Models of pigment-protein interactions in photosynthetic systems: Tetraphenylporphyrin complexes with polycationic sequential polypeptides. Absorption, circular dichroism and fluorescence properties

Chemical Physics 147 ( 1990) 40 l-41 3 North-Holland

Models of pigment-protein interactions in photosynthetic systems: tetraphenylporphyrin complexes with polycationic sequential polypeptides. Absorption, circular dichroism and fluorescence properties Petr Pa&o&a =*I,Marie UrbanovA ‘, Lucie BednArovB a, Karel Vacek a, Vladimir Z. Paschenko b, Sergej Vasiliev b, Petr Malofi c and Miroslav KrAl c ’ Department of Chemical Physics, Faculty o$Mathematics and Physics, Charles University, I21 16 Prague 2. Czechoslovakia b Faculty ofBiology, Moscow State University, Moscow, USSR ’ Institute of Organic Chemistry and Biochemistry. Czechoslovak Academy o/science, 166 10 Prague 6, Czechoslovakia Received 27 January 1989; in final form 9 July 1990

Absorption and circular dichroism spectra and steady state and time-resolved fluorescence of complexes of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin with sequential helical polypeptides (LLys-LAla)., (GLys-LAla-L-Ala), and (LLys-LAla-L-Ala-L-Ala), are studied. Changes of electronic transition energies, induction of optical activity and two-component fluorescence decay with components t, = 2-3 ns and t2= 180 ps characterize the complexed pigment. Dependence of these changes on relative number of lysine amino acids in polypeptide primary structures was observed and is discussed in terms of perturbational influence of polypeptide molecular field on mixing of porphyrin electronic states.

1. Introduction

The relationship between the molecular components of basic building blocks of the photosynthetic apparatus - pigment-protein complexes - is supposed to ensure their proper biological functions, especially for pigments involved in primary photophysical processes of photosynthesis. The importance of the protein matrix in this adaptation of pigment (especially porphyrin) physical properties is generally accepted on the qualitative level but the evidence about physical nature of the adaptation process is rare. We have shown recently [ l-41 that the complexes of tetraanionic tetraphenylporphyrins and polycationic polypeptides poly ( Llysyl-{ Galanyl} ,_3) are able to simulate structural fixation of the pigment to a regular (a-helical) protein matrix. The regularity of both primary and secondary structures in utilized structural model polypeptides forms the specific molecular fields, generated in a water environment, by ’ Author to whom correspondence should be addressed. 0301-0104/90/$03.50

positively charged a-amino groups of the lysine side chain. This structural periodicity of the polypeptides generates also the specific regular environment which accents the conformationally dependent perturbational influence of the protein models on the fixed porphyrins. As shown in our previous papers [ l-4 1, the induction of pigment optical activity indicates the large changes in structure of electronic levels of bonded porphyrin. Variations in spatial density of the E-NH: groups caused by the coil-helix conformational transition of poly (Llysyl-Lleucyl-Lalanine) were also shown to induce a variation in the relative contribution of the new, short component (r= 240 ps) in the fluorescence decay kinetics of the complexed pigment [ 41. In this paper we describe absorption, circular dichroism and fluorescence spectra of a tetraanionic tetraphenylporphyrin complexed with regular poly (di-), poly(tri-) and poly(tetra-) peptides of the above type. These complexes represent the model systems where spatial density of cationic groups (yielding the dominant, Coulombic part of the mo-

0 1990 - Elsevier Science Publishers B.V. (North-Holland)

402

P. Pa&o&a et al. /Spectroscopy of porphyrin-polypeptide complexes

lecular field of these polypeptide matrices) is precisely controlled by the primary structure of the polypeptide. The study was designed to evaluate the role of the protein molecular field in the formation of the “spectral forms” of the complexed porphyrin and to discuss the possible mechanisms of the pigment electronic structure alterations after complexation. 2. Materials and methods The synthesis and HPLC purification of a, P,y,G-tetrakis ( Ccarboxyphenyl ) porphyrin (TPPC ) is described in ref. [ 51. Poly (L-lysyl-Lalanine) MW 6500 [LA],,, poly(L-lysyl-L-alanyl-Lalanine) MW 6900 [ LAA ] ,,, poly ( L-lysyl-L-alanyl-L-alanyl-Lalanine) MW 6500 [ LAAA]. and poly (Llysyl-Lalanyl-glycine) MW 7500 [LAG]. are described in ref. [ 61. TPPC-polypeptide complex formation is spontaneous in the 0.02 M phosphate buffer, pH= 7.2, but is limited to a relative narrow concentration and pH interval (pH=6.9-8.3, maximal concentration of porphyrin Cr,, x 10T3 M) by the solubility of both components and the complex. Constant porphyrin concentration (C,,, = 5 X 1Ow4 M) and variable polypeptide concentration C,, with pigment to pep tide mean molar ratios c[=Crpp/Cpp= 1 and 4 were selected for detailed study, because under these conditions there is the lowest interference of the free pigment. From the analysis of concentration dependence of absorption and circular dichroism spectra over the e ranged from 0.1 to 10, we have estimated the standard free stabilization energy AG= 34 kJ/mol for TPPC- [ LAA] ,, complex [ 281. The free pigment concentrations evaluated from the above value are 1.2x 1O-7 and 8.1 x 1O-7 M for &l and 4 respectively (i.e. 1.2 and 8% in these samples). Absorption, circular dichroism (CD) and fluorescence spectra were measured as in ref. [4] at room temperature. The intensities of CD spectra induced in porphyrin absorption bands were recalculated to AE values using analytical concentration of the pigment in the sample. This treatment neglects the fraction of the free (optically inactive) pigment in the solution with the above estimated uncertainty in the presented intensities. For the time-resolved fluorescence measurements the apparatus described in ref. [ 7 ] was used (2 ps

exciting pulse duration, A,,= 534 nm, fluorescence detected for 1> 600 nm ) . The deconvolution procedure is described in ref. [ 41.

3. Results 3.1. Characterization of polypeptide matrices

On the basis of characteristic amide UV spectra (two negative maxima at 208 and 222 nm) which are observed with minor differences for all studied polypeptide and porphyrin-polypeptide samples, we can conclude that our polypeptide matrices (both free or in complex) are predominantly in regular righthanded a-helical conformation. Taking into account standard geometrical parameters for an a-helix, known primary structures and mean molecular weights of our polypeptides, we can calculate the local “concentration” of positively charged (protonated) a-amino groups on the lysine side chains. Based on the molecular dimensions of both components (see fig. 1 ), the volume in which the peptide-fixed porphyrins can be localized and the charged side chains are also situated, is the cylinder with the radius rzz 100 A and height x 120 A. Local “concentrations” (C+ ) of the charged side chain groups in this volume (summarized in table 1) can be calculated. They are proportional to the reciprocal of the distances (d+ ) between the a-amino groups (C+ x 1/w2d+ ) and can be expressed relatively to the [ LAln (symbol p + (charge density) is used below for this relative quantity). 3.2. Induced circular dichroism spectra in visible spectral region The formation of conformationally well defined complexes of polycationic polypeptides [LA] ,,, [ LAA] n and [ LAAA ] ,, with TPPC is confirmed by observation of strong induced circular dichroism in the Soret AX*transition region (300-500 nm - see fig. 2). The sign pattern of these induced dichroic spectra depends on the primary structure of the particular polypeptide. Differing (odd and even) periodicity of the lysine residues in the primary structures of our sequential polypeptides ( [ LAA ] n and [LA ] ,,, [ LAAA ] ,, respec-

P. PanfoSka et al. /Spectroscopy of porphyrin-polypeptide complexes

tively ) seems to provide the decisive structural dif-

a) A

(=f-)n

1

r

b-‘n

403

1 -T

w--)n

ference on the secondary structure level: assuming common right-handed u-helical backbone conformation for all three polypeptides, it defines the sense of the superhelix of the charged lysine side chain groups (see fig. lc) which is left-handed for (odd) [ LAA]. while right-handed for (even) [ LAln and [ LAAA] ,, sequences. In this sense, the sign changes in the induced CD or porphyrin in the Soret region reflect the chirality difference of the polypeptide matrix molecular fields.

I T

b)

3.3. Absorption and fluorescence spectra As compared to properties of the TPPC in the free state ( 10m5- 10e6 M solutions), the complexation of porphyrin to our polypeptides induces the following changes in the TPPC absorption and fluorescence spectra (see figs. 2 and 3 ). For all complexes the absorption bands of the ‘So-+‘S, transition (Q,) as well as the corresponding fluorescence maxima are red-shifted (see table 2)) depending on the nature of the polypeptide matrix. TPPC fluorescence quantum yields (see table 2 ) decrease to 16-55% of the free pigment value, again depending on the polypeptide used in the complex. In the Soret absorption region, broadening and blue shifts ( 8- 10 nm ) are observed of the apparent maximum both in the absorption and fluorescence excitation spectra. Scanning the excitation wavelength throughout the Soret region for samples, where complexed and uncomplexed porphyrins are simultaneously present (this situation occurs for @- 4, when either the limited number of pigment bonding sites on the polypeptide or the equilibrium concentrations

R%tOH

I

Fig. 1. (a) Schemes of primary structures (A) and localization of lysine side chains in a-helical secondary structures of studied lysine, 0 alanine. sequential polypeptides. Dashed arrows indicate the sense of the side chain superhelices. The relevant distances are indicated. (b) Scheme of TPPC structure with distances of anionic groups. (c) Edge-on (A) and planeon (B ) fixation of TPPC on a-helical polypeptide matrices. The volume used for determination of C+ and p + is indicated.

Table I Local “concentrations” C+ and relative spatial densities of lysine a-amino groups for studied polypcptides Peptide

C+ x10-r

lLA1. [Ml. WAAl”

7.0 4.7 3.5

P

+ b)

[Ml a)

WI.

[J-AA].

lLAAA1”

1.0 1.489 2.0

0.677 1.0 1.343

0.500 0.745 1.0

‘) Local molar concentration of s-NH: groups for individual polypeptides as calculated from their primary structures,chain lengths and approximate volume occupied by one complex molecule. b, Relative ratios of C+ for a-helical polypeptides indicated. The values related to LA are used in tigures.

P. Panfoskaet al. /Spectroscopyof porphyrin-polypeplide complexes

404

450

500 A EmI

350

x, nm

450

350

x,

noi,

450

Fig. 2. Absorption and induced CD spectra of TPPC-polypeptide complexes for C= 1 and 4 in the Soret region.

for complexation are exhausted), the characteristic red-shifted fluorescence spectra of complexed pigments are preferentially excited not only for the blueshifted absorption maximum (e.g. at 380 nm in fig. 4) but also on the low-energy edge of the Soret region (at 430 nm in fig. 4). In fig. 4 the typical example of this set of fluorescence spectra is shown for the TPPC[LA In complex ((~4). We have observed this lowenergy, complex-specific absorption band in the Soret region previously [4] for the TPPC complexed with another polypeptide, poly (L-lysyl-L-leucyl-L Fig. 3. Fluorescence spectra of the free TPPC and its complexes b with [LA],,, [Ml,,, [LAM].and [LAG],excitedbyl,=413, 398,400,403 and 409 nm, respectively; molar ratio used clpr/ cpp= 1.

-7

“““” I I

TPPC ,. .. .._. FREE ,:’ ‘;

m

800

650

700

750

A,(nm)

800

P. Pa&o&a et al. /Spectroscopyofporphyrin-polypeptidecomplexes

405

Table 2 Absorption and fluorescence characteristics of TPPC and its complexes with polypeptides Peptide in complex free

649 15400

414 24140

650 15380

1.0

0

WI.

660 15150

404 24740

675 14815

0.55

1.0

1~1”

656 15250

405 24680

665 15038

0.16

0.68

v-‘@Al”

651 15350

406 24640

655 15267

0.27

0.5

‘) Position of long wavelength - tirst entry in nm, second entry in cm-‘. w Soret maxima -first entry in nm, second entry in cm-‘. ‘) Fluorescence maxima - first entry in nm, second entry in cm-‘. d, Relative fluorescence quantum yields (calculated as in ref. [ 41). e, Relative charge density for given polypeptide matrix.

alanyl-L-alanine) [ LLA] ,,. In TPPC- [ LLA] n systern this band is dominant and accompanied by the sign change of the induced CD.

600

650

The linear dependence of the transition energy differences on the reciprocal distance between the perturbing charges is to be expected for the charged mo-

700

Aemism)

Fig. 4. Dependence of fluorescence spectra of the TPPC- [ LA] ,, sample (& 4) on the excitation wavelength in the Soret region. Arrows indicate the red shifted fluorescence of complexed pigment.

P, Pa&o&a et al. /Spectroscopyof porphyrin-poIypeptidecomplexes

406

3.4. Time-resolvedjluorescence

E

la’;;1FREE

II

LAAA

LAA

LA

wdH

250t

\ \

\

\

\

\ \

\ \ \

\ \ 00 ,hI

242 -c '--\

i 153

The fluorescence decay curves of TPPC complexes with studied polypeptides were measured for <= 1 and 4. This selection was based on the concentration study for the TPPC-[ LA],, complex. In fig. 6 and in table 3 it can be seen that generally new, short-lived components characterize the complexed pigment fluorescence decays as compared to those of the free pigment. In TPPC- [LA I,, samples with <= 1, 4, 6 and 8, the TPP fluorescence decay kinetics should generally be deconvolved using a three-exponential formula:

A

Cd-

/ / -I // -_.-.-.-.-.-.-.-.-.-.-.-.-.-.-.-,-..'\_

\ \ \

151

(1)

1491

-.

with ‘A, = 4A, = 0 and r in nanoseconds.

147 t

II 0

I

0,5

I

I

0,67

IO

Q+

Fig. 5. Correlation of frequencies of the Soret absorption maximum (A) and fluorescence maximum (B) with polypeptide charge densityp+. The low-energy values for the Soret region are estimated from the observed bandwidths. The supposed interaction with forbidden (B,) state (- - - ) is shown schematically.

lecular systems, where the first-order correction should describe the greatest part of the observed changes [ 8 ] if the perturbational approach is acceptable. It is shown in fig. 5 that both in red and Soret regions such a linear relationship exists.

Fig. 6. Fluorescence decay curves of the TPPC- [ LA] ,, complex with variable
Table 3 Results of deconvolution of fluorescence decay curves for TPPC- [LA]. samples with variable t c

Relative amplitudes ‘)

Contribution of component to global quantum yield (%) ‘)

r= 4.2 =)

rz2.4

~~0.18

rz4.2

rz2.4

~0.18

free 8

1.0 0.2

0.25

1.0

100 52

37

6 4 1

0.01

0.28 0.25 1.0

1.0 1.0 1.0

11 19 23 7

‘) Calculated from cAiin eq. ( 1). ‘) Calculatedas_feAi exp( -t/r,) dt/ZJCAi exp( -t/r,) dt. ‘) 7 in ns.

5

76 77 93

P. PanEoSka et al. /Spectroscopy of porphyrin-polypeptide complexes

TIME

Fig. 7. Dependence of contributions of fluorescence decay components with given lifetime on pigment to polypeptide molar ratio t for TPPC- [ LA] n complex.

The contributions of individual components to the global quantum yield vary for different < (see fig. 7 ) , the shortest 180 ps component contributes maximally for <= 4. For @ 4 the increase of the free pigment concentration in the sample is manifested by the increase of the 4.2 ns contribution. Two-component decays were observed for TPPC[LA] n, TPPC- [ LAA] ,, and TPPC- [ LAAA] ,, complexes, prepared with r= 1 and 4 (see fig. 8 and table 4). The contribution of the shortest invariant 180 ps component 7, to the global quantum yield depends linearly on p + for C;=4 but not for c= 1 (see fig. 9a). Table 4 Results of deconvolution

of fluorescence

Fig. 8. Fluorescence decay curves for TPPC-polypeptide plexes with & I (a) and C=4 (b).

t

r2 (ns)

A2

ILAIn

4 1

2.4 2.5

0.25 1.0

fLA‘41.

4 1

2.7 3.0

If-A.4‘41.

4 1

3.0 3.6

WAGI,

4 1

4.2 4.2

L

com-

The longer component Q is characterized by the lifetime shorter than that of free pigment and decreases approximately linearly with increasing p+ for both discussed pigment to polypeptide molar ratios < (see fig. 9b). Shortening of the porphyrin fluorescence lifetime

decay curves for TPP-polypeptide

P a’

‘) Peptide in complex. ‘r Contribution of given component

TIME

complexes with <= 1 and 4 ~3 (-1

AS(~)

9 ret

71 91

0.18 0.18

1.0 1.0

29 9

0.3 0.35

82 a5

0.18 0.18

1.0 I.0

18 15

0.5 0.6

a9 92

0.18 0.18

1.0 1.0

11 8

(mu)

4rd

b’

100 100

to global fluorescence quantum yield (see table 3, footnote b). Values in 96.

b)

408

P. Panfosko et al. /Spectroscopyofporphyrin-polypeptidecomplexes

Fig. 9. (a) Dependence of the relative contribution of the TZcomponent to the global fluorescence quantum yield on the peptide chargedensity p + for TPPC-pol~ptide complexes for C= 1 ( 0 ) and & 4 ( 0 ). (b ) Dependence of the T, lifetime on the peptide charge density p + and Cfor TPPC-polypeptide complexes.

is connected critically with polypeptide regular conformation. If [LAG],, is used as the model matrix (glycine in the primary structure does not favor the u-helix formation), a monoexponential decay with a lifetime comparable to that of free pigment is observed for TPPC complexed by this unordered polypeptide (see fig. 8 and table 4).

4. Discussion Disparity between the operator and/or state symmetry selection rules [ 9,101 specific for the rotational strength and the “forbidden” high symmetry ( DZh) of TPPC molecule allows us to utilize the induction of porphyrin optical activity as reliable start-

ing point for discussion of the changes of porphyrin electronic structure in the complex. There are two necessary conditions for the efficient induction of optical activity of originally achiral porphyrin. The first of them is slructurd To avoid the cancellation of induced dichroic bands by conformational averaging, the pigment position should be uniformly faed relative to the peptide. Consistently with our previous conclusions [ l-41, we can suppose that structurally homogeneous and defined molecular species are formed due to the favorable geometrical arrangements of cationic groups on the polypeptide and anionic groups with fixed mutual distances ( z 22 A and 28 8, respectively) on TPPC (see fig. lb). The second condition is electronic: Mixing of BZu (B,,) states providing the porphyrin electric dipole xrr* transitions (e.g. in terms of configurational interaction) with other appropriate states should yield the necessary nonzero magnetic dipole transition moment. Generally [ 11,12 ] this is possible by: (a) lowering of porphyrin symmetry by chiral conformational change (induction of the inherent chirality), (b) the interaction of the XX*transition dipoles localized on at least two chirally oriented, uniformly and sufficiently closely fixed porphyrin molecules (degenerate coupled oscillator mechanism ) , (c) admixture of peptide state(s) of B, (B,,) symmetry (static ,u-m mechanism), (d) admixture of porphyrin state(s) of BZg(B,,) symmetry (static one-electron mechanism) (for the last two mechanisms, the external static perturbational field should have the nonzero chiral component with A, transformational properties [ 13]), (e) the interaction of porphyrin BZu(B,,) electrically dipole allowed xx* states with the effective bond moments on the polypeptide (nondegenerate coupled oscillator mechanism) [ 28 1, (f) the dynamic perturbation of porphyrin Bzg ( BJ,) states through the polarizability-type interaction with polypeptide [ 9, lo]. Case (a) can be realized either by the fixation of TPPC meso-phenyl substituents in propeller-like structure (which seems to be incompatible with the dependence of the observed spectral changes on p+ see the discussion below) or by the distortion of the tetrapyrrole macrocycle planarity. The porphyrin skeleton is generally considered as rigid although some

P. Pantoika et al. /Spectroscopy of porphyrin-polypeptide complexes

flexibility has been reported [ 14- 17 1. The nonplanarity of natural porphyrins introduced directly by their molecular structure [ 181 generates CD spectra with simple (unisignate in Soret) sign pattern and weak intensity which contrasts with the multiple signs and high intensity for the Soret CD of TPPC induced in our complexes. We therefore suppose that inherent chirality is not a dominant mechanism for the CD in the systems under study. For the degenerate coupled oscillator mechanism the clustering of the pigment molecules on the polypeptide is necessary. This mechanism has been considered by several authors as the source of induced optical activity for various achiral pigments (cationic porphyrins [ 19-221, aromatic dyes [23,24] ) complexed with chiral molecular matrices (polynucleotides [ 19,201, nucleic acids [ 2 11, cellulose [ 241, polylysine [ 23,241). In many of these systems, the pigment to matrix bonding specificity, possibility of dye intercalation and/or other factors, formed structural basis for such an explanation. In our complexes, for small pigment to polypeptide molar ratios (<< 1) the CD intensity induced is maximal and also sign patterns, overall shapes and relative band intensities are comparable to those observed for higher <. No bisignate CD couplets were found in long-wavelength red (500-700 nm) 1 ‘So-l ‘S, porphyrin transition region: e.g. for the TPPC- [ LAA] ,, complex the single positive bands at 529 nm (A&=6.4), 565 nm (A&=6.3), 597 nm (As= 1.6) and655 nm (A&= 1.2) were seen. Despite of this, the presence of TPPC dimers or oligomers cannot be ruled out because of undetermined distribution of the pigment on the polypeptide. We have therefore studied [ 291 the complexes formed by the same polypeptides with a,y-bis (4-carboxyphenyl ) porphyrin (TPPCZ ). This pigment is largely aggregated under the conditions of our experiments. The induced circular dichroism in Soret region of TPPCZ after complexation with [LA],,, [LAA],,, [LAAA]. and [LLA],, was (i) (qualitatively) insensitive to the character of the peptide matrices (positive dichroic band at 430 nm with positive shoulder at 407 nm, negative band at 443 nm, zero crossing at 437 nm). The intensity differences for individual TPPC2 complexes varied within Q of factor z 2. (ii) (Quantitatively) it was by an order of magnitude larger in overall CD intensities than the

409

most intense CD spectra of TPPC complexes. (iii) The position of red shifted Soret absorption maximum of TPPCZ (relatively to TPPC) remains unchanged after the complexation. No peptide dependent blue/red shifts of this maximum were observed. These differences in behaviour of the two types of pigments support our hypothesis that the monomeric TPPC is the dominant form in the systems discussed in this paper and therefore the degenerate coupled oscillator mechanism does not dominate the CD induction for our TPPC-polypeptide complexes. For better understanding of the role of remaining mechanisms we decided to employ the theoretical formalism derived in refs. [ 9,10,29]. Its application allows us to use the approximative molecular parameters and known information about the structural features of components of our TPPC-polypeptide components (see below). For the characterization of the changes in TPPC electronic and in particular chirooptical spectra we were inspired by ab initio calculations of Petke et al. [ 25 ] on (isosymmetric) porphine. They showed the exclusive localization of 1 ‘Bs, and 1 ‘B, (magnetic dipole) transitions in the Soret region. Just these states are required for the effective induction of optical activity here. The equations for rotational strength in refs. [ 9,10,29] allowed us to use for this purpose the pairs of x, y, in-plane polarized electric and magnetic transition dipoles (c and m) of 1 D [ 281, as the spectroscopic representation of porphyrin. The polypeptide chain has been represented by 100 7crr*electric transition dipole moments of about 1 D oriented along the line connecting nitrogen and carbonyl oxygen at individual amide groups. The amide groups have been arranged in space to form the polypeptide a-helical secondary structure using standard a-helix parameters [ 3 1 ] and the primary sequences of our polypeptides. The transition energies corresponding to the experimentally found maxima of supposed electronic transitions have been used (1.1 x lo-l8 and 7.6~ lo-l9 J for polypeptide AZ* 4.6~ 10-19, 5.1 X 10-19, and nx* transitions, 4.8~10-‘~and5.6~10-‘~JforTPPCm,,~~,~,,and my dipoles respectively). The last values were obtained as averaged maxima positions from simultaneous Soret band deconvolution of families of absorption and CD spectra measured as a function of temperature within the range 25-65°C [ 301 where

P. PanEoSka et al. /Spectroscopy of porphyrin-polypeptide complexes

410

the multiple spectra analysis reduces the ambiguity of parameters for deconvolved bands. The assignment is arbitrary but provided us with the correct sign pattern in the reference case (see below). The localization of both electric dipole allowed and magnetic dipole allowed transitions in the Soret region is the key feature for the induction of CD in them by all remaining mechanisms. The basic differences between the possibilities (d) and (f ) are in the character of the chiral factor which induces the optical activity - for static (one-electron mechanism) it can be the asymmetric (Coulomb) static molecular field generated by the helically arranged a-amino groups of lysine side chains on a-helical backbone, for dynamic mechanisms it is the chiral transient polarizability change induced in the (again helically arranged) amide groups. The qualitative consequence of this fact is the difference of “sector rules” describing the sign response of the induced CD to the various relative orientations of pigment molecular and polypeptide helical “rod”. For the static one-electron mechanism, this rule is governed by the porphyrin symmetry and is described by the transformational properties of A,, irreducible representation of D2,, group. In the case of dynamic mechanisms we applied our computational model to get some feeling about the distortions of the sectors. We used a system consisting of TPPC and ahelical lOO-peptide. The center of the tetrapyrrole was placed at 20 A from helix axis. The TPPC plane was parallel to helix. The peptide was rotated about the normal to the TPPC plane. The obtained location of

the nodal surfaces and relative signs of sectors for dynamic mechanisms are shown in scheme 1. Resulting distortion of the nodal planes from the one-electron “reference” case is the dominant difference. The extent of this distortion (e.g. angle 19) varies periodically when the above calculations are repeated after rotation of the TPPC center about the helix axis by some angle. We have therefore no unambiguous basis for the interpretation of sign of the induced CD in absolute structural terms. Despite of this, the conclusion about the equivalence of pigment bonding sites arranged regularly along the lysine side chain superhelices (which we have formulated empirically on the basis of insensitivity of induced CD sign patterns to the variation of < in various complexes) is fully supported by this model calculation. The signs of sectors and layout of the no&l planes remain constant for rotation of porphyrine center by an angle of ~60” followed by translation along the helix axis for x 45 A. Considering the semi-quantitative aspects of the calculation, the form of the equations makes it also possible to visualize the contributions of individual amide groups to the net induced CD. The resulting rotational strength is a difference (imposed by the chirality of the structure) between the positive and negative contributions from odd-even amide groups in the peptide sequence. These differences are typically = 10% of the absolute values of the individual amide group contributions. The envelope of these 51 contributions - when plotted as a function of amino acid residue number in the polypeptide sequence -

+ + + =;jm

static

electron

one-

dynamic

pm y-polarization

dynamic

IJP x-polarization Scheme 1.

dynamic

I+ y-polarization

P. PanEoska et al. /Spectroscopy ofporphyrin-polypeptide

has Lorentzian-like bandshape with the “full width at half maximum” equal to = 28 amide groups. The most important contribution to the induced CD is therefore governed by 14 amino acids in the sequence around the TPPC position onto the helix. Quantitative results of our simple computations have the least reliability for conclusive reasoning. We only mention here the calculated ratios of the orders of magnitude for the respective contributions to net rotational strength of static pTpp-mpp, nondegenerate dynamic pTpp-,upp,dynamic ppp-mTppand pTppmTPP which are 1: 1: 1: 100 for comparable TPPCPP geometries. The order of magnitude calculated for the last (dynamic pTpP-mTPP) is moreover comparable to that found experimentally. The strong contender for the mechanistic explanation of the observed induction of CD is the mixing of electrically and magnetically allowed porphyrin transitions, mediated by the strongly chiral polarizability-type perturbation induced dynamically in the helically arranged amide groups of polypeptide. For further discussion, the following structural possibilities provided by the studied model complexes can be used: TPPC molecules with two different fixed distances between adjacent and opposite anionic groups can be in principle fixed on our u-helical sequential polypeptides (with superhelical structure of spatially uniformly localized cationic bonding sites) in two favorable orientations “edge-on” and “plane-on” (see fig. 1c ). Preliminary molecular mechanic calculations for TPPC- [ LAA] 20complex favored the latter conformation. We have used this energy-optimized model as the reference case during the assignment of p and m character to the observed absorption bands - the assignment mentioned above yielded the correct sign pattern for calculated rotational strengths of this complex geometry. The common feature of all considered structures is the fixation of pigment relatively to peptide. In a theoretical discussion we can use this fact and choose a common coordinate system for both molecular components of the complex. Polypeptide molecular field can be then described locally by the perturbational potential V expanded in terms of symmetry adapted components Vr, obtained by application of projection operators Pr, for individual irreducible representations Ii of TPPC symmetry group DZh. Such a partition opens the possibility to discuss the studied

complexes

411

observables in terms of symmetry selection rules and provides also the tool to get some qualitative information about the conformational dependence of the changes studied. The “duality” of the complexed pigment behavior in the red and Soret regions is now understandable when the presence and/or absence of magnetic dipole allowed transition in respective regions is taken into account. The state mixing under any mechanism provides nonzero oscillator strengths for originally forbidden transitions through terms in which the components Vri play in some form the decisive role. This makes these induced contributions as well as the intensity redistributions in the Soret band conformationally dependent. No such structurally sensitive effects are observed in the electronically “homogeneous” red region, where the p+dependent shifts can be explained by the effect of increased charge density on TPP transition energies as described previously [ 26,271. The same reasoning can be used to rationalize the qualitative difference in multisignate versus monosignate character of induced CD in Soret and red regions. We suppose that another type of conformational change can explain the nonlinearity of r3-p+ dependence. Inspection of induced CD spectra of the TPPC- [LA ] ,, sample reveals the difference between CD curves for & 1 and &4. Only the last one is comparable with CD of [LAAA], containing complex. So as for the previously reported TPPC- [ LLA ] n system [ 41, the higher porphyrin concentration is necessary to “fold” the TPPC- [ LA]” complex into the conformation corresponding to TPPC- [ LAAA ] ,, model system, in which the p+ influence on fluorescence kinetics is then similar and more comparable. These results provide evidence that shortening of fluorescence lifetime for porphyrin fixed on the regular polypeptide molecular matrix is: (a) directly proportional to the intensity of polypeptide matrix molecular field, characterized in our model systems by p+; (b) exhibits definite conformational dependence.

5. Conclusions The presented results of our spectroscopic study of model porphyrin-polypeptide complexes with structurally different peptide parts confirm the earlier ob-

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servation [ 41 of large changes in porphyrin electronic properties induced by its fixation on model polypeptide matrix. The similarity of the results for structurally different polypeptide, nucleic acid [ 182 1 ] and polymeric [ 22-241 matrices supports the notion that this phenomenon is not specific for a few specially selected molecules, but is of more general character. This is connected primarily with the following structural features of the matrix: regularity of its conformation, intensity and transformational properties of its molecular field and possibility of the defined fixation of the pigment on it. The correlation between the fluorescence lifetime shortening and the intensity of the peptide perturbing molecular field, well defined here by the synthesized primary structure, has been shown to be approximately linear if the conformations of pigmentpolypeptide complexes are comparable on CD spectroscopy “sensitivity level”. The observables, describing the transition probability distribution of studied spectroscopic properties of porphyrin (oscillator and rotational strengths, fluorescence quantum yields and lifetimes) were found to be in general more conformationally sensitive than “energy’‘-related observables. Changes of electronic state energies of porphyrin visible transitions correlate linearly with the estimated reciprocal distances of the cationic polypeptide groups. The molecular fields of u-helical polypeptides with 2550% of basic amino acids in their primary structures can be considered as upper limit of the scale of protein field intensities e.g. in natural photosynthetic pigment protein complexes. The perturbational formalism seems to be therefore safely applicable for the theoretical treatment ofthe protein induced changes of bonded pigment properties in vivo. Various discussed mechanisms of the induced spectral changes have the similar basis - the mixing of porphyrin electronic states. As a consequence, in pigment-protein complexes the electronic states loose their “free pigment” identity. This process is also related to the energy separation of the states capable by symmetry to interact and can be therefore different in different spectral regions (viz. differences in Soret and red regions for our complexes). Moreover, due to the interconnection between the operator and state selection rules, the manifestation of this mixing can

be different for various experimental observables, depending on the symmetry properties of corresponding operator.

Acknowledgement

Authors thank Mrs. H. Janesova (Institute of Organic Chemistry and Biochemistry, Czechoslovak Academy of Sciences) for skillful technical assistance. The important comments of referees which contributed substantially to the presentation of the paper are gratefully acknowledged.

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