyanobacterial phytochrome Cph1 has explored thelight-activated function of phytochromes.8,18-21 The linear tet-rapyrrole chromophore is bound to the PAS (BPh) or GAF(Cph1) domain at ring A through covalent linkage to a conservedcysteine. BV is largely engulfed by the GAF domain, whichprovides most of the hydrogen bonding networks and steric andhydrophobic interactions to secure the chromophore in position.The PHY domain forms an extension to the photosensory core ofphytochromes that works in tandem with the GAF domain fortuning of spectral properties and implementing photochemicaleffectiveness. In the Pr state the chromophore assumes a ZZZssaconfiguration (Figure 1) and is positioned in its binding pocketthrough steric interactions and hydrogen bonds from proteinresidues to the pyrrole rings and propionate side chains. Recent

studies have indicated that 15Za to 15Ea isomerization of thechromophore at the C15dC16 double bond, which causes a flip ofpyrrole ring D, accompanies formation of the Pfr state.8 Theprimary photoproduct, denoted Lumi-R, is formed on the10-100 ps time scale and adopts the 15Ea configuration.22-27

The X-ray crystal structure of a classical Bph from Deinococcusradiodurans DrBphP and cyanobacterial phytochrome Cph1shows a single hydrogen bond between a conserved histidineresidue and the bilin chromophore ring D carbonyl in thechromophore binding pocket (Figure 1B).18,28,29 In the P3X-ray crystal structure (Figure 1A), besides the conserved histidine,Lys-183 and Ser-297 are within hydrogen bonding distance to thering D carbonyl.20 Importantly, P3 is the only Bph with threepotential H-bonding partners interacting with D-ring carbonylgroup in the Pr state, as shown in the sequence alignment(Figure 1D). These different aspects of classical Bph and P3 presentan opportunity to study the influence of the chromophore bindingpocket on the phytochrome photochemistry.

In our previous femtosecond time-resolved absorption studieson P2 and P3 PAS-GAF-PHY constructs, we have shown that theexcited-state lifetimes and the spectra of P3 are very differentfrom P2 and other classical Bphs. Strikingly, the BV excited stateof 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 theBV chromophore.27We determined that the two additional polarresidues, lysine and serine located at the immediate vicinity of BVring D, are responsible for a lowering the Lumi-R quantum yieldand increasing the BV excited-state lifetime. In addition, weidentified excited-state proton transfer (ESPT) from the BVpyrrole rings to the protein backbone or a bound water as theprocess that deactivates the BV excited state to the Pr state.27

Taken together, the fluorescence quantum yield of P3 is sig-nificantly higher than that of classical Bph and with detailedknowledge about its excited-state dynamics, P3 forms an attrac-tive starting material to generate a highly fluorescent deep-tissuefluorescent probe by means of rational structure- and mechanism-based engineering.27

The vibrational spectrum of a protein or a protein-boundchromophore 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 revealmany of the dynamic structural and physical-chemical propertiesof chromophores involved in (photo)biological reactions.30,31 Inaddition, it can reveal the involvement of those parts of the proteinthat partake in the ongoing reactions. As the primary reactions inbiological photoreceptors proceed on the ultrafast time scale,femtosecondmid-IR spectroscopy is a method of choice to identifyreaction mechanisms of biological photoreceptors.32-42 The fem-tosecond time-resolved infrared absorption study on Cph1 hadshown that methine bridges, ring A/D of the phycocyanobilin(PCB) chromophore, were involved in structural changes in itsprimary photochemistry.24 The application of femtosecond IR onanother bacteriophytochrome, Agp1, had given a similarconclusion.42 Structural changes related to the methine bridgeswas also inferred by steady-state resonance Raman studies on thecryo-trapped intermediate states of various phytochromes.43 How-ever, information about the structural evolution of BV in the earlyphotochemistry of P2 and P3 is lacking.

In this work, we extend our previous studies by comparing theearly photochemistry of P2 and P3 in the Pr state using ultrafastmid-IR spectroscopy. We have studied the full photosensory

Figure 1. (A) Biliverdin (BV) binding site in the X-ray structure ofRhodopseudomonas 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 ringand atom numbering. (D) Partial protein sequence alignment ofbacteriophytochromes P3 and P2 from Rhodopseudomonas palustris,DrBphP from Deinoccocus radiodurans, AtBphP1 from Agrobacteriumtumafeciens, PaBphP from Pseudomonas aeruginosa, and cyanobacterialphytochrome Cph1 from Synechocystis sp. pcc 6803. Numerical valuesindicate positions of amino acids in P3 primary sequence.



PAS-GAF-PHY core of P2 and P3, as well as their short PAS-GAF constructs. Our results support our earlier observation thatexcited-state decay is significantly slower in P3 as compared toP2. Comparison of time-resolved IR spectra and FTIR spectra ofP2 and P3 and site-directed mutants where hydrogen bonding toring D was modified provides direct spectroscopic evidence thathydrogen bonding to the ring D carbonyl is indeed significantlystronger in P3 than in P2.

’MATERIAL AND METHODS

Sample Preparation. The detailed preparation of wild typeP2 (PAS-GAF), P2 (PAS-GAF-PHY), and the P2 (PAS-GAF-PHY)M169K/A382S (P2KS)mutant and of wild type P3 (PAS-GAF), P3 (PAS-GAF-PHY), and the P3 (PAS-GAF-PHY)K183M/S297A (P3MA) mutant bacteriophytochrome proteinswas described previously.20 For the ultrafast mid-IR experiments,the proteins were dissolved in D2O buffer (20mMTris 3HCl, pD8 at room temperature). For the FTIR experiments, the proteinswere dissolved in H2O buffer (20 mM Tris 3HCl, pH 8 at roomtemperature).Femtosecond Mid-IR Spectroscopy. The experimental set-

up is a home-built spectrometer based on a 1 kHz amplified Ti:sapphire laser system operating at 1 kHz (Spectra PhysicsHurricane) that allows visible pump/mid infrared probe in a timewindow from 180 fs to 3 ns, as previously described.30,32 The redexcitation pulse was generated by means of a noncollinear opticalparametric amplifier and centered around 680 nm, at an excitationenergy of 150-250 nJ. The infrared probe had a spectral width of200 cm-1, was spectrally dispersed after the sample, and wasdetected with a 32-element array detector, leading to a spectralresolution of 6 cm-1. Vibrational spectra between 1780 and1450 cm-1 were taken in two intervals and simultaneouslyanalyzed. Spectra were recorded at 100 time delay points bet-ween-20 ps andþ2.8 ns. During the experiments, the sample cellwas continuously translated with a Lissajous scanner, whichensured sample refreshment after each laser shot and a time intervalof 1 min between successive exposures to the laser beams. Back-ground illumination to photorevert the Bph sample to Pr wasprovided with a LEDwith a center wavelength at 750 nm (P2 PAS-GAF-PHY, P2 PAS-GAF and P3 PAS-GAF) or 650 nm (P3 PAS-GAF-PHY).Data Analysis. The time-resolved data can be described in

terms of a parametric model in which some parameters, such asthose descriptive of the instrument response function (IRF), arewavenumber-dependent, whereas others, such as the lifetime of acertain spectrally distinct component, underlay the data at allwavenumbers. The presence of parameters that underlay the dataat all wavenumbers allow the application of global analysistechniques, which model wavenumber-invariant parameters asa function of all data.44 The femtosecond transient absorptiondata were globally analyzed using a kinetic model consisting ofsequentially interconverting evolution-associated differencespectra (EADS), i.e., 1 f 2 f 3 f ... (Figures 2, 3 and 5A) inwhich the arrows indicate successive monoexponential decays ofincreasing time constant, which can be regarded as the lifetime ofeach EADS. The first EADS corresponds to the time-zerodifference spectrum. This procedure enables us to clearly visua-lize the evolution of the (excited and intermediate) states of thesystem. It is important to note that a sequential analysis ismathematically equivalent to a parallel (sum-of-exponentials)analysis.44 The analysis program calculates both EADS and

decay-associated difference spectra (DADS), and the time con-stants that follow from the analysis apply to both. In general, theEADS may well reflect mixtures of molecular states such as mayarise, for instance, from heterogeneous ground states or branchingat any point in the molecular evolution or inverted kinetics.45-52

Throughout the manuscript, the EADS are shown in the maintext and the corresponding DADS are shown in the SupportingInformation. The advantage of showing EADS over DADS is thatthe former are intuitively more easily interpreted. A detailedaccount of the global analysis methodology is given in theSupporting Information.

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



To account for unresolved fast relaxation processes within theinstrument response that become apparent as a sharp peakaround zero time delay, a pulse follower was included in theglobal analysis procedure. To avoid any effect of prezero signalsarising from perturbed free induction decay (FID)53 and un-resolved relaxation dynamics around zero delay on the outcomeof global analysis procedure, the prezero to 0.5 ps spectra weregiven a low weight.Differential Fourier-Transform Infrared (FTIR) Spectros-

copy. The differential FTIR data were recorded at room tem-perature using a FTIR spectrometer (IFS 66s Bruker) equipped

with a nitrogen cooled photovoltaicMCT detector (20MHz, KV100, Kolmar Technologies, Inc.). Two LEDs, emitting at 680 and750 nm, were used to convert P2 to its light or dark states,respectively. For P3, LEDs emitting at 680 and 650 nmwere usedinstead. The light minus dark FTIR data were obtained, bysubtracting an initially recorded protein dark-state spectrum asthe background spectrum, from the light activated (using the 680nm LED) protein spectrum. Background and sample interfero-gram data were averaged from 500-2000 interferogram scans, at4 cm-1 spectra resolution. Measurements were repeated byilluminating the sample with a 750 nm (on P2) or a 650 nm(on P3) light to deactivate the light state of the protein and bytaking a background and a light activated spectrum. The FTIRsample was prepared using a drop of 2 mL of sample at OD700 nm

of ∼100 (in 20 mM Tris/HCl pH8 buffer) and spread betweentwo tightly fixed CaF2 windows.

’RESULTS AND DISCUSSION

Ultrafast Mid-IR Spectroscopy of P2. The reaction dy-namics of P2 (PAS-GAF-PHY) in the Pr state was investigatedfrom a subpicosecond time scale up to 3 ns by means of ultrafastmid-IR spectroscopy. The sample was excited at 680 nm, and aspectral range of 1470-1780 cm-1 that covers the CdO andCdC vibration regions was monitored. The data were globallyanalyzed in terms of a kinetic scheme with sequentially inter-converting species, where each species is characterized by anEADS that has a specific lifetime. One decay lifetime of 58 ps anda nondecaying component were required for an adequate fit ofthe data. The EADS are shown in Figure 2A, the correspondingDADS are shown in Figure S1 of Supporting InformationFigure 2B shows kinetic traces at selected wavenumbers. Notethat prezero signals in the kinetics, most apparent in the 1702cm-1 trace in Figure 2B, arise from perturbed FID53 and do notrelate the Bph photophysics. In some of the kinetics, anunresolved fast relaxation within the instrument response be-comes apparent as a peak around zero delay. It is taken intoaccount by including a pulse follower in the global analysisprocedure 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 excited-state decay component of 50 ps, consistentwith the present data. Inaddition, 0.4, 4, and 250 ps components were observed with visibletransient absorption spectroscopy.27 The absence of these compo-nents in our femtosecondmid-IR data is likely due to limited signal-to-noise, or to a relative insensitivity of the IR spectra to thedynamics associated with these time constants. The 58 ps EADSshows major bleach bands in the range 1570-1640 cm-1,attributed to the BV chromophoreCdCmethine bridges vibrationalbands.24,42 These bands are located at 1591, 1613, and 1635 cm-1.Their frequencies closely resemble those in the Pr state of plantphytochrome A (PhyA), DrBphP, and Agrobacterium bacterio-phytochrome 1 (Agp1), as observed with resonant Ramanspectroscopy.54,55 The largest bleach band at 1591 cm-1 in ourfemtosecond mid-IR data is assignable to the ring C-Dmethinebridge stretch band56 (see Figure 1C for BV ring and atomnumbering). The bleach band at ∼1630 cm-1 is assigned to thering A-Bmethine bridge (C4dC5) vibration band.56 Due to thereduction of bond orders in the excited states, these bleach bandsare expected to be downshifted in the S1 state.

24

The 58 ps EADS shows major bleach bands at 1728 and1702 cm-1 (Figure 2A). In PhyA, the high-frequency carbonyl

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



band at 1730 cm-1 was assigned to ring A C1dO stretchingthrough 18O isotope labeling of PCB at this site.57,58 Thus, the1728 cm-1 band can confidently be assigned to the BV ring AC1dO stretch vibration. It follows that the 1702 cm-1 band isassociated with the BV ring D C19dO vibration, which isconsistent with assignments made for Agp1 by Diller and co-workers26 and Bartl and co-workers.59 The CdO stretchingbands observed here have a lower frequency than those found inCph1 (located at 1738 and∼1720 cm-1 respectively), probablydue to a different environment of the chromophore. Also, thedifferent conjugation at ring A between BV and PCB may play a

role. The 58 ps EADS shows a bleach band at 1540 cm-1 that wasobserved previously in femtosecond mid-IR spectroscopy onphytochromes but not interpreted.24,42

The final nondecaying component is formed from the BVexcited state in 58 ps and persists through our experimental timescale of 3 ns. It is assigned to the primary photoproduct Lumi-R.The nondecaying component has a low amplitude andmost of itsfeatures do not rise above the noise level, in keeping with the lowquantum yield of Lumi-R formation of 0.13.27 Surprisingly,however, is the occurrence of a very sharp absorption band at1541 cm-1, at the same frequency as the bleach in the 58 psEADS. Figure 2B shows the kinetic trace that demonstrates therise of this positive-amplitude feature. Its possible origin will bediscussed further on in the paper.We also performed ultrafast IR experiments on the P2 PAS-

GAF construct that undergoes limited photoconversion20 underidentical experimental conditions. Two time constants of 4 and175 ps and a long-lived component were required for anadequate fit of the data. Figure 3A shows the resulting EADS,while kinetic traces at selected wavelengths are shown inFigure 3B. Figure S2 (Supporting Information) shows theDADS. The BV excited-state lifetime is significantly longer at175 ps than in the P2 PAS-GAF-PHY construct, which agreeswith our findings from ultrafast visible spectroscopy.60 Theinfrared signature of excited-state BV is essentially the sameas that in P2 PAS-GAF-PHY, with CdO bleaches at 1734 and1708 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, thelong-lived photoproduct shows a pronounced induced absorptionband at 1541 cm-1.In the P2 PAS-GAF construct, a 4 ps component was resolved.

Inspection of the corresponding DADS (Figure S1, SupportingInformation) reveals a pattern of alternating negative andpositive bands at similar amplitudes, with a negative band at1690 cm-1, a positive band at 1655 cm-1, and a broad negativeband near 1590 cm-1. The pattern of the 4 ps DADS does notresemble BV excited-state decay, as the negative feature at1690 cm-1 does not correspond to a BV CdO vibration in the Prstate, and the broad negative feature near 1590 cm-1 does notresemble the BV methine bridge stretches in Pr as observed for the175 ps DADS. Hence, we conclude that the 4 ps componentrepresents a relaxation process in the excited state.27

Ultrafast Mid-IR Spectroscopy of P3. We investigated thereaction dynamics of the P3 PAS-GAF-PHY construct in the Prstate by means of ultrafast mid-IR spectroscopy to uncoverstructural aspects of its photoreaction. The sample was excitedat 680 nm and a spectral range of 1470-1780 cm-1 that coversthe CdO and CdC vibration regions was monitored. Globalanalysis indicated that three lifetime components of 37 and 362ps and a nondecaying component were required for an adequatefit of the data. The EADS are shown in Figure 4A, whereas kinetictraces 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 visiblespectroscopy. Its origin will be discussed below. It evolves intothe EADS that has a lifetime of 362 ps (Figure 4A, soliddiamonds). The 362 EADS corresponds to a relaxed form ofthe BV excited state similar to, although somewhat shorter than,that observed with ultrafast visible spectroscopy, which had alifetime of 450 ps.27 It has an overall shape similar to that of theBV excited state of P2 (Figure 2A) but differs in some important

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



aspects. It shows strong bleach bands at 1588, 1603, and1630 cm-1 that represent the BV chromophore CdC methinebridges vibrational bands. The strongest bleach band at1588 cm-1 is assigned to the BV ring C-D methine bridge(C15dC16) stretching. The 1630 cm

-1 bleach band is assignedto the ring A-B methine bridge (C4dC5) stretching. Thesebands are similar to those observed in P2 (Figure 2A), Cph1 andAgp1.24,42 Inspection of the carbonyl region (1680-1740 cm-1)reveals a pattern that is quite different from that of P2: The ring ACdO stretching band is located at 1736 cm-1 (Figure 4A, soliddiamonds), a frequency comparable to those found in P2, Cph1and Agp1.24,42 Strikingly, a pronounced bleach is observed at1678 cm-1, a frequency where P2 and other phytochromes showno such signal. Given its frequency and its prompt rise within theinstrument response, it must correspond to a CdOmode of theBV chromophore. As the ring A C1dO was already firmlyassigned to the 1736 cm-1 band,58 the 1678 cm-1 band mostlikely corresponds to the BV ring D C19dO stretch mode. Wewill substantiate this assignment below by means of FTIRspectroscopy on P2, P3, and site-directed mutants. This observa-tion implies that in P3, the ring D CdO stretch mode has asignificantly lower frequency than in other phytochromes. Wewill demonstrate below that the downshift of the ring D CdOfrequency results from the increased hydrogen bond strength toring D in P3. As in P2, a strong and broad bleach band at 1541cm-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 primaryphotoproduct Lumi-R. This EADS has a very low amplitude, inkeeping with the low Lumi-R quantum yield of P3 of 0.06.27 As inP2, a sharp induced absorption band at 1541 cm-1 is observed.Figure 4B shows the rise of this product band. Other bands in thisEADS are mostly buried in the noise and will not be furtherconsidered.The spectral evolution shows a 37 ps component in addition to

the 362 ps and nondecaying components. Inspection of theDADS (Figure S3, Supporting Information) shows negativebands at 1730, 1690, and 1655 cm-1, a broad negative featurebetween 1580 and 1630 cm-1, and positive bands at 1675 cm-1

and at frequencies below 1530 cm-1. The mostly negativepattern suggests that the 37 ps component mainly represents aBV excited-state decay process. However, the negative signals at1690 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 tothose of Pr, and we conclude that the 37 ps does not representdecay of the Pr excited state. We conclude that this componentremains difficult to interpret in specific molecular terms.We also performed ultrafast IR experiments on the P3 PAS-

GAF construct under identical experimental conditions. P3 PAS-GAF does not form the Pnr state and undergoes photoconver-sion to a Meta-R like state.20 Figure 5A shows the resultingEADS, kinetic traces at selected wavelengths are shown inFigure 5B. Figure S4 (Supporting Information) shows theDADS. The BV excited-state lifetime is significantly longer at435 ps than in the P3 PAS-GAF-PHY construct, which agreeswith our findings from ultrafast visible spectroscopy.60 In addi-tion, the 9.5 ps component observed in the P3 PAS-GAF-PHYconstruct does not appear in these data. The infrared signature ofexcited-state BV is essentially the same as that in P3 PAS-GAF-PHY, with the ring A CdO bleaches at 1741 cm-1, the ring DCdO bleach at 1677 cm-1, methine bridge stretches at1588, 1612, and 1635 cm-1, and a bleach at 1546 cm-1. As in

the PAS-GAF-PHY construct, the long-lived photoproductshows 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 of1678 cm-1 (Figures 4A and 5A) as compared to P2 (Figures 2Aand 3A) and other classical (bacterio)phytochromes24,42 andassigned it to the ring D carbonyl stretch mode. The lowering ofthe ring D carbonyl frequency is most likely due to the strongerhydrogen bonding at this site: the P3 X-ray structure shows 2-3amino acids hydrogen bonding to the carbonyl of ring D in darkstate, i.e., the conserved His-299, Lys-183, and Ser-297.20 Inclassical phytochromes, only a conserved His hydrogen bonds toring D18,28,61 (Figure 1). To investigate this idea, we performed

Figure 5. Time-resolved spectroscopy of theRps. palustris P3 PAS-GAFconstruct. (A) Evolution-associated difference spectra (EADS) and theircorresponding lifetimes resulting from global analysis of ultrafast mid-IRexperiments upon excitation at 680 nm. (B) Kinetic traces at 1677, 1588,and 1541 cm-1.



light-minus-dark FTIR spectroscopy on wild type P3 and theP3MA mutant,27 where the two polar amino acids Lys-183 andSer-297 are mutated to methionine and alanine, respectively,eliminating the two hydrogen bonds. Also, FTIR spectra weretaken on the classical Bph P2 and its P2KS mutant, where Met-169 and Ala-382 were replaced by polar residues Lys and Ser. AllFTIR experiments were performed on PAS-GAF-PHY con-structs. We note that the P2KS and P3MA mutants retain theirrespective wild-type photoconversion properties, i.e., P2KS con-verts to Pfr and P3MA converts to Pnr.20

Figure 6A shows the light-minus-dark FTIR spectra of P3 wildtype (solid line) and the P3MAmutant (crossed symbol line). InP3 wild type, carbonyl bleaches are observed at 1734 and 1685cm-1 (Figure 6A, solid line), assigned to ring A C1dO and ringD C19dO, respectively. This result is consistent with those ofultrafast IR spectroscopy, which indicated ring A and D carbonylfrequencies at 1736 and 1678 cm-1 (Figure 4A) (note that theFTIR spectra were taken in H2O and ultrafast IR spectra in D2O,giving rise to slightly different frequencies). In the P3MAmutant,the bleach at 1685 cm-1 has disappeared and a new bleach at1711 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 andLys 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). InP2 wild type, which forms only a single hydrogen bond from aconserved His to ring D, bleaches are observed at 1732 and 1703cm-1 (Figure 6B, solid line), assigned to the ring A C1dO andring D C19dO, respectively. With ultrafast IR, similar frequen-cies are observed at 1728 and 1702 cm-1 (Figure 2A). In theP2KS mutant, where M169 was replaced by Lys and A283 by Ser(the equivalent amino acids in P3), it is expected that twoadditional 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 C19dOstretching frequency from 1703 (in WT) to 1676 cm-1 (inP2KS) 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 inP2, consistent with the X-ray structures of P3 and classical (B)ph(Figure 1).20,28,61 This result further corroborates our earlierwork,27 where we identified the factors that determine the BVexcited-state lifetimes and isomerization quantum yields of wild-type andmutants of P2 and P3.We concluded that the hydrogen-bond strength to ring D is rate-limiting for isomerization and theexcited-state lifetime and that the quantum yields of fluorescenceand isomerization are determined by excited-state deprotonationof biliverdin at the pyrrole rings, in competition with hydrogen-bond rupture between the D-ring and the apoprotein.27

Origin of 1540 cm-1 Band. An interesting observation of thepresent work is a band near 1540 cm-1 that appears as a broadbleach in the BV excited state (thus corresponding to Pr) and anarrow induced absorption at essentially the same frequency inthe primary photoproduct in all Bph variants studied here, i.e., P2PAS-GAF-PHY, P2 PAS-GAF, P3 PAS-GAF-PHY, and P3 PAS-GAF. Because the 1540 cm-1 band in the primary photoproducthas an amplitude that exceeds all other product bands, it mightcorrespond to a molecular state that plays an integral role in theprimary photochemistry of bacteriophytochrome. The questionarises what specific vibrational mode(s) of BV these bandsbelong to. We first note that the negative (Pr) and positive

(photoproduct) bands do not necessarily relate to the exact samevibrational mode. In previous ultrafast mid-IR experiments onCph1 and Agp1, a similar but smaller bleach band at ∼1540cm-1 was observed.24,42 With resonant Raman spectroscopy onplant PhyA in D2O, Pr shows a band at 1547 cm-1, and cryo-trapped Lumi-R exhibited a sharp band at 1541 cm-1.43 InDrBphP, a band at 1546 cm-1 was observed in D2O withresonant Raman spectroscopy.55 In neither of these studies werethe bands near 1540 cm-1 interpreted. It does not belong to theN-H in-plane bending vibrational band (at ∼1570 cm-1 inPhyA, Agp1 and DrBphP in H2O

10,54,55) because this banddownshifts to below 1100 cm-1 upon deuteration.43,55

As a first possible origin of the 1540 cm-1 band, calculationsand IR absorption experiments on the model compound bili-verdin dimetyl ester have indicated a mode at 1543 cm-1 in D2Othat corresponded to the CdC ring stretch and C-vinyl stretchband of ring D.57 As a second possibility for the origin ofthe ∼1540 cm-1 band, recent DFT calculations have indicatedthat in a pyrrole-N deuterated PCB chromophore in a ZZZssaconfiguration, a band near 1540 cm-1 arises that includes mainlystretching coordinates from ring B and, to a minor content, fromthe B-C and A-B methine bridges. In the ZZEssa configura-tion, this band slightly shifts to 1542 cm-1.62 It is difficult to

Figure 6. (A) Light-minus-dark FTIR spectroscopy of wild type P3PAS-GAF-PHY (solid line) and the P3MA PAS-GAF-PHY mutant(crossed line). (B) Light-minus-dark FTIR spectroscopy of wild typeP2 PAS-GAF-PHY (solid line) and the P2KS PAS-GAF-PHY mutant(crossed line).



understand how such a band assignment would relate to Lumi-Rformation because no significant changes are thought to occur atring B upon isomerization about the C15dC16 double bond. Wenote, however, that the long-lived 1540 cm-1 absorption doesnot necessarily relate to Lumi-R and may correspond to aground-state intermediate on the pathway of Pr reformation.Such ground-state intermediates were recently observed forvarious phytochromes.26,63 At this stage the origin of the1540 cm-1 bands remain unclear and will be the subject offurther studies.

’CONCLUSIONS

Here, we have reported an ultrafast mid-IR study of tworelated bacteriophytochromes: P2, which shows classical Pr-Pfrphotochemistry and P3, which shows an unusual Pr - Pnrphotochemistry. In P2, BV excited-state decay occurs with a timeconstant of 58 ps, largely consistent with our results from visibletransient absorption spectroscopy which indicated a biexponen-tial decay with a main decay component of 60 ps. Excited-statedecay in P3 is significantly slower with a time constant of 362 ps,which is also consistent with visible transient absorptionresults.27 In our previous work, we proposed that the slowerexcited-state decay of P3 is related to an increased hydrogenbond strength at ring D, with three amino acid side chains (His,Lys, and Ser) competing for hydrogen bonding to the ring Dcarbonyl in P3.20,27 In P2, only one such hydrogen bond can formfrom conserved His to ring D.18,61 Here, we obtained directspectroscopic evidence for increased hydrogen bond strength atring D in P3: ultrafast IR spectroscopy on P2 and P3, and FTIRspectroscopy on the P2 and P3 wild types and P3MA and P2KSmutants indicated that in P3, the ringDC19dO stretchmode hasan unusually low vibrational frequency at 1685-1678 cm-1. Incontrast, in P2 the ring D C19dO stretch mode is located at1703 cm-1, which demonstrates that P3 has one or two additionalhydrogen bonds to ring D.

’ASSOCIATED CONTENT

bS Supporting Information. DADS spectra. Discussion ofmodel based data analysis. This material is available free of chargevia 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 UniversityBerlin for sharing unpublished results. We thank Jos Thieme fortechnical support. K.C.T. and J.T.M.K. were supported by theEarth and Life Sciences Council of The Netherlands Foundationfor Scientific Research (NWO-ALW) through a VIDI grant to J.T.M.K. E.A.S and K.M. were supported by an NIH grantGM036452 to K. M.

Bph, bacteriophytochrome; BV, biliverdin; PCB, phycocyanobi-lin; EADS, evolution-associated difference spectrum;DADS,decay-associated difference spectrum; ESPT, excited-state protontransfer

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1750 1700 1650 1600 1550 1500

-5

-4

-3

-2

-1

0

1

2

DADS P2 PAS-GAF-PHY

∆A

(1

0-3 )

Wavenumber (cm-1)

58 ps

non-decaying

Fig. S1. Decay-associated difference spectra (DADS) that follow from global analysis of

time-resolved IR data of Rps. palustris P2 PAS-GAF-PHY bacteriophytochrome. The

excitation wavelength was 680 nm.

1750 1700 1650 1600 1550 1500

-3

-2

-1

0

1

2

DADS P2 PAS-GAF

∆A

(1

0-3 )

Wavenumber (cm-1)

4 ps

175 ps

non-decaying


time-resolved IR data of Rps. palustris P2 PAS-GAF bacteriophytochrome. The


1750 1700 1650 1600 1550 1500

-2

-1

0

1

2

DADS P3 PAS-GAF-PHY

∆A

(1

0-3 )

Wavenumber (cm-1)

37 ps

362 ps

non-decaying


time-resolved IR data of Rps. palustris P3 PAS-GAF-PHY bacteriophytochrome. The


1750 1700 1650 1600 1550 1500

-1

0

1

DADS P3 PAS-GAF

∆A

(1

0-3 )

Wavenumber (cm-1)

435 ps

non-decaying


time-resolved IR data of Rps. palustris P3 PAS-GAF bacteriophytochrome. The


S.1

Supporting Information to

The primary reactions of bacteriophytochrome observed with

ultrafast mid-infrared spectroscopy

K.C. Toh, Emina A. Stojković, Alisa B. Rupenyan, Ivo H.M. van Stokkum, Marian

Salumbides, Marie-Louise Groot, Keith Moffat, John T.M. Kennis

Model based data analysis

The aim of data analysis is to obtain a model-based description of the full data set in

terms of a model containing a small number of precisely estimated parameters, of which

the rate constants and spectra are the most relevant. The basic ingredient of kinetic

models, namely the exponential decays, will be described first, followed by use of these

ingredients for global and target analysis1-3

of the full data. Our main assumption is that

the time and wavelength properties of the system of interest are separable, which means

that spectra of species or states are constant. For details on parameter estimation

techniques the reader is referred to1-4

. Software issues are discussed in5.

A. Modeling an exponential decay

Here an expression is derived for describing an exponentially decaying component. The

instrument response function (IRF) i(t) can usually adequately be modeled with a

Gaussian with parameters µ and ∆ for, respectively, location and full width at half

maximum (FWHM):

))/)(2)(2log(exp(2

~1

)( 2∆−−∆

= µπ

tti

where ))2log(22/(~

∆=∆ . The convolution (indicated by an *) of this IRF with an

exponential decay (with rate k) yields an analytical expression which facilitates the

estimation of the IRF parameters µ and ∆:

S.2

2 21 ( ))( , , , ) exp( ) ( ) exp( )exp( ( )){1 ( }

2 2 2

I k t kc t k kt i t kt k erf

µµ µ

∆ − + ∆∆ = − ∗ = − + +

∆

% %

%

The wavelength dependence of the IRF location µ can be modeled with a polynomial.

max

1

( ) ( )c

jj

j c

j

aλµ λ µ λ λ=

= + −∑

Typically, a parabola is adequate and the order of this polynomial ( maxj ) is two. The

reference wavelength cλ is usually at the center of the spectrograph.

B. Global and target analysis

The basis of global analysis is the superposition principle, which states that the measured

data ),( λψ t result from a superposition of the spectral properties )(λε l of the

components present in the system of interest weighted by their concentration )(tcl .

∑=

=compn

l

ll tct1

)()(),( λελψ

The )(tcl of all compn components are described by a compartmental model that consists

of first-order differential equations, with as solution sums of exponential decays. We

consider three types of compartmental models: (1) a model with components decaying

monoexponentially in parallel, which yields Decay Associated Difference Spectra

(DADS), (2) a sequential model with increasing lifetimes, also called an unbranched

unidirectional model6, which yields Evolution Associated Difference Spectra (EADS),

and (3) a full compartmental scheme which may include possible branchings and

equilibria, which yields Species Associated Difference Spectra (SADS). The last is most

often referred to as target analysis, where the target is the proposed kinetic scheme,

including possible spectral assumptions. In this paper we did not attempt a target

analysis. Instead, throughout the manuscript, the EADS are shown in the main text and

the corresponding DADS are shown in the Supporting Information.

(1) With parallel decaying components the model reads

1

( , ) ( ) ( )compn

I

l l

l

t c k DADSψ λ λ=

=∑

The DADS thus represent the estimated amplitudes of the above defined exponential

S.3

decays ( )I

lc k . When the system consists of parallel decaying components the DADS are

true species difference spectra. In all other cases, they are interpreted as a weighted sum

(with both positive and negative contributions) of true species difference spectra.

(2) A sequential model reads

1

( , ) ( )compn

II

l l

l

t c EADSψ λ λ=

=∑

where each concentration is a linear combination of the exponential decays,

1

( )l

II I

l jl l

j

c b c k=

=∑ , and the amplitudes6 jlb are given by 11 1b = and for j l≤ :

1

1 1,

/ ( )l l

jl m n j

m n n j

b k k k−

= = ≠

= −∏ ∏

When the system consists of sequentially decaying components 1 2 ... compn→ → → the

EADS are true species difference spectra. In all other cases, they are interpreted as a

weighted sum (with only positive contributions) of true species difference spectra.

Equivalence of the parallel and the sequential model

It is important to note that the fit is identical when using a parallel or a sequential model.

Both the estimated lifetimes and the residuals from the fit are identical. This can be

demonstrated as follows. Since the concentrations of the sequential model are a linear

combination of the exponential decays we can write for the matrix of concentrations

(where column l corresponds to component l)

II IC C B=

where the upper triangular matrix B contains the elements jlb defined above.

Furthermore, in matrix notation the parallel model reads

I TC DADSΨ =

where TDADS is the transpose of the matrix that contains the DADS of component l in

column l. Likewise, in matrix notation the sequential model reads

II TC EADSΨ =

Combining these three equations we obtain the relations

TDADS EADS B= ⋅

S.4

TEADS DADS B−= ⋅

where the coefficients of the lower triangular matrix TB− are given by 1

1 11 T

l lb b− −= = and

for j l≤ :

11

1

( )jTn l

jl lj

n n

k kb b

k

−− −

=

−= =∏

Thus the DADS are linear combinations of the EADS, and vice versa. Thus, the lth

EADS is a linear combination of the lth and following DADS. In particular, the first

EADS, which corresponds to the time zero difference spectrum, is the sum of all DADS;

and the final EADS is proportional to the final DADS.

In systems where photophysical and photochemical processes occur the sequential model

with increasing lifetimes provides a convenient way to visualize the evolution of the

(excited and intermediate) states of the system. Therefore, the EADS are shown in the

main text and the corresponding DADS are shown in the Supporting Information.

References

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