ABSTRACT. The IR spectra of the bacteriochlorophyll a and b cations and the bacteriopheophytin a and b anions were obtained by using an ER and optically ...
Proc. Nati. Acad. Sci. USA Vol. 85, pp. 8468-8472, November 1988 Biophysics
Infrared spectroelectrochemistry of bacteriochlorophylls and bacteriopheophytins: Implications for the binding of the pigments in the reaction center from photosynthetic bacteria (photosynthesis/bacterial reaction center/chlorophyll-protein interactions/infrared spectroscopy/electrochemistry)
W. G. MANTELE*t, A. M. WOLLENWEBER*, E.
NABEDRYKO, AND J. BRETONt
*Institut fur Biophysik und Strahlenbiologie der Universit~it Freiburg, Albertstrasse 23, D-7800 Freiburg, Federal Republic of Germany; and *Service de Biophysique, DMpartement de Biologie, Centre d'Etudes Nucldaires de Saclay, 91191 Gif-sur-Yvette Cddex, France
Communicated by Pierre Joliot, June 9, 1988
ABSTRACT The IR spectra of the bacteriochlorophyll a and b cations and the bacteriopheophytin a and b anions were obtained by using an ER and optically transparent electrochemical cell. Prominent effects of radical formation on the vibrational spectra were found for bands assigned to the ester, keto, and acetyl C=O groups and for vibrations from macrocycle bonds. The (radical-minus-neutral) difference spectra are compared to the light-induced difference spectra ofthe primary donor photooxidation and the intermediary acceptor photoreduction in the reaction center of photosynthetic bacteria. Light-induced absorbance changes from bacteriochlorophyll a-containing reaction centers bear striking similarities to the electrochemically induced absorbance changes observed upon formation of bacteriochlorophyll a' in vitro. Comparison ofthe radical formation in vitro in a hydrogen-bonding or a nonhydrogen-bonding solvent suggests an ester C=O group hydrogen bonded in the neutral state but free in the cation state. For the keto C=O group, the same comparison indicates one free carbonyl group. The (anion-minus-neutral) difference spectra of bacteriopheophytin a and b exhibit a single band in the ester C=O frequency range. In contrast, two bands are observed in the difference spectra of the intermediary acceptor reduction in the reaction center of Rhodopseudomonas viridis. The higher frequency band exhibits a sensitivity to 'H-2H exchange, which suggests a contribution from a protonated carboxyl group of an amino acid side chain.
chromatophores concomitant with P photooxidation (10) as well as with H reduction (11). By using Fourier transform infrared (FTIR) difference spectroscopy, sensitivity can be high enough to detect single bonds. The light-induced FTIR difference spectra consist of a number ofcharacteristic bands that are highly reproducible in frequency and amplitude (10, 11). Since IR spectroscopy does not monitor just the pigments, bands might arise from the polypeptides, lipids, and water molecules as well. Thus, a comparison to modelcompound spectra is essential for the interpretation of the difference bands. IR spectroscopy has established the role of the C=O groups in the intermolecular interaction of aggregated forms of chlorophylls (Chls) (9, 12). These model studies of Chl have covered only the neutral species. However, cation and anion IR spectra have to be included in order to account for the radicals formed upon charge separation in vivo.
Cations and anions of pigments can be generated chemically (13, 14). IR spectroscopic investigation of the chemically oxidized BChl-a shows bands that are diagnostic for molecular vibrations of the cation. However, overlapping IR bands of oxidants and the instability of BChl-b considerably limit this access to model-compound spectra. A much more appropriate approach would be the electrochemical generation of radicals in a cell suited to IR spectroscopy (15, 16). In a previous report (17), we have described an optically and IR transmitting, anaerobic, thin-layer electrochemical cell that allows Chl radicals to be generated and investigated in situ. We have shown that the selectivity of this FTIR spectroelectrochemistry of isolated pigments is as high as the selectivity already demonstrated for the light-induced FTIR difference spectroscopy of large Chl-protein complexes (10, 11, 18). Here, we present FTIR spectra of the BChl cation and BPheo anion radicals generated electrochemically in situ, which can serve as model-compound spectra for the lightinduced FTIR difference spectra of the P photooxidation and the H photoreduction in RC from photosynthetic bacteria. By comparison of the FTIR difference spectra of the P aprotic solvents with those of the pigments in the RC, conclusions are drawn on the interactions of the pigments with their native environment in their neutral and radical form.
In the primary events in photosynthesis, absorption of light leads to charge separation between electron donor and acceptor molecules. In the bacterial reaction center (RC), the primary electron donor (P) is a bacteriochlorophyll (BChl)-a or -b dimer, the intermediary electron acceptor (H) is a bacteriopheophytin (BPheo)-a or -b monomer, and the primary acceptor is a quinone. Spectroscopic data on these components have been reported (1-3), and, together with the high-resolution structure analysis (4-7), electron transport pathways can be visualized. Specific interactions of the pigment molecules with their polypeptide environment, in addition to pigment-pigment interactions, account for the spectral and redox properties of BChls in vivo. Among these interactions are external liganding to the Mg atom of the BChl (8) as well as hydrogen-bonding from pigment groups to polypeptide side chains (7, 8). Although such interactions have been studied by resonance Raman (RR) and IR spectroscopy in model systems (8, 9), details about the interactions in vivo both in the neutral and charge-separated state still have to be collected. In previous studies, we have used IR difference spectroscopy to investigate the molecular changes in bacterial RC and
MATERIALS AND METHODS BChl-a and BChl-b were isolated and purified by using an HPLC step to remove lipids (19). BPheo-a and BPheo-b were prepared with an excess of oxalic acid and were repurified by Abbreviations: H, intermediary electron acceptor; P, primary electron donor; RC, reaction center; RR, resonance Raman; FTIR,
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Fourier transform IR; Chl, chlorophyll; BChl, bacteriochlorophyll; BPheo, bacteriopheophytin. tTo whom reprint requests should be addressed.
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Biophysics: Mdntele et al.
Proc. Natl. Acad. Sci. USA 85 (1988)
HPLC as described (20). The pigments were dried by repeated evaporation of added dichloromethane, followed by drying under reduced pressure (10-' mmHg). They were stored under argon at -200C until use. Optical and IR spectra were obtained in the optically transparent thin-layer electrochemical cell described previously (17). Sample handling and transfer to the cell were made anaerobically under argon. Fully deuterated methanol (C2H40) and tetrahydrofuran (C42H80) were used as solvents for IR spectroelectrochemistry. Methanol was dried for 48 hr over molecular sieves and activated aluminium oxide. Tetrahydrofuran was dried by condensation under reduced pressure onto aluminium oxide (21). The supporting electrolyte tetrabutylammonium hexafluorophosphate was purified as described (22). FTIR spectra were recorded as in ref. 17. Controlled potential electrolysis and coulometry were performed with a home-built potentiostat. Films of bacterial RC were prepared according to refs. 10 and 11. Light-induced FTIR difference spectra of these films, as well as the corresponding controls in the visible spectral range, were obtained as described (10, 11).
RESULTS An optical absorbance spectrum of BChl-a in tetrahydrofuran recorded in the spectroelectrochemical cell together with the spectrum obtained after several minutes of electrolysis at U = +0.5 V (the potential at the working electrode was corrected for the internal reference drop according to a procedure described in ref. 17) is shown in Fig. 1 Inset. At this potential, the ir-monocation radical is generated according to cyclic voltammograms (20, 23). The time course of oxidation as followed by the absorbance at 772 nm (data not shown) indicates that a quantitative conversion is achieved within a
few minutes. Simultaneous coulometric measurements show that at this potential one electron per BChl-a is removed. The spectrum of the electrolysis product is in close agreement with published spectra of the cation radical of BChl-a generated by electrochemical (23) or chemical (13) oxidation. Upon reversal of the potential, about 80-90% of the neutral BChl-a are regenerated, and little degradation product is formed, as is seen by the almost complete absence of a band at 680 nm (data not shown). The corresponding IR absorbance spectra of the same neutral and fully evolved radical cationic BChl-a are shown in Fig. la. The time course of oxidation and rereduction allows consecutive spectra of the evolving cation to be recorded, as has been demonstrated for the anion of BChl-a (17). The IR difference spectrum (BChl-a' minus BChl-a) is shown in Fig. lb (solid line). Such spectra will be referred hereafter as BChl-a' spectra to allow direct comparison with the light-minus-dark spectra of the P photooxidation in bacterial RC (10). They were termed P+ spectra and can only be obtained as difference spectra. With this definition, the appearing cation bands are positive, while the disappearing BChl bands are negative. Starting from the fully evolved cation, the neutral species can be regenerated by reversal of the polarity. The difference spectrum thus obtained is shown in Fig. lb (dotted line) and indicates a remarkable reversibility. A light-induced P+ spectrum from Rhodobacter sphaeroides RC is shown in Fig. 2a together with the BChl-a' spectra obtained in tetrahydrofuran (Fig. 2b) and in methanol (Fig. 2c). The band frequencies measured in the difference spectra and their tentative assignment are summarized in Table 1 for the neutral and cation state. BChl-b is known to be much more susceptible to degradation than is BChl-a, which limits the choice of solvents (24,
a
e0
S
8469
VI~~~~~~~~L .0~~~~~~~~~~~~c U
5~~~~~~~L
U) I 0D C-.~~~7
A
0~~~~~~~~~~~~~~~~ ---------t------1 D--------
400
700
Wavelength (nm)
.0~~~~~~~~~~~~~~~.0
180
1000
1650
150
135
12
I-au.
tUQ 0
C~~~~~~~~~~D
c
ul~~~~~~~~~ 1800
1700
1600
1500
1400
l
1300
Wavenumbers (cmrf)
FIG. 1. (Inset) Optical spectra of neutral BChl-a (-) in deuterated tetrahydrofuran before electrolysis and after cation formation at U - +0 5 V ( ). (a) Corresponding IR absorbance spectra. (b) IR difference spectra of the BChl-a cation formation [BChl-a' minus BChl-a (-)] and of the re-reduction of the BChl-a cation [BChl-a minus BChl-a' (.)] after reversal of the potential. (T = 295 K; 4-cm-' resolution.) a.u., absorbance units.
Wavenumbers (cm') FIG. 2. (a) Light-induced IR difference spectrum of the primary
donor photooxidation in Rb. sphaeroides RC (T-=255 K; excitation
wavelength = 715-1100 nm). Redox-induced IR difference spectra of the BChl-a cation formation (BChl-a+ minus BChl-a) in deuterated tetrahydrofuran (b) and methanol (c). (T = 295 K; 4-cm-1 resolution.) a.u., absorbance units.
Proc. Natl. Acad. Sci. USA 85 (1988)
Biophysics: MAntele et al.
8470
Table 1. Comparison of peak frequencies (cm-') in light-induced difference spectra from RC (P and P+; H and H-) and in radical difference spectra from isolated BChl (BChl and BChl') and BPheo (BPheo and BPheo-)
THF Rp. vir. RC THF Rp. vir. RC Methanol THF P+ BChl-b BChl-b+ H H- BPheo-b BPheo-bBChl-a BChl-a' BChl-a BChI-a+ P
Tentative assignment C=0 ester
Rb. sphaer. RC
C=O keto
1683 s 1715 s 1684 s 1716 s 1652 s 1721 s 1674m 1712s 1682s 1701 s 1662 sh 1710 sh 1702 sh 1669 w 1703 s 1636 sh 1650 s 1658 s 1653 w 1655 m 1645 w 1658 m 1659 m 1597 sh 1636 w 1634 w 1624 w 1622 m
C=0
or or or
P
P+
1750 s
acetyl peptide quinone
1737 s
1750 s
1756 s
1745 m 1755
m
1731
s
1747
s
1747 s 1743 1732 s 1714 s 1709 sh 1683 s 1667s 1703
s
1727 m
s
1677 sh 1658 s 1627 s
O-H (H20)
C=C or C-C (mainly pigment)
1570 m 1544
1523
s w
1593 s 1545 m 1570 w 1579 w 1566 m 1593 m 1565 w 1523 s 1541 w 1552 m 1526 m 1552 m 1560 m 1552 m 1500 w 1531 w 1483 w 1483 m 1485 w 1477 s
1544 m 1524 s 1502 w
1418 w 1419 w C=C or C-C 1425 w 1477 s 1456 sh (mainly 1401 m quinone) Band intensities: s, strong; m, medium; w, weak; sh, shoulder. Rb. sphaer., Rb. sphaeroides; Rp. vir., Rp. viridis. THF, tetrahydrofuran.
25). The electrochemical oxidation of BChl-b was thus performed in tetrahydrofuran at the same potential as that used for BChl-a. The BChl-b+ IR spectrum (data not shown) is similar to that of BChl-a+ as seen by comparable band frequencies (see also Table 1). The reversibility of the reaction is only about 70-80%, due to partially irreversible degradation, most probably at the out-of-cycle double bond (24). By using tetrahydrofuran as a solvent, the BPheo-b anion radical was generated almost quantitatively at U = -0.8 V. Reoxidation to the neutral species was performed at U = +0.3 V, with a reversibility of only 60-70%o. An IR difference spectrum (BPheo-b- minus BPheo-b) of the BPheo-b reduction is shown in Fig. 3a. The spectrum of the photochemical reduction of the intermediary electron acceptor (H-) in the Rhodopseudomonas viridis RC (11, 26) is shown in Fig. 3b. The frequencies and a tentative assignment of the main bands are listed in Table 1.
4~ ~
~
n 0 t
~~C
n
s
3
o
b
