5536
J. Phys. Chem. B 2001, 105, 5536-5545
Electrochemical Oxidation of Bacteriochlorophyll a in Reaction Centers and Antenna Complexes of Photosynthetic Bacteria Tatyana N. Kropacheva† and Arnold J. Hoff*,‡ Chemistry Department, Udmurt State UniVersity, 426037 IzheVsk, Russia, and Biophysics Department, Huygens Laboratory, Leiden UniVersity, P.O. Box 9504, 2300 RA, Leiden, The Netherlands ReceiVed: September 19, 2000; In Final Form: March 21, 2001
The oxidation of BChl in several pigment-protein complexes involved in bacterial photosynthesis was investigated by optical spectroelectrochemistry, with the aim to obtain information on the tuning of the BChl oxidative midpoint potential (Em) through BChl-BChl and BChl-protein interactions. The two accessory BChls in reaction centers (RCs) of Rhodobacter sphaeroides (R-26 and wild-type) have Em of 0.73V and 0.83V ((0.01V), appreciably higher than the Em for pentacoordinated monomeric BChl in nonprotic solvents (∼0.66V). The self-aggregation of BChl in vivo leads to a lowering of the oxidation potential: Em ) 0.50 V (the primary donor P865 in Rb. sphaeroides RCs), Em ) 0.57 V (the light-harvesting complex LH1 of Rhodopseudomonas acidophila), Em ) 0.60V (LH1 complex of Rb. sphaeroides). Only 20-35% of the total absorbance at 875 (880) nm in LH1 complexes corresponds to reversible BChl oxidation, whereas further bleaching is irreversible. Complete irreversibility of BChl oxidation was observed in LH2 complexes of Rps. acidophila for both monomeric (B800) and aggregated (B850) pigments, with the B850 component bleached somewhat more easily than the B800 component. One of the products of degradative oxidation is 2-desvinyl2-acetyl Chl. The following order of susceptibility to oxidation is observed: P865 (RCs) > B880 (LH1) = B875 (LH1) > B850 = B800 (LH2) > B800 (RCs). It is suggested that irreversible oxidation of BChl in antenna complexes results from dismutation of BChl and carotenoid (Car) cation pairs (BChl.+- BChl.+ and BChl.+- Car.+), resulting in the formation of the highly reactive BChl dication. The remarkable difference in LH1 and LH2 oxidation could be due to the presence of oxidized Car in LH2 at early oxidation stages of BChl. The results are discussed in terms of the X-ray diffraction structures of the RC and antenna complexes.
Introduction One tool for studying the structural arrangement and interactions in photosynthetic pigment-protein complexes is the determination of the in situ redox characteristics of the cofactor pigments and the comparison of these characteristics with corresponding in vitro data. With respect to electrochemical characteristics of a pigment, the protein matrix differs considerably from a homogeneous medium in solution. In addition to creating a local environment with a low dielectric constant, the protein provides the possibility for various specific interactions with a pigment, such as metal coordination, hydrogen bonding, etc. Another cause for changing the pigment redox behavior in vivo is the often rather short distances between adjacent cofactors (5-15 Å) compared to isolated molecules in solutions. For example, the primary electron donor in a bacterial reaction center (a BChl dimer) has an oxidation potential ranging from 0.45 to 0.50 V (purple bacteria) to 0.25-0.35 V (green sulfur bacteria), which potential is much lower than that found for BChl a in organic solvents.1 Strong coupling between the two BChl molecules of the dimer presumably accounts for most of the potential decrease relative to in vitro values. A strong modulation of the oxidation potential of the primary donor by the protein matrix was demonstrated for a number of Rhodobacter (Rb.) sphaeroides mutants designed to change the number of hydrogen bonds to the primary donor. The oxidative midpoint * Author for correspondence. Phone +31-71-5275955, Fax +31-715275819, E-mail
[email protected]. † Udmurt State University. ‡ Leiden University.
potential was shown to increase from 500 mV (wild-type) to 765 mV by the formation of up to four hydrogen bonds to the two BChls of the dimer.2 In contrast, the primary donor in Photosystem II (a Chl monomer or dimer) has a very high Em (>1 V), higher by several hundreds of mV than in any model system studied so far.1 The large range in variation of the oxidative midpoint potentials measured in vivo clearly makes it of interest to study the redox behavior of a given pigment in different photosynthetic preparations and to relate the obtained results to the known structure. Thus, the redox characteristics of the pigment may be used to probe its interactions with the local environment. In this communication we report on the oxidative electrochemistry of BChl in several pigment-protein complexes involved in bacterial photosynthesis: RCs of Rb. sphaeroides (R-26 and wild-type), the light-harvesting complexes LH1 and LH2 of Rhodopseudomonas (Rps.) acidophila, and the LH1 complex of Rb. sphaeroides. In contrast to the primary electron donor in reaction centers (RCs), whose oxidation behavior is well characterized,3,4 no data are available on in situ oxidation of accessory BChls in RCs. The oxidation behavior of antenna BChl in various purple bacteria was reported earlier for oxidation by chemical agents only.5-12 With such titration it is difficult to obtain accurate measurements of Em and reversibility. In the work presented here the oxidation behavior of BChl in vivo was studied by optical spectroelectrochemistry. This method offers advantages over chemical titration in that it allows one to determine accurate redox midpoint potentials as well as to follow the kinetics and reversibility of the electrode process
10.1021/jp003381b CCC: $20.00 © 2001 American Chemical Society Published on Web 05/16/2001
Electrochemical Oxidation of Bacteriochlorophyll a and possible concomitant reactions.13,14 Our results show that the one-electron oxidation potential of BChl in the pigmentprotein complexes studied can vary over the range 0.5-0.8 V. Comparison of the data obtained for monomeric BChl in vivo with the reported Em values for the BChl.+/BChl couple in various solvents shows that the influence of the protein matrix on the Em does not exceed 0.1-0.2 V. Aggregation of BChl in natural systems leads to a significant lowering of the oxidative Em. The stability of the BChl cation radical formed upon oxidation of accessory BChl in RCs and of BChl in LH1 complexes is lower compared to that of the oxidized primary donor, P865.+. Despite the similar structure of LH1 and LH2 complexes, for the latter no reversible oxidation of BChl is observed. This difference in reversibility at the early oxidation stage is attributed to the simultaneous presence of oxidized carotenoid and BChl in LH2 but not in LH1. Materials and Methods Reaction centers of Rb. sphaeroides R-26 were isolated in TL-buffer (0.1% lauryldimethylamine N-oxide (LDAO), 10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA,) according to ref 15. Wildtype Rb. sphaeroides RCs were isolated by incubating chromatophores (OD800 ) 50 cm-1) with 0.3% LDAO at 30 °C for 40 min, followed by centrifugation for 1.5 h at 200 000 g. To the supernatant 45% (NH4)2SO4 was added to make a final concentration of 25%, and the LDAO concentration was increased up to 0.3%. After 10 min of low-speed centrifugation the pellet was collected; resuspended in 0.025% LDAO, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA; and desalted on a Sephadex G-50 column. Finally the RCs were washed from a DEAE Sephacel column with 100-300 mM NaCl, 0.025% LDAO, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA. The LH2 (B800-850) and LH1-RC (B880-RC) complexes from Rps. acidophila (strain 10050) were isolated as follows. The cells broken by sonication were solubilized for 4 h at 4 °C by 2% LDAO in 20 mM Tris-HCl (pH8.0). Individual complexes were isolated from chromatophores by sucrose density gradient centrifugation. The gradient was prepared by layering on 4 mL of 0.8 M sucrose, 8 mL each of 0.6 M, 0.4 M, 0.2 M sucrose (all in 0.1% LDAO, 20 mM Tris-HCl, pH8.0). The chromatophore solution (6 mL) was layered on top, and the tubes were centrifuged at 220 000 g for 16 h at 4 °C. Three distinct bands appeared: the lower and the upper bands containing LH1-RC and LH2 complexes, respectively, were collected. The fractions were dialyzed extensively against TLbuffer and concentrated with an Amicon 70 kDa membrane filter. LH1-only complexes, not containing RCs and LH2, were isolated from the Rb. sphaeroides mutant M2192. The redox mediators (tris(1,10-phenantroline)iron(II) sulfate, [Fe(phen)3]SO4, dicyanobis(1,10-phenantroline)iron(II), [Fe(phen)2(CN)2], and potassium tetracyanomono(1,10-phenantroline)ferrate(II), K2[Fe(phen)(CN)4], were synthesized according to ref 17. The redox potentials of the mediators under our experimental conditions (100m KCl, 50 mM phosphate buffer, pH6.8, 0.1% n-dodecyl-β-D-maltoside (DM)) were determined by chemical redox titrations with Ce(IV) and Sn(II) as oxidant and reductant, respectively. The resulting values (vs. NHE) are: [Fe(phen)(CN)4]-/[Fe(phen)(CN)4]2- (Em ) 0.59 V), [Fe(phen)2(CN)2]+/[Fe(phen)2(CN)2] (Em ) 0.80V), [Fe(phen)3]3+/ [Fe(phen)3]2+ (Em ) 1.03 V). These values differ by 10-20 mV from those reported in the literature for other experimental conditions.18 In the visible spectral region the reduced forms of the mediators absorb in the range 400-550 nm with
J. Phys. Chem. B, Vol. 105, No. 23, 2001 5537 extinction coefficients [Fe(phen)3]3+: 510∼11mM-1 cm-1; [Fe(phen)2(CN)2]: 509∼6mM-1 cm-1; [Fe(phen)(CN)4]2-: 462∼4mM-1 cm-1. Upon oxidation all mediators give practically colorless compounds.17,18 The optically transparent thin-layer cell (see e.g.13) with optical path length ∼0.2 mm was constructed with a gold minigrid working electrode sandwiched between two quartz windows fixed in a Teflon body. The platinum circular wire serving as a counter electrode surrounded the working electrode outside the light beam. A silver-silver chloride in saturated KCl reference electrode was connected to the cell through a salt bridge. The potential of the Ag/AgCl reference electrode was periodically measured against saturated calomel electrode, yielding reproducibly 198 ( 2 mV vs NHE. The cell was checked by determining the midpoint potential of the [Fe(CN)6]3-/[Fe(CN)6]4- redox couple in 0.1 M KCl. The Em was obtained from cyclic voltammograms recorded in the thin-layer cell and also from monitoring the changes in mediator absorbance at 420 nm as a function of applied potential. The electrochemical titrations were performed at room temperature under aerobic conditions. For the titrations, the concentrated stock solutions of RCs or antenna complexes were diluted 10 to 40-fold into a solution containing 100 mM KCl, 50 mM phosphate buffer (pH6.8), 0.1% DM, and a mediator (40-400 µM). The surface of the gold electrode was cleaned and chemically modified before each titration as follows. To remove organic contaminants the electrode was washed successively with 95% ethanol, water, and 2 M nitric acid (70 °C, 1 min) and generously rinsed with water. The freshly cleaned surface was treated for 10 min with a 4 mM solution of 4,4′dithiodipyridine (Aldrich). The stable monolayer of modifier formed on the gold prevents protein from adhering to the gold and promotes electron transfer. Values of Em obtained in repeated titrations of pigment-protein complexes usually agreed within (10 mV. Optical spectra were recorded with a Perkin-Elmer Lambda 900 UV/VIS/NIR spectrometer. A Princeton Applied Research model 173 potentiostat with model 175 Universal Programmer was used for electrochemical measurements. Results Electrochemical Oxidation of Pigment Cofactors in Rb. sphaeroides Reaction Centers. Figure 1 shows the typical results of electrochemical titration of Rb. sphaeroides RCs over a wide range of potentials. A mixture of mediators was used for poising the potential, as the effective working range over which a given mediator controls the solution potential is close to the mediator Em (about Em ( 100 mV).19 A slight contribution of mediators to the absorbance can be observed in the initial spectrum around 450-550 nm. Upon oxidation of all mediators, the decrease of the absorbance in this spectral region is estimated to be less than ∼0.01, so that redox reactions of the mediators do not interfere with the spectral features of RC pigment oxidation. Oxidation of the primary donor P865 results in a bleaching of the 865 nm band together with small shifts of the accessory BChl (802 nm f 800 nm) and BPheo (757 nm f 760 nm) maxima, and a slight increase of the absorbance in the near-infrared. The same spectral changes were observed for photochemical or electrochemical3 oxidation of the bacterial RCs. The redox titration of P865 mediated by K4[Fe(CN)6] or K2[Fe(phen)(CN)4] was performed by changing the potential of the working electrode in intervals of 20 mV in both oxidative and reductive directions. The kinetics of the absorbance changes (Figure 2A) shows that less than 5 min is required for the
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Figure 1. Optical absorption spectra of Rb. sphaeroides R-26 reaction centers during electrochemical titration at various potentials. (The feature at about 870 nm is an instrumental artifact.) Mediators: Mixture of K2[Fe(phen)(CN)4] (140 µM), [Fe(phen)2(CN)2], (40 µM), [Fe(phen)3]SO4 (80 µM). RCs: OD802 ∼13 cm-1.
equilibration of a sample at each potential step. The oxidationreduction cycle is highly reversible and may be repeated many times. The redox titration curve (Figure 2B) obtained with both mediators is approximated with the one-electron Nernst function, yielding Em ) 495 ( 5 mV for the P865.+/P865 couple (wildtype and R-26 mutant). At potentials higher than ∼600 mV, spectral changes associated with accessory BChl and BPheo oxidation are observed (Figure 1). First, the maxima at 800 nm and 598 nm (Qy and Qx transitions of accessory BChl, respectively) are bleached. At more oxidizing potentials there is a bleaching of the 760 and 533 nm bands characteristic of the Qy and Qx transitions of BPheo in the RCs. Simultaneously the absorbance in the 400450 nm region and around 700 nm increases. To ascribe the observed spectral changes to known oxidation products, we first consider the literature data on in vitro optical spectra of BChl and BPheo cation radicals.20-22 The BChl.+ spectrum reported by different authors is characterized by a weak absorbance at the Qy (∼770 nm) and Qx (580-605 nm) maxima of the initial neutral BChl. The absorbance at the Soret band (∼360 nm) is almost unchanged, and an intense new band in the 400-440 nm region appears as well as a weak broad absorbance in the near-infrared (870-970 nm). The BPheo.+ spectrum has essentially the same features as that of the BChl cation radical: a small residual absorbance at the Qy (∼750 nm) and Qx (∼525 nm) bands of the initial neutral BPheo, some decrease of the absorbance at the Soret maximum (∼360 nm), and the appearance of a new strong band at 400-420 nm. Figure 1 shows that some of the spectral changes observed upon oxidation of the RC pigments resemble those attributed to the formation of monomeric BChl and/or BPheo cation radicals in vitro. Due to the high reactivity of these species, the appearance of a number of secondary products is not excluded, thus the maximum at ∼700 nm belongs to a green BChl oxidation product (2desvinyl-2-acetyl Chl23).
Figure 2. (A) Kinetics of the potential-induced absorbance changes of the primary donor in Rb. sphaeroides R-26 reaction centers. The potential was changed in steps of 20 mV (shown by arrows). Mediator: K2[Fe(phen)(CN)4], ∼40 µM; RCs: OD802 ∼3 cm-1. (B) Redox titration curve of the primary donor P865 of Rb. sphaeroides R-26 reaction centers. The line is drawn for the one-electron Nernst function with Em ) 495 mV.
To obtain information on the BChl.+ concentration, the reversal technique can be used: The forward potential step that causes the build-up of reaction products is followed by a sudden potential shift to a value resulting in a rereduction of accumulated BChl.+. Figure 3A shows the kinetics of BChl oxidation at a constant potential (0.74 V) with rereduction performed by stepping the potential to a cathodic region (0 V). In general, both the oxidation of accessory BChl and its rereduction are much slower processes compared to those of the primary donor. Figure 3A shows that most of the accessory BChl is oxidized reversibly, i.e., it is transformed into the cation radical, whereas a smaller amount is converted into other oxidation products. To determine the spectral contribution from different pigments the spectra were deconvolved into Gaussian components in the range 600-1100 nm. The area of the Gaussian band was used to estimate the amount of each chromophore. As an example, Figure 3B shows the spectra taken during an oxidationreduction cycle. Oxidation at 0.76 V results in ∼42% bleaching of the initial BChl absorbance, which can be restored back up to ∼82% from the starting value. BPheo is more stable toward oxidation and also less recoverable than the accessory BChl. Under the same conditions the bleaching of BPheo corresponds to ∼29% of the initial amount, from which ∼15% is oxidized irreversibly. The band arising at ∼700 nm is eliminated upon rereduction; at the same time the featureless absorbance increase
Electrochemical Oxidation of Bacteriochlorophyll a
J. Phys. Chem. B, Vol. 105, No. 23, 2001 5539
Figure 4. Redox titration curve of the accessory BChl in Rb. sphaeroides reaction centers. The BChl absorbance of the different samples was normalized at the initial value at 0 V. The arrows show the absorbance level obtained upon rereduction at a number of potentials. The solid line represents the sum of the two one-electron Nernst functions with Em ) 725 mV and Em ) 830 mV, shown also by dashed lines. RCs: R-26 mutant (b, 9, 2), wild-type (0, O); OD802 5-13 cm-1. Mediators: (2) mixture of K2[Fe(phen)(CN)4] (140 µM), [Fe(phen)2(CN)2], (40 µM), [Fe(phen)3]SO4 (80 µM); (b, O) mixture of K2[Fe(phen)(CN)4] (64 µM), [Fe(phen)2(CN)2], (30 µM); (9, 0) K2[Fe(phen)(CN)4] (420 µM). Inset: Redox titration curve of the BPheo in Rb. sphaeroides R-26 reaction centers. Mediators: the same as for (2).
Figure 3. (A) Kinetics of absorbance changes of the accessory BChl in Rb. sphaeroides wild-type reaction centers upon oxidation at a constant potential (0.74 V) followed by rereduction. Mediator: K2[Fe(phen)(CN)4], ∼420 µM; RCs: OD802 ∼5 cm-1. (B) Gaussian deconvolution of optical absorption spectra of Rb. sphaeroides R-26 reaction centers obtained during the oxidation-reduction cycle at 0.76 V. Mediator: K2[Fe(phen)(CN)4], ∼420 µM; RCs: OD802 ∼6 cm-1. The baseline was approximated by a sloping absorbance linear in the 9501150 nm range.
in the whole spectral region is irreversible. Part of this increase may be due to light-scattering caused by protein adhesion or denaturation at the electrode; another part may represent the broad absorbance of a mixture of BChl and BPheo oxidation products. The determination of the oxidation midpoint potential of the accessory BChl (B800) in the RCs is difficult because the B800 cation radical is not stable and undergoes secondary irreversible reactions: k1
k2
BChl - e S BChl.+ w secondary products k-1
Experimentally, only the concentration of neutral BChl can be monitored directly (for example, from the amplitude of the Qy band) because the absorbance of BChl.+ or that attributable to secondary products is weak and not clearly defined. The rate of forward reaction rises with the applied potential up to ∼0.8 V and then remains almost constant due to mass transport limitation. At potentials lower than ∼0.85 V, all absorbancetime curves approach to potential-determined quasi-equilibrium values and not to completion, which proves that the first step in the above scheme is faster than the second step (estimated (k1 + k-1)/k2 ∼10). The contribution of the secondary reaction as determined by reversing the potential is approximately the same in the range 0.6-0.9 V: about 60-70% of oxidized BChl is transformed into cation radical. At higher potentials (>0.9 V) the reversibility of BChl oxidation becomes much smaller (20-30%), which indicates that the degradation of BChl.+ then occurs more rapidly (estimated (k1 + k-1)/k2 ∼1). The titration curve of accessory BChl obtained with different mediator combinations is shown in Figure 4, where for a number of points arrows indicate the absorbance level obtained upon rereduction. Essentially the same results were obtained for both Rb. sphaeroides wild-type and the R-26 mutant. Because BChl oxidation is not completely reversible, fitting the experimental data with the Nernst equation can be done only tentatively. Nevertheless, no good approximation to the experimental points is possible by using a one-component one-electron Nernst curve, so that at least two different redox components must be considered. In Figure 4, dashed lines represent the theoretical one-electron Nernst curves for two redox components (Em ) 725 mV and Em ) 830 mV) with equal contribution to the total absorbance; their sum (solid line) is a satisfactory fit to the experimental data. Figure 4 (inset) shows that BPheo is more stable toward oxidation than BChl: under identical conditions the BPheo oxidation curve is about 60 mV anodically shifted compared to the BChl curve. About 30% of the initial BPheo absorbance at the Qy band still remains, even at potentials around 1 V. The
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Figure 6. Kinetics of the potential-induced absorbance changes of BChl in LH complexes. The potential was increased stepwise (shown by arrows) with intermediate reduction steps at 0 V (shown by v). Curve a: The B875 complex from Rb. sphaeroides. Mediator: K2[Fe(phen)(CN)4], ∼126 µM; OD875 ∼1 cm-1, λ ) 874 nm. Curve b: The LH1RC complex from Rps. acidophila. Mediator: K2[Fe(phen)(CN)4], ∼126 µM; OD883 ∼1 cm-1, λ ) 883 nm. Curve c: The B800-850 complex from Rps. acidophila. Mediator: K2[Fe(phen)(CN)4], ∼126µM; OD802 ∼1.5 cm-1, λ ) 802 nm.
Figure 5. (A) The Qy absorption band of the B875 complex of Rb. sphaeroides at various potentials. Mediator: K2[Fe(phen)(CN)4], ∼126 µM; OD873 ∼4 cm-1. Inset: The full initial spectrum. (B) The difference spectra (oxidized-initial) obtained at different potentials corresponding to Figure 5(A). Near isosbestic points are shown by V. (C) The optical absorption spectra of the LH1-RC complex of Rps. acidophila at various potentials. The potential was increased stepwise from 0.5 V (initial) to 0.66 via 20 mV steps; at each step oxidation was performed for 5 min, after which the spectrum was recorded. Mediator: K2[Fe(phen)(CN)4], ∼126 µM; OD883 ∼3 cm-1.
small degree of BPheo recovery after oxidation (5-15% from the initial absorbance) does not allow the determination of its Em. Electrochemical Oxidation of LH1 Complexes. The absorption maximum of membranes of the Rb. sphaeroides mutant lacking both the LH2 complex and the RC is centered at 874 nm, close to the value for the B875 complex isolated from wildtype Rb. sphaeroides.12,24 The availability of this mutant allows one to examine the oxidation of antenna BChl without com-
plications arising from the oxidation of the RC. The spectra measured at different potentials (Figure 5A) show a gradual bleaching, which is accompanied by a shift of the absorption maximum to shorter wavelengths (874 nm f 856 nm), attributed to a gradual loss of electronic interactions between neighboring antenna BChl upon oxidation. To check the reversibility of absorbance changes at different potentials, sets of oxidation-reduction cycles were studied. Figure 6 (curve a) shows the experimental results upon progressive oxidation of antenna BChl at increasing potentials with intermediate return to the starting point. At potentials up to ∼0.62 V the absorbance bleaching is highly reversible, and a sample can be cycled for many times without any absorbance loss. The fast kinetics of reversible oxidation reminds one of the oxidation of the RC primary donor (Figure 2A). Going to more positive potentials, notwithstanding the higher degree of oxidation achieved, the amount of recovered absorbance remains the same. The amplitude of this reversibly oxidized component is stable up to ∼0.70 V; at higher potentials the degree of rereduction becomes smaller. The reversible oxidation corresponds to approximately of 30-35% of the total absorbance at 874 nm and occurs with Em ) 0.60V (n ) 1). As can be seen from the difference spectra (Figure 5B), the reversible B875 oxidation (observed at potentials up to the range 0.62 to 0.64 V) is associated with the bleaching centered at a wavelength 6-8 nm longer than that of the bulk pigment. The absorption spectrum of the isolated LH1-RC complex from Rps. acidophila has a minor band near 800 nm due to the presence of the RC (Figure 5C). The band at 883 nm is dominated by antenna BChl absorbance. The location of the
Electrochemical Oxidation of Bacteriochlorophyll a antenna BChl band in the LH1-RC complex from Rps. acidophila is close to that observed for Rhodospirillum (Rs.) rubrum chromatophores (880-882 nm) containing only the LH1 complex associated with the RC and not LH2. At potentials between 450 and 550 mV, the long-wavelength absorbing pigment (887-888 nm) is bleached first; at higher potentials (>0.6 V) the absorbance decrease is centered at shorter wavelengths (883-884 nm). The oxidation of the LH1-RC complex performed at a number of potentials (Figure 6, curve b) differs from that found for the LH1 complex from Rb. sphaeroides. Even at low potentials (0.5-0.6 V) the oxidation is not completely reversible, so that reversible and irreversible oxidation reactions take place simultaneously. Moreover, once started, irreversible oxidation occurs even without applying a potential. In the Rb. sphaeroides LH1 complex at the early stage of oxidation, the reversibly oxidized component is accumulated quite selectively. When it reaches its maximum yield (at 0.660.68 V) the irreversible losses are only about 10-20% of the total absorbance. In contrast, the reversible component in Rps. acidophila LH1-RC complex is accumulated to a maximum (at 0.60-0.62 V) corresponding to only 30-40% of the bleaching of the 883 nm band. Significant accumulation of the BChl green oxidation product is evidenced by the appearance of the 690 nm band; for the Rb. sphaeroides LH1 complex this byproduct was a minor one. Estimation of the Em for reversible oxidation of antenna BChl in Rps. acidophila LH1-RC is further complicated by the presence of RCs whose oxidations partially overlap with antenna BChl. The maximum contribution of a reversibly oxidized antenna BChl component to the maximum achievable B883 bleaching is 20-25% with Em ) 0.57 V (n ) 1). As for the Rb. sphaeroides LH1 complex, the amplitude of the reversibly bleached component decreases upon going to more positive potentials (>0.62 V). In both LH-1 complexes Car is oxidized to a smaller extent compared to antenna BChl. At potentials when a high proportion of BChl is bleached reversibly, only slight reversible oxidation of Car is observed (Figure 5C). At higher potentials, the oxidation of Car is 80-20% reversible depending on the potential and oxidation time. Electrochemical Oxidation of LH2 Complexes. The spectra taken during the oxidation of the B800-850 complex from Rps. acidophila show a bleaching of both infrared bands with simultaneous accumulation of the 685 nm-absorbing product typical for degradative BChl oxidation (Figure 7A). The carotenoid bands in the 450-550 nm region disappear concomitantly with the BChl bands, at practically the same potentials. To check whether the BChl cation radical is formed upon oxidation, a number of oxidation-reduction cycles were studied by monitoring the absorbance at both BChl bands (Figure 6, curve c). At all potentials no recovery of 800 nm and 850 nm bands was found, whereas the absorbance of carotenoid was partially regenerated. Because of the irreversibility of the oxidation process, different degrees of BChl oxidation can be achieved at a given potential depending on the reaction time, and no thermodynamic values characterizing the oxidation process can be determined. To compare the kinetic susceptibility of BChl to oxidation in different antenna preparations, its bleaching as a function of applied potential was measured under identical conditions. The potential was changed in steps of 20 mV, keeping the sample for 5 min at each step. The selected mediators allow the rate of the process to be controlled by the BChl oxidation rather than by mediator electrode reactions. Figure 7B shows that both LH1
J. Phys. Chem. B, Vol. 105, No. 23, 2001 5541
Figure 7. (A) The optical absorption spectra of the B800-850 complex from Rps. acidophila. The potential was increased stepwise from 0.6 V (initial) to 0.86 V via 20 mV; at each step oxidation was performed for 5 min. Mediator: K2[Fe(phen)(CN)4], ∼126 µM; OD802 ∼12 cm-1. (B) The oxidation of BChl in different antenna complexes. The oxidation was performed by gradual increase of the potential in steps of 20 mV. The absorbance measured at the maximum of the Qy band after 5 min of oxidation at each potential step was used to calculate the bleaching (in % with respect to the initial absorbance). The results are the average from the data obtained with different mediators (K2[Fe(phen)(CN)4], 420 µM; mixture of K2[Fe(phen)(CN)4] (105 µM) and [Fe(phen)2(CN)2] (35 µM)). The OD of all samples at the Qy band was 1-2 cm-1.
preparations are easier to oxidize than the B800 and B850 components in the LH2 complex. For the latter, the loss in absorption starts about 20 mV earlier for the 850 nm maximum compared to that of the 800 nm band. The somewhat lower stability of the B850 component toward oxidation can be also deduced from comparing the relative bleaching of both spectral bands (Figure 7A). Discussion Oxidation of Monomeric BChl in vivo. The two Rb. sphaeroides RCs accessory BChl molecules (BA and BB) are generally considered to be monomeric, although they are in van der Waals contact with the P865 as well as with the two BPheo.25 The reversible one-electron oxidation of monomeric BChl in aprotic solvents is characterized by the following oxidation potentials: Em1 ) 0.64 V (CH2Cl2) ;21 Em1 ) 0.66 V (CH2Cl2)26; Em1 ) 0.66 V (MeCN).26 The presence of small amounts of water ( 0.60 V;6 Em ) 555 mV;8 Em ≈ 570 mV;12 chromatophores from Rb. sphaeroides: Em ≈ 550 mV.12 Remarkably, although the configuration of the B850 pigment in LH2 complex is very similar to that in LH1, no initial reversible oxidation of B850 BChl of Rps. acidophila was observed in the present study. The degradation of B850 takes place at practically the same potentials as for monomeric B800;
Electrochemical Oxidation of Bacteriochlorophyll a both are harder to oxidize compared to the LH1 BChl pigments (Figure 7B). Thus, the order of the susceptibility of BChl toward oxidation is the following: P865 > B880 (LH1) = B875 (LH1) > B850 = B800 (LH2) > B800 (RC), suggesting a direct relationship between the oxidative midpoint potential and the degree of short-range coupling of the BChls (see, for example, ref 28). Reversibility of Oxidation in vivo. The essential reason for BChl.+ instability is its further oxidation to the dication, which is very susceptible to subsequent reactions. Compared to π-cations, metalloporphyrin π-dications more readily undergo an irreversible reaction with nucleophiles leading to metalloisoporphyrin formation.39,40 Though this reaction is not excluded for tetrahydroporphyrins (bacteriochlorins), the more common stable products of their two-electron oxidation are chlorins.41 The dehydrogenation of the 3,4 bond of BChl to form 2-desvinyl-2-acetyl Chl was observed even with mild oxidants (FeCl3, I2), whereas with stronger oxidants (2,3-dichloro-5,6-dicyanop-benzoquinone, chloranil) this compound was obtained selectively with a high yield.23 The formation of 2-desvinyl-2-acetyl Chl upon electrochemical oxidation, detected due to its absorbance in 685-700 nm region (Figures 1, 5, 7), is a result of BChl 2+ formation via the following reactions: -e
-e
-2H+
BChl 98 BChl.+ 98 BChl2+ 98 Chl Further oxidation of chlorophyll into porphyrins -e
-e
-2H+
Chl 98 Chl.+ 98 Chl2+ 98 Por can take place,42 though the oxidation potentials of Chl (Em1 ) 0.86 V; Em2 ) 1.10 V (CH2Cl2)1) are higher compared to those of BChl. The formation of the BChl dication can take place through direct oxidation BChl.+f BChl2+ or through disproportionation BChl.+ + BChl.+ f BChl + BChl2+. The latter reaction demands a close association of BChl molecules and thus is likely to occur in BChl antenna complexes. The formation of BChl2+ seems not very likely when in vitro Em values for the BChl2+/ BChl.+ couple are considered (Em2 ) 1.02 V (MeCN); 1.06 V (CH2Cl2)26). However, a follow-up reaction can lead to a fast consumption of the dication, thus making its formation favorable. Our results show that only 20-35% of the total BChl in LH1 complexes can be bleached reversibly, while further oxidation gives rise to destructive losses, including the formation of the green pigment. These results agree with the data obtained for Rs. rubrum and Rhodopseudomonas capsulatus chromatophores where ∼30% and 15-35% of the total BChl, respectively, was reversibly oxidized.8,9,11 Assuming that the LH1 ring contains 32 BChl molecules and that all of them contribute equally to the Qy band, the ∼25% reversible bleaching corresponds to ∼8 reversibly oxidized BChl molecules in a ring. Thus, up to ∼8 positive charges can be stored within the LH1 ring without them mutually interacting, which otherwise would lead to BChl.+BChl.+ dismutation. It follows that the charge/electron delocalization length extends over four BChl molecules. Interestingly, theoretically43 and experimentally44 a similar exciton delocalization length (4 ( 2 BChl) was estimated for the B850 complex. A spin density delocalized over 10-12 BChl molecules in different LH1 complexes was suggested to explain line narrowing of the EPR signal (∼3.8 G).8,12 For a distribution such line narrowing emphasizes components with longer delocalization length, which might explain the difference between the value we obtain and that quoted in refs 8 and 12. With
J. Phys. Chem. B, Vol. 105, No. 23, 2001 5543 progressive oxidation, the probability of having more unpaired electrons within a single antenna aggregate increases, which then leads to BChl.+ disproportionation making the oxidation process irreversible. The essential difference between the two LH1 complexes studied here is that in LH1 of Rb. sphaeroides the initial oxidation stage is completely reversible, while for LH1 of Rps. acidophila accumulation of the reversible component is concomitant with irreversible bleaching (Figure 6). Whether the lower stability of the BChl cation radical in Rps. acidophila is due to the presence of RCs or to differences in the antenna structure presently is not clear. Another reason for variations in BChl oxidation among the LH1 complexes, as discussed below, may be the different types of Car presented in these complexes (rhodopin-glucoside in Rps. acidophila and spheroidene/spheroidenone in Rb. sphaeroides45). No reversible oxidation of BChl was found to occur in LH2 antenna complexes, both for monomeric (B800) and aggregated (B850) pigment. Among the products obtained upon irreversible oxidation of LH2 complexes the green pigment corresponds to only 10-20% of the original BChl (Figure 7A), as can be estimated by comparing the extinction coefficients of BChl and 2-desvinyl-2-acetyl Chl (770 ) 96 mM-1 cm-1, diethyl ether and 677 ) 65 mM-1 cm-1 in acetone, respectively23). Obviously, other unidentified oxidation products, including bile pigments, are also formed. The degradative oxidation of the 800 nm and 850 nm bands in various chromatophores has been described earlier;5,6 the instability of the BChl cation radical obtained upon chemical oxidation of B800-850 complexes was also demonstrated by the absence of an EPR signal.12 In addition to BChl.+-BChl.+ dismutation, hole transfer via BChl.+ + Car.+ f BChl2+ + Car also could lead to BChl2+ formation in antenna complexes. The observed reversibility of Car oxidation suggests the formation of the Car cation radical as a primary oxidation product. The formation of cation radicals of different carotenoids has been reported in solutions upon oneelectron oxidation (Car - e- f Car.+) or comproportionation (Car2+ + Car f 2 Car.+).46-48 The stability of the Car cation radical was found to be higher in chlorinated solvents (CH2Cl2, C2H4Cl2) compared to THF.46 The formation of Car.+ was also favored by the presence of electron accepting substituent groups in the Car structure.46,47 The oxidation potentials for some Car (β-carotene and its synthetic derivatives) were determined.47,48 In LH2 complexes of Rps. acidophila, the Car molecule (rhodopin-glucoside) is in contact with the two BChl rings; the closest approach to B800 is 3.42 Å and to B850 3.57 Å.32 As discussed below, recent theoretical work suggests that dismutation through hole transfer between the BChl+ cations and between BChl+ and Car+ cations is indeed a plausible mechanism to explain irreversible BChl degradation and its remarkable difference between the LH1 and LH2 antenna complexes. Hole transfer occurs between the HOMO orbitals of two adjacent cofactors, and is governed by the overlap integral of the two HOMOs. Thus, it is similar to triplet energy transfer, which can be viewed as simultaneous hole transfer between the HOMOs and electron transfer between the two LUMOs, and singlet energy transfer through the Dexter mechanism. Although hole transfer in antenna complexes has not been studied much,49 recently considerable attention has been given to the two latter transfer processes. For gaining insight in the characteristics of hole transfer it is therefore of interest to summarize key results of these theoretical studies. Scholes et al.50,51 have evaluated the long-range and short-range Coulomb couplings between the B850R,R′ (the prime refers to the BChlR on the adjacent R,β
5544 J. Phys. Chem. B, Vol. 105, No. 23, 2001 polypeptide) and B850β monomers in LH2 complexes of Rps. acidophila. The latter coupling depends on interchromophore orbital overlap and is found to be comparable to or larger than the long-range dipole-dipole coupling for distances shorter than 4-5 Å.52 This result suggests that for the distances involved also the exchange coupling (which likewise depends on orbital overlap) would be important. Damjanovic et al.,53 however, find for Rs. moleschianum that for singlet energy transfer, the exchange interaction is more than 100-fold less than the Coulomb interactions (including all higher order Coulomb terms). For triplet energy transfer from the BChls to the lycopene carotenoid, the exchange interaction does account for a transfer time of 0.7 µs between 3B850R′ and lycopene, well within the lifetime of 3BChl (10 µs in solution). Transfer times from 3B850β or 3B800 to lycopene, however, are 1011 and 106 times slower, respectively. Taking the results of Damjanovic et al.53 as a lower estimate of the triplet transfer times in LH2, and assuming for holes similar transfer times, it appears that efficient hole transfer between the oxidized carotenoid and the B850 ring, and among the B850 pigments themselves, is entirely possible. Hole transfer among the B800 chromophores (mediated by superexchange interactions via the protein matrix) and, following ref 57, between B800 and the carotenoid is expected to be several orders of magnitude slower than in the B850-carotenoid system. Likely, however, it is still in the seconds range, well within the time scale of our experiments. Other factors may also contribute to efficient hole transfer. For example, there still may be a second carotenoid molecule in LH2 of Rps. acidophila (not well-resolved in the X-ray structure) linked to each Rβ heterodimer.32 Second, the calculated exchange interaction between lycopene and B800 may be underestimated because of the neglect of dynamic interactions, leading to fast fluctuations in the distance. Interestingly, Damjanovic et al.53 found that the triplet transfer time is governed by the distance between the conjugated systems of lycopene and the BChl and fluctuations of 1-1.5 Å in the distance would be sufficient to bring the hole transfer time of, for example, the B850β-Car pair in the range of our experimental observations (seconds to a few minutes). Third, optimization of the reaction center structures of Rps. Viridis and Rb. sphaeroides36 shows that considerable deviations exist between the X-ray structures and the optimized structures. Distance changes of 0.5-1.0 Å are common and likely will also be found when the X-ray structures of LH2 are optimized. Also, it should be noted that for a refined crystal structure the error in the atom positions is on average about one-tenth of the resolution (for LH2, 2.5 Å), so about 0.3 Å, adding a considerable uncertainty to calculations of wave function overlap integrals. The above considerations suggest that in both LH1 and LH2 complexes efficient hole transfer can occur among the BChl and Car. Thus, it is likely that the striking difference in stability of BChl cation radical in LH1 and LH2 complexes is at least in part due to the presence of Car coupling the BChl rings. The reversible step of BChl oxidation in LH1 complexes studied here corresponds to low potentials (less than ∼0.62 V), when almost no Car oxidation occurs (Figures 5 and 6). At the higher potentials (>0.64 V) necessary to oxidize BChl in LH2 complex, Car also undergoes oxidation (Figure 7A), thus leading to simultaneous appearance of both BChl.+ and Car.+ at an early oxidation stage. Of course, possible variations in BChl-BChl and BChl-Car couplings due to the different structures of antenna complexes may also contribute to the differences in reversible BChl oxidation found for LH1 and LH2.
Kropacheva and Hoff Conclusions The data on electrochemical oxidation of BChl in the different photosynthetic pigment-protein complexes studied here revealed a wide variation in oxidation characteristics of this pigment cofactor. Complete reversibility of oxidation was observed for BChl functioning as RCs primary donor (Rb. sphaeroides) and for 20-35% of the total BChl in LH1 complexes (B875 in Rb. sphaeroides, B880 in Rps. acidophila). Oxidation of RCs accessory BChl was found not to be completely reversible: about 70% of the original pigment could be regenerated, much higher than for RCs BPheo chromophores (∼15%). No reversibility of the bleaching was detected for the B800 and B850 chromophores in LH2 (Rps. acidophila). It is suggested that irreversible BChl oxidation in RCs and LH1 and LH2 complexes is due to dismutation of BChl.+-BChl.+ and BChl.+-Car.+ cation pairs created by hole transfer in the CarBChl pigment system resulting in formation of the BChl2+ dication, which undergoes different subsequent reaction including formation of 2-desvinyl-2-acetyl Chl. The remarkable difference in reversibility of BChl oxidation for LH1 and LH2 complexes is suggested to be due to the presence of oxidized Car in LH2 but not in LH1 at early stages of BChl oxidation. The influence of the protein matrix on the oxidation characteristics of BChl was estimated by comparing the behavior of monomeric BChl in vivo with the literature Em values for the BChl.+/BChl couple. One-electron oxidation of the two accessory monomeric BChls in Rb. sphaeroides RCs is characterized by different Em of 0.73 and 0.83 V, which are higher than was found for pentacoordinated BChl in solutions (Em ∼ 0.66 V). The difference of ∼0.1 V is attributed tentatively to a difference in site energy of the two BChls. The RC BPheo is oxidized more easily than expected from oxidation potentials of BPheo in vitro, at potentials anodically shifted by about 60 mV with respect to the corresponding potentials for the accessory BChls. Dimeric and oligomeric forms of BChl in vivo are characterized by a lowering of the oxidation potential. We are indebted to Dr. John Olson for a gift of the LH1only mutant. We thank Saskia Jansen and Dre´ de Wit for preparing the reaction center and antenna complexes, Dr. Jan Raap for helping with mediators synthesis and Willem Versluys for assisting in the construction of the electrochemical cell. T.N.K. was supported by grant 47-006-003 of The Netherlands Foundation for Scientific Research (NWO) for Russian-Dutch scientific collaboration and by grant 93-2894-ext from INTAS. References and Notes (1) Watanabe, T.; Kobayashi, M. Chlorophylls; Scheer, H., Ed.; CRC Press: Boca Raton, 1991; p 287. (2) Lin, X.; Murchison, H. A.; Nagarajan, V.; Parson, W. W.; Allen, J. P.; Williams, J. C. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 10265. (3) Moss, D. A.; Leonhard, M.; Bauscher, M.; Mantele, W. FEBS Lett. 1991, 283, 33. (4) Williams, J. C.; Alden, R. G.; Murchison, H. A.; Peloquin, J. M.; Woodbury, N. W., Allen, J. P. Biochemistry 1992, 31, 11029. (5) Goedheer, J. C. Biochim. Biophys. Acta 1960, 38, 389. (6) Loach, P. A.; Androes, G. M.; Maksim, A. F.; Calvin, M. Photochem. Photobiol. 1963, 2, 443. (7) Beugeling, T. Biochim. Biophys. Acta 1968, 153, 143. (8) Gomez, I.; Sieiro, C.; Ramirez, J. M.; Gomez-Amores, S., del Campo, F. F. FEBS Lett. 1982, 144, 117. (9) Gomez, I.; Picorel, R.; Ramirez, J. M.; Perez, R.; del Campo, F. F. Photochem. Photobiol. 1982, 35, 399. (10) Gomez, I.; del Campo, F. F. AdVances in Photosynthesis Research; Sybesma, C., Ed.; Kluwer: Dordrecht, 1984; p 229. (11) Gomez, I.; Sanchez , A.; del Campo, F. F. Physiol. Veg. 1985, 23, 583. (12) Picorel, R.; Lefebvre, S.; Gingras, G. Eur. J. Biochem. 1984, 142, 305. (13) Kuwana, T.; Heineman, W. R. Acc. Chem. Res. 1976, 9, 241.
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