J. Phys. Chem. B 2010, 114, 17155–17161
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Photoreaction of Plant and DASH Cryptochromes Probed by Infrared Spectroscopy: The Neutral Radical State of Flavoproteins Dominik Immeln,† Richard Pokorny,‡ Elena Herman,† Julia Moldt,‡ Alfred Batschauer,‡ and Tilman Kottke*,† Department of Chemistry, Biophysical Chemistry, Bielefeld UniVersity, UniVersita¨tsstrasse 25, 33615 Bielefeld, Germany, and Faculty of Biology, Department of Plant Physiology and Photobiology, Philipps UniVersity, Karl-Von-Frisch-Strasse 8, 35032 Marburg, Germany ReceiVed: August 12, 2010; ReVised Manuscript ReceiVed: NoVember 10, 2010
Flavoprotein radicals are important intermediates in many biochemical processes. In the blue light sensor plant cryptochrome, the radical state acts as a signaling state. An isolation and assignment of infrared bands of flavin radicals in the most relevant spectral region of carbonyl stretches is missing because of their overlap with absorption of water and the protein moiety. In this study, the neutral radical state of flavoproteins was investigated by Fourier transform infrared difference spectroscopy. The light-induced conversion of oxidized to neutral radical state was monitored in a plant cryptochrome and that of radical to fully reduced state in a DASH cryptochrome. A pure difference spectrum of flavin radical minus oxidized state was obtained from a point mutant of a phototropin LOV (light-, oxygen-, or voltage-sensitive) domain. The analysis of the spectra revealed a correlation between the frequencies of carbonyl vibrations of the flavin radical state and those of its visible absorption. Plant cryptochrome shows a very low frequency of the carbonyl stretch in the radical state. It is postulated that the downshift is caused by the charge of an adjacent aspartate, which donated its proton to flavin N5. Contributions from the protein moiety to the spectra were isolated for DASH and plant cryptochromes. As a conclusion, the photosensitive domain of plant cryptochromes shows changes in secondary structure upon illumination, which might be related to signaling. Introduction Cryptochromes are flavoproteins that regulate the central responses of many organisms. These responses include the setting of the biological clock by blue light in plants1 and flies.2 In plants, cryptochromes additionally control light-dependent development, i.e., photomorphogenesis and flowering.3 Furthermore, cryptochromes are the most promising candidates for the magnetoreceptor of migratory birds.4,5 Some cryptochrome-like proteins, the DASH cryptochromes, act as repair enzymes for photodamaged single-stranded6 and loop-structured duplex DNA,7 similar to the homologous photolyases. All cryptochromes noncovalently bind a flavin adenine dinucleotide (FAD) as a chromophore inside their photolyase homology region (PHR),8–11 which spans about 500 amino acids. Additionally, methenyltetrahydrofolate (MTHF) has been found to bind to plant11,12 and DASH cryptochromes13–16 functioning as an antenna pigment,17–19 and being photoreduced to methylenetetrahydrofolate.20 Although cryptochromes and photolyases form a large superfamily, members of each subclade and sometimes even within the same subclade show different photochemistry of the FAD depending on their origin.21 In plant cryptochromes, FAD undergoes a transition from the oxidized to the neutral radical state upon illumination in Vitro and in ViVo22,23 and oxidizes back in the dark (Figure 1, pathway I). In contrast, the FAD in purified DASH cryptochromes is present in all redox states with dominant contributions from oxidized and fully reduced FAD.19 * Corresponding author. Phone: +49-521-106-2062. Fax: +49-521-1062981. E-mail:
[email protected]. † Bielefeld University. ‡ Philipps University.
Figure 1. In plant cryptochromes, the flavin chromophore is present in its oxidized state in the dark. By blue light illumination, it is photoreduced to the neutral radical state (pathway I) and decays back in the dark. In DASH cryptochromes, the purified form contains a mixture of all three depicted redox states. Conversion to the fully reduced form takes place without significant accumulation of the neutral radical either along pathways I and III or directly via pathway II. In the dark, the oxidized state is slowly regained. Formation of the flavin anion radical under certain conditions has been omitted for clarity.
Upon illumination, the fully reduced state is formed similar to conventional DNA photolyases.24,25 The photoreduction takes place without detectable accumulation of the neutral radical state19 except in the case of zebrafish DASH cryptochrome.26 Some formation of anion radical has been found as an intermediate in cyanobacterial DASH cryptochrome.27 It is unclear whether the photoreduction proceeds directly (Figure 1, pathway II)19 or in two fast consecutive steps. For the back reaction, a direct oxidation along pathway II has been suggested.19,26
10.1021/jp1076388 2010 American Chemical Society Published on Web 12/03/2010
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The formation of the flavin neutral radical in plant cryptochromes has been studied by time-resolved UV/vis spectroscopy. The electron transfer from a nearby tryptophan triad28,29 proceeds before 100 ns and is followed by a microsecond proton transfer to the flavin anion radical.30 The tryptophan radical decays within milliseconds,28 leaving behind the flavin neutral radical as the signaling state.22,23 Information from UV/vis experiments on light-induced changes in the protein moiety is limited to aromatic residues. Alternative techniques are needed to resolve the steps of signal transfer from photoexcited FAD to the outside presumably proceeding via alterations in the protein shell. Infrared difference spectroscopy is a valuable tool to resolve responses of the protein moiety to photoexcitation of the chromophore. For instance, light-induced deprotonation of an aspartic or glutamic acid has been detected in the plant cryptochrome 1 from Arabidopsis (AtCry1) by Fourier transform infrared (FT-IR) spectroscopy.31 The observed signal was postulated to originate from an aspartic acid close to flavin N5. Further valuable information might be obtained from the amide I spectral region from 1615 to 1695 cm-1, where changes in secondary structure of the protein backbone can be monitored.32 However, this region contains additional contributions from carbonyl stretches of flavin and is overlapped by strong water absorption. The separation of bands originating from the flavin neutral radical from those of the protein moiety in the infrared difference spectra is further complicated by the fact that reference data are rare. To our knowledge, there are only two FT-IR spectra available on neutral radical minus oxidized state of flavoproteins: first, the aforementioned study on AtCRY1,31 where the amide I region was not well resolved, and second, a spectrum from the LOV2 domain from oat phototropin 1, which was limited to the spectral region >1670 cm-1.33 Radical states of free flavin have been characterized in acetonitrile by time-resolved spectroscopy but with a limited spectral resolution of 16 cm-1.34,35 In addition, the photoreaction from neutral radical to reduced state has been analyzed by infrared spectroscopy on E. coli DNA photolyase (EcPHR)36 including 15N global isotopic labeling. In this study, we set out to record and analyze FT-IR spectra of the flavin neutral radical from three different proteins, the light-sensitive PHR domain of the plant cryptochrome CPH1 (Chlamydomonas photolyase homologue 1) from the green alga Chlamydomonas reinhardtii, the DASH cryptochrome 3 from Arabidopsis thaliana (AtCry3), and the LOV1-C57S mutant from C. reinhardtii phototropin (CrPhot). The comparison of the obtained data revealed similarities and differences in the band positions of the flavin CdO modes that allowed one to isolate those from contributions of the protein moiety. As a result, responses of the protein to the flavin photoreaction were identified in the plant cryptochrome as well as in the DASH cryptochrome. Materials and Methods Protein Expression, Purification, and Buffer Exchange. CPH1-PHR,37 AtCry3,15 and LOV1-C57S38 were expressed in E. coli and purified as described before. CHP1-PHR was in 20 mM phosphate buffer, pH 7.5, 100 mM NaCl, 1% glycerol. The protein was concentrated by ultrafiltration using a Vivaspin 500 filter device (Sartorius) with a 10 kDa cutoff to OD450 > 10. For H/D exchange of CPH1-PHR, the concentrated protein solution was shock-frozen in liquid nitrogen and lyophilized for 12 h at -31 °C reaching a pressure of 0.08 mbar (ALPHA 2-4, Christ). The yellow powder was dissolved in the original volume of D2O (Sigma) and incubated for at least 3 h at 4 °C.
Immeln et al. AtCry3 was concentrated to 40 mg/mL in 50 mM phosphate buffer pH 7.5, 200 mM NaCl, 10 mM 2-mercaptoethanol, and 10% glycerol. LOV1-C57S was transferred via ultrafiltration into 10 mM phosphate buffer, pH 8, containing 70 mM 2-mercaptoethanol and 50 mM NaCl, and was concentrated to OD450 ) 8. Sample Preparation. For the FT-IR experiments on CPH1PHR, a droplet of 2 µL of protein solution was applied to a BaF2 window and the water content was slowly reduced for 4 min at a pressure of 700 mbar. This procedure is more gentle than the established procedure of drying and rehydration, which might interfere with the structural integrity of the protein. The sample was sealed by a second window. The optical path length of the sandwich cuvette was 5-15 µm. For studies on the deuterated sample, the same procedure was applied but under a nitrogen atmosphere. Sandwich cuvettes with deuterated samples were incubated at 4 °C for at least 12 h before measurements. For experiments with AtCry3, 4 µL of the sample was concentrated on the window at 300 mbar for 10 min. For LOV1-C57S, 9 µL of the concentrated solution was applied to a BaF2 window and the sample was gently deoxygenated and dehydrated by several cycles of applying vacuum of 60 mbar and flushing with nitrogen. FT-IR Experiments. FT-IR spectroscopy was performed on Bruker IFS 66v and 66v/S spectrometers with a spectral resolution of 2 cm-1. The temperature of the sample was maintained ambient for LOV1-C57S, and adjusted to 10 or 4 °C by a circulating water bath for CPH1-PHR and AtCry3, respectively. Absorbance spectra were recorded across the whole mid-infrared range. For difference spectroscopy, a long wave pass filter (OCLI) was placed in front of the detector to improve the signal-to-noise ratio and to block stray light. The filter restricted the recording range to below 1975 cm-1. For light-induced difference spectroscopy, CPH1-PHR was illuminated for 15 s with a blue LED (Luxeon Star, Lumileds) with an emission maximum at 455 nm (20 nm full width at half-maximum (fwhm)) and an intensity of ca. 20 mW/cm2 at the sample. LOV1-C57S was illuminated for 30 s by the same light source. AtCry3 was exposed for 40 s to light from an orange LED with an emission maximum at 594 nm (20 nm fwhm) and an intensity of ca. 4 mW/cm2. Light-minus-dark difference spectra were collected after illumination. Representative difference spectra of two independent preparations were selected and averaged to a total of 25088 scans for CPH1-PHR in H2O, 4096 scans for CPH1-PHR in D2O, 4096 scans for AtCry3, and 6144 scans for LOV1-C57S. Results FT-IR Difference Spectrum of Flavin Neutral Radical Minus Oxidized State. To facilitate the assignment of flavin bands, contributions from vibrational modes of the protein moiety had to be minimized. Therefore, the C57S mutant of CrPhot-LOV1 was investigated where the replacement of the reactive cysteine with a serine prevents adduct formation with flavin and leads to an unphysiological photoreaction of the oxidized state to the flavin neutral radical.39,40 To further suppress motions and responses of the protein moiety to photoexcitation of the flavin chromophore, the amount of water in the sample was reduced to a minimum (Figure S1, Supporting Information). The infrared difference spectrum of the mutant was obtained in the presence of the reducing agent 2-mercaptoethanol (Figure 2). Only bands of those vibrational modes are visible that change upon illumination. Negative bands correspond to the dark state that contains fully oxidized flavin.
Infrared Spectroscopy on Plant and DASH Cryptochromes
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Figure 2. Light-induced infrared difference spectrum of the C57S mutant of the CrPhot-LOV1 domain. Negative bands originate from vibrations of the dark state, and positive bands represent the photoproduct. The mutant forms an unphysiological flavin neutral radical from the oxidized state upon illumination in the presence of the external reducing agent 2-mercaptoethanol. Contributions from the protein moiety were suppressed by a low hydration of the sample.
Bands at 1550 and 1584 cm-1 originate from the ring II of the isoalloxazine ring.41 The C2dO stretching vibration is detected at 1681 cm-1.42 The other two negative bands at 1693 and 1711 cm-1 are assigned to the C4dO stretching vibration with strong and weak hydrogen bonding, respectively.43 Positive bands are induced by illumination and represent the flavin neutral radical state. The bands at 1535 and 1611 cm-1 are characteristic ring stretching modes with contributions from N5-H bending. They have been assigned in the resonance Raman spectra of photolyases.44,45 Higher frequency modes have not been detected in the resonance Raman spectra. Accordingly, the bands at 1623 and 1656 cm-1 of LOV1-C57S are assigned to the CdO stretching vibrations of the flavin neutral radical, which have a low Raman cross section. FT-IR Difference Spectrum of the Plant Cryptochrome CPH1-PHR. The protein sample was prepared by gentle lowering of the water content under reduced pressure (Figure S2, Supporting Information). This procedure ensured a full flexibility of the protein to resolve potential changes in secondary structure as opposed to protein film preparation by drying and rehydration. Light-minus-dark difference spectra were recorded after illumination with blue light (Figure 3A, black line). In the dark, the bands decreased in intensity in the order of minutes (data not shown), reflecting the decay of the radical state. In direct comparison to the band pattern of LOV1C57S (Figure 3A, red line), more bands were resolved, which points to additional contributions from the protein moiety. The negative bands of oxidized flavin at 1545, 1577, 1692, and 1708 cm-1 are downshifted up to 7 cm-1 as compared to the LOV1 domain (Table 1). Notably, a band corresponding to that of C2dO in LOV1 at around 1681 cm-1 was not detected. This loss might be explained by an overcompensation by a strong positive contribution in CPH1-PHR at 1671 cm-1, which hides the negative band of the C2dO stretch. The negative bands at 1633 cm-1 and at around 1661 cm-1 might contain contributions from the protein moiety. The prominent band at 1733 cm-1 is assigned to deprotonation of a carboxylic acid side chain. It has been observed before in AtCRY1 but close to the noise level.31 Its strong intensity is a peculiarity of CPH1-PHR that allows for its unambiguous detection. Positive bands typical for the reaction product, the flavin neutral radical, were observed at 1530 and 1611 cm-1. Again, the former mode is downshifted by 5 cm-1 as compared to LOV1 (Table 2). Additionally, three positive contributions are
Figure 3. (A) Light-induced FT-IR difference spectrum of the photosensitive PHR domain of the plant cryptochrome CPH1 (black) in comparison to that of LOV1-C57S (red). The band pattern shows formation of the FAD neutral radical from the oxidized state. Contributions at around 1671 cm-1 are assigned to changes in the CPH1-PHR secondary structure (highlighted in gray). (B) Difference spectrum of CPH1-PHR in D2O. Due to limited isotope exchange in the flavin binding pocket, the band pattern is mostly preserved. A loss of the band at 1671 cm-1 is observed, which is interpreted as suppression of secondary structural changes by the lyophilization procedure.
TABLE 1: FT-IR Difference Bands of Oxidized Flavin in Plant Cryptochrome and Their Assignmenta CPH1-PHR H 2O
D 2O
1733 1732 1708 1706 1692 1689 1673 1661 1633 1577 1578 1545 1544
AtCRY1-PHRb H2O
D2O
∼1735 1710
1734 1705
1578 1543
1638 1577 1544
CrPhot LOV1-C57S H 2O 1711 1693 1681 1584 1550
assignment Asp/Glu C4dOb,c,d C4dOe C2dOc,d protein moiety protein moiety/band If band II band III
a Band positions are given in cm-1. Band numbering according to Bowman and Spiro.61 b Kottke et al.31 c Ataka et al.41 and references therein. d Iwata et al.42 e Alexandre et al.62 f Resonance Raman experiments by Li et al. on (6-4) photolyase.44
found at 1625, 1642, and 1671 cm-1. Of these, two represent the carbonyl modes of flavin and the remaining signal is assigned to a contribution of the protein moiety. A more detailed analysis of these results is given in the Discussion section.
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TABLE 2: FT-IR Difference Bands of the Flavin Neutral Radical in Plant Cryptochrome, DASH Cryptochrome, and Photolyase and Their Assignmenta CPH1-PHR AtCry1b EcPHRc,d AtCry3 CrPhot plant cryptochrome plant cryptochrome DNA photolyase DASH cryptochrome LOV1-C57S H 2O 1671 1642 1625 1611 1530
D2 O e
1642 1626 1609 1529
H2 O
1524
D2O e
1609 1525
H2O/D2O
H2O
1693d
1696
1660c
1663
1606c 1532,d 1528c
1530
H 2O
assignment
1656 1623 1611 1535
protein moiety amide I CdOf CdO ν(ring I), δ(N5sH), ν(N1dC10a), ν(C4asN5)c,g ν(ring I), δ(N5sH)c
a Band positions are given in cm-1. b Kottke et al.31 c Resonance Raman experiments by Murgida et al.45 and references therein. d Schleicher et al.36 e N5sH was not deuterated due to limited exchange in the chromophore binding pocket. f Martin et al.34 g Resonance Raman experiments by Li et al.44
To aid in the assignment of the band pattern, the accessible acidic protons of CPH1-PHR were exchanged to deuterons. The protein was lyophilized, redissolved in D2O, and incubated for several hours. Exchange of water to D2O is evidenced by the infrared absorption spectrum (Figure S2, Supporting Information). Despite the solvent exchange, the light-induced difference spectrum exhibits only a small downshift of most bands by
