Photochemistty and Photobiology, Vol. 57, No. 2, pp. 391-395, 1993 Printed in the United States. All rights reserved

003 1-8655193 $05.00+0.00 0 1993 American Society for Photobiology

RAPID COMMUNICATION

ULTRAVIOLET RESONANCE RAMAN SPECTRA OF PHYTOCHROME: A COMPARISON OF THE ENVIRONMENTS OF TRYPTOPHAN SIDE CHAINS BETWEEN RED

LIGHT-ABSORBING AND FAR-RED LIGHT-ABSORBING FORMS

A U R A TOYAMA’, MIKI NAKAZAWA’, KATSUSHI MANABE2‘, HIDE0 TAKEUCHI’, and ISSEI HARADA”

‘Pliarmaccutical Institute, Tolioku University, Aobayama, Scndai 980, Japan; arid ’Department of Biology, Yokohama City University, Seto 22-2, Kanazawa-ku, Yokohama 236, Japan

(Received 23 October 1992; accepted 17 November 1992)

Abstract --Ultraviolet resonance Raman spectra of pliytoclirome in the red light-absorbing form (Pr) and the far-red light-absorbing form (Pfr) are reported. The spectra excited at 210-nm pro- vide structural information about the protein part of phytochrome. The protein contains only a very small amount of fl-slieet structure and most of the tyrosiiic side chains arc located in hydrophobic environments. Iiidole rings of tryptophan (Trp) interact with ncigliboriiig groups in the Pr form arid these interactions become weaker with the conversion from Pr to Pfr. Some Trp side cliains of Pfr arc surrouridcd by aliphatic groups but such is not the case in Pr. Tlicsc cliarigcs in tlic cn\.iron- men1 occur at the same time as cliarigcs in orientation of Trp side chains. Our observations suggest that interactions between Trp residues and the tctrapyrrolic cliromopliorc occur in tlic Pr form and that the strenglh of tliese interactions diminishes in the Pfr form.

INTRODUC’I’ION

Phytochromc is a dimcric chromoprotcin that rcgulatcs photomorplioge~iesis in higlicr plants. It contains one tutrapyrrolc chromophore per subunit and exists in two photo-iiitcrconvcrtible fcrms: a red light-absorbing form (Pr) and a physiologically active, far-red light-absorbing form (Pfr). Only limited information is available about the structure of the protein part of phytocliromc arid the structura1 changes that are induccd by llie photoconversion (see Song’ for review). Some preliminary information about the secondary structure of tlic protein part of pliytocliromc has bceii obtained by infrared spectroscopy‘. With respect to tlie surface topography of aromatic amino acids, Sing11 et (11.~ and Siiigli & Song4 showed by ultraviolet difference spectroscopy and fluoresccncc-qiicncliiiig experiments that the extent of exposure of some aromatic amino-acid residues changes during the photoconversion of Pr to Pfr. In this paper, n.e report the ultravi- olet resonance Raman (UVRR) spectra of phytocliromc in llie Pr and PTr forms. This is the first time such spectra have been reported even though UVRR was sliow~n several years ago to be a very useful tool in structural studies of proteins’. We have obtained some information about tlic main-chain structure arid the environments in which tryptophan (Trp) arid tyrosine (Tyr) side cliains are located. Changes i n the environments around Trp residues occurred with the trarisitiori of Pr to Pfr.

MATERIALS AND METHODS

Native (121-kDa) pea phytocliromc was purified from 6-day-old etiolated pea seedlings (Pisurn satiiwm L. cv. Alas- ka: seeds supplied by Snow Brand Seed, Co., Sapporo, Japan) by the procedure described previously6. The pliytoclironic used for the experiments had a specific absorbance ratio (A6 nm/AZROn of Pr form) that ranged from 0.90 to 1.00.

UVRR spectra were recorded with a Jasco CT-&D clout& monochromator (Japan Spectroscopic Co., Ltti., Tokyo, Japan) equipped with a D/SIDA-700 multichannel detector (Princeton Instruments Inc., Trenton, NJ, USA). Excitation light (240 nm) was provided by an H,-Raman-shifted Nd:YAG laser (Quanta Ray DCR-3; Spectra-Physics Iasers Inc. Mountain View, CA, USA) and it was’focused into a spot of ca 0.1 x 0.2 mm. Tlie incident energy from the

To whom correspondence should be addressed. + Deceased on August loth, 1992. Abhreviariom: Pr and Pfr, red light- and far-red light-absorbing forms of pliytocliromc; UVRR, ultraviolct resonance Raman.

19 1

392 A ~ R A TOVAMA et al.

laser was 15 +J/pulse at the sample wdiicli was in a spiiiiiiiig quart7 cell. The UVRR apparatus has bcen dcscribed elsc- wlicre7. The spectral rcsolution was 8 cm-', and tlie pcak wavenumbers of the Raman bands wcrc reproducible to within +2 cm-'. l l i c Pfr form was generated by illuminating tlic Pr form with tlic 633-iim radiation (30 mW) from an He-Nc laser. The sample solution was replaced with a fresh onc aftcr each spcctral mcasuremciits for a maximum of 8 miii and the Raman spectra from scvcral recordings werc combined. Absorption spcctra of tlie solutions wcrc rccordcd before and aftcr tlic UVRR mcasurcmcnts. The percentage of Pfr was calculatcd from tlic difference spcctrum of phytoclirome. The Pfr ratio i n tlie photostationary state under red-light irradiation was estimatcd to be 0.88'. No significant change was dctccted for Pr in the absorption spectra. By contrast, the Pfr contciit dccrcascd to 30% (with 30% Pr and 40% bleaclicd protcin) soon aftcr the start of the UV excitation (within 1 miii), but further change was not dctcctcd during tlie recording. Thus, tlie Raman spectrum of Pfr was obtained by subtracting the spcctra of Pr (30%) and the bleaclicd protciri (40%) from tliat recordcd under rcd light-irradiation. Absorbance at 666 nm of the bleachcd protcin was assumed to be 0.

RESULTS AND DISCUSSION

Pliytoclirome is a dimcr composcd of two identical subunits9. Each suburiit of pca phytoclirome corisists of 1123 amino acid residues, which include 7 0 tryptoplian (Trp) and 21 tyrosine (Tyr) residues, as deduccd from tlie nnclcotidc scqucncc of the corresponding cDNA'". Figure 1 shows tlic UVRR spectrum of Pr excitcd at 2-10 nm (a) and that of a mixture of aqueous solutions of Trp and Tyr at a molar ratio o f 1021. Thc spectrum of Pr is dominated by rcsonaiice- cnhanccd vibrational bands of the aromatic rings o f Trp arid Tyr sick chains. In tlie spcctrum of Pr (Fig. la), the main- cliaiii amidc I and I11 bands are observcd at 1660 and 12-18 cm-', rcspcctivcly. Tlicse bands can bc assigricd to a mixturc of random coils and a-lieliccs and to random coils, rcspcctively, on the basis of the rclationships betwecn tlic sccoridary structure arid Raman frequc~icy~. No promincnt bands assigiiablc to p-sliccts, namely, at 1675-1 665 cm-' for amidc I arid 1240-1230 cm-' for amide 111, can he dctcctcd. Howcvcr, shouldcrs around thcse rcgions may iiidicate some P-shcct structure. Siricc the 240-nm cxcited Raman scattcriiig cross scctions of ~-slieel amidc tmds arc cxpcctcd to bc Iargcr

, / ' I I l l i i i

l€OG :Loo 1200 ' RAMAN SiiiFT / cm-l

Figure 1: Ultraviolct resonance Raman spectrum of a 1-1 pM solution of tlie Pr form of pliyto- chrome (a) and of a mixture of 250 lul.l tryptophan and 525 pkf tyrosinc (at a molar ratio of 1021) (b), dissohzd in 10 mM potassium pliospliatc buffer (pH 7.8) that contained 1 mM EDTA, 13 mM 2-mcrcaptocthanol and 10% glycerol. Excitation was cffectcd at 230 nm. Iiiteiisitics were normalized to the 980-cm-' phospliatc band (not shown). Ordinate represents Ramari iiitcnsity.

Rapid Communication 393

I

CD 'D I

I 1 1

1

1600' ' ' 1400 ' ' " ' 12co ' RAMAN SHIFT I cm-'

Figure 2: Ultraviolct resonance Raman spectrum of Pfr obtained by subtracting tlie spectra of Pr and the blcaclied protein from that recordcd under red-light irradiation. Excitation was effected at 230 nm. For details. see the text.

than those of a-hclices and random coil^"^'^, thc present observations indicate that the amount of 0-sheet structurc is \'cry low in Pr. Relati\dy high lcvels of a-hclis and random coil and the absence of 0-sheet structures have been rc- ported from tlie measiiremciits of circular d i c l i r o i ~ m ' ~ ~ ' ~ .

The 1209- and 1177-cm-' bands in Fig. l a can bc acsigncd to the v~~ and vg, modes of Tyr phenol ring vibra- tions, respecti~cly'~. Yic intensities of thcsc bands are about lwicc tliose obtairicd from the aqucous mixture of amino acids (Fig. Ib). Most of Ihc Tyr sidc chains of Pr appcar to bc located in hydrophobic environments since tlic intensity of the Tyr band i n hydrophobic environmcnts i5 expcctcd to bc twice tliat in an aqucous cnvironmciit from data on acctylry- rosiiic ethyl ester in H 2 0 and in solution in mcthanol or chloroform".

The strong band at 1618 cm-' is duc to the overlap of Tyr vSa and Trp WI bands'. Thc intcnsity of the vSa band of Pr is also expected to increase together with tliosc of v , ~ and v d. The 1618 cm-' band i n Fig. l a is, however, only slightly stronger than that i n Fig. lb, suggesting that tlic iritciisity of tlic overlapping W1 band is weaker in Pr than in the mixture of amino acids. By contrast, the 1553-cm-' band of Pr, which ariscs from anothcr Trp modc W3, is stronger than that in Fig. lb. I n another words, tlic ratio of iiitensities of W3 and W1, Zu'3/lR,I, for Pr is very much larger than that for aqueous Trp. It is know~ii tliat the ratjo of intensities increases concomitant with a red shift of the strong B, absorption of indole ring at around 218 11m'~. Such a rcd shift is concidcrcd to be caused by hydrogen bonding at thc intlole NH or clectronic pcrturbatiori to thc indole ring. Hencc, the present obscrvations indicate that strong interaction between Trp and neighboring groups occur in Pr. Interactions between the tctrapyrrole chromophorc arid aromatic amino acids i n proximity lo the cliromopliore have been suggeslcd from measiiremciits of circular dicliroi~m'~*'~.

Figurc 2 shows tlic UVRR spectrum of phytocliromc in the Pfr form. Although the signal-to-noise ratio in lhc spectrum is not as good as in that of Pr (because of the instability of Pfr during the measurements), changes in iiiteiisity of thc Trp bands are clearly apparent. Since the intensity ratio IuJIw,l dccreascs upon the transition of Pr to Pfr, thc interac- tions of Trp sidc cliairis in Pfr are weaker than tliosc in Pr.

Two Trp bands around 1360 and 1330 cm-' are due to Fcrmi resonance and the ratio of intensities, 113J~1q40, increases, in particular when aliphatic groups lie under or over a Trp iridolc plane'. The ratio is small i n Fig. l a , si igcst- iiig non-aliphatic eiivironmerits for most indole rings of Trp residues. The ratio obtained from the spcctriim of pliyto- chrome aftcr red-light irradiation is larger than that for Pr, and it indicates that some Trp side chains become surroundcd by aliphatic groups. This result is consistcrit with the quenching of Trp fluorescc~icc~ in oat phytochrome. But it may appear inconsistent with thc UV difference spectrum obtained with oat phytocliromc3 and with the data obtaincd aftcr cficmical modification of Trp in pea phytochrome (M. Nakwawa and K. Manabc, submitted), which indicated Ilia1 tlie numbcr of exposed Trp rcsidues increases with tlic conversion of Pr to Pfr. Howcver, it should bc noted that tlic acccssi- bility of the solvent and of chemical reagents to Trp side chains cannot be related dircctly to the hydrophobicity of the ein~iroiiment around tlic indole rings.

Thc W3 frequency is known to be correlated with tlic absolute value of tlic torsion angle x2*' around the C -C, linkagem. The peak frequencies, 1553 cm-' i n Pr and 1557 cm-' in Pfr, correspond to x"' values of 100" and 120°, re&:

394 AKIRA TOYAMA et a/.

tively. The different frequencies indicate that the conformations of Trp rcsidues change upon the conversion o f Pr to Pfr. I n summary, our analysis of the local cnlironmeiits of Trp side chains in Pr indicates that many iiidole rings

are located in lion-aliphatic environments and their electronic states are perturbed by ncigliboring groups (possibly iia hydrogen-bonding or J C - - ~ t interactions). Upon the conversion of Pr to Pfr, the environments of some Trp residues become aliphatic. Conformational changes around the C -C, bonds arid decrcascs of the clectronic perturbation of tlie Trp resi- dues also occur with the photoconversion. It has beeii reported that changes in the configuration of tlie tctrapyrrole group occur with tlie transformation of Pr to Pfr and that its D-ring becomes esposcd at the surfacc of the protein2’*”~””. Strong- er interactions between protein and chromophorc in Pr than in Pfr 1iaX.e also been suggested from experiments with fluo- rcsccnt hydrophobic probe^*^*."^ arid observations of differential oxidation of the ~Iirornopliore~”.”’. We suggest that the perturbation of the Trp clcctronic states in Pr is caused, at Icast i n part, by iiitcractioiis between Trp sidc chairis and the tctrapyrrole group, and that such perturbation diminishcs i n Pfr as a rcsult of structural cliangcs in both groups.

P

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Rapid Communication 395

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