J . Phys. Chem. 1993,97, 13216-13222
13216
Long-Range Electron Spin-Spin Interactions in the Bacterial Photosynthetic Reaction Center Donald J. H i d and Gary W. Brudvig' Department of Chemistry, Yale University, 225 Prospect Street, New Haven, Connecticut 0651 1 Received: July I , 1993; In Final Form: September 25, 1993' The electron spin-lattice relaxation behavior of the oxidized bacteriochlorophyll a dimer in reaction centers from Rhodobacter sphaeroides has been examined by the method of saturation-recovery EPR over the temperature range 3.8 K I T I 22 K. Its spin-lattice relaxation is nonexponential due to an orientation-dependent dipolar interaction with the non-heme Fe(1I) of the reaction center. The saturation-recovery EPR traces were fit by using an equation which models the recovery in terms of a sum of isotropic (scalar) and orientation-dependent (dipolar) rate constants. The center-to-center distance between the bacteriochlorophyll a dimer and the nonheme Fe(I1) is 28 A and it is found that the Heisenberg exchange interaction is too small to make a measurable contribution to the scalar relaxation rate of the oxidized bacteriochlorophyll a dimer. The scalar relaxation rates for the oxidized bacteriochlorophyll a dimer show a T1temperature dependence which differs significantly from that of model porphyrin radicals. It appears that the unusually rigid protein environment surrounding the bacteriochlorophyll a dimer produces a strong coupling between the spin transitions of the radical and the low-frequency vibrational modes of the lattice. The dipolar rate constants of the oxidized bacteriochlorophyll a dimer and those of the stable tyrosine radical, YD', in Mn-depleted photosystem I1 show the same temperature dependence. This confirms the assignment of the non-heme Fe(I1) as the source of relaxation enhancement for YD'in Mn-depleted photosystem 11 and shows that the spin relaxation properties of the non-heme Fe(I1) species in the two proteins are very similar. Using the relative magnitudes of the dipolar rate constants in the two proteins and the distance between the bacteriochlorophyll a dimer and the non-heme Fe(I1) in the bacterial reaction center, we calculate a YD'-Fe(II) distance of 37 f 5 A in photosystem 11. This agrees well with the distance predicted from the structure of the bacterial reaction center.
Introduction Electron transfer mediates the conversion of sunlight to carbohydrates and the conversion of carbohydrates back to chemical energy for the cell. The successfulapplication of Marcus theory to a variety of chemical systems has focused the attention of chemists on long-range electrontransfer in biological systems.*.2 The rate of electron transfer is influenced, among other things, by exchange interactions between redox active species and their vibrational coupling to the immediate "environment". Since these same factors also influence the spin-lattice relaxation properties of paramagnetic species and since most redox-active species are paramagnetic in at least one of their oxidation states, magnetic resonance can beused to investigatethese interactions. Our group has been studying the long-range pairwise spin-spin interactions between paramagnetic species in pr0teins.3-~ In this work, we report investigations of the magnetic interaction between the oxidized primary electron donor, (BChla)z+, and the high-spin non-heme Fe(I1) in the reaction center protein of the bacterium Rb. sphaeroides strain R26.1. The reaction center protein is the site of the primary events of photosynthesisfor the purple nonsulfur bacteria. In the reaction center, the energy of a photon of light is converted into a oneelectron charge separation across the membrane bilayer. Excitation of the primary electron donor, (BChl)Z, a bacteriochlorophyll dimer, leads to its oxidation by the primary electron acceptor, BPheo, a bacteriopheophytin. Electron transfer proceeds from BPheo- to quinone A, QA, and then quinone B, QB. The high-spin non-heme Fe(I1) lies between the two quinones. The chemical potential produced by the reduction and protonation of the quinone in the Qe site provides energy for the chemical reactions of the cell. Since the bacterial reaction center is well characterized, it is an attractivesystemtostudy. ThestructureoftheRb. sphaeroides reaction center is known to atomic resolution- and the magnetic properties of the non-heme Fe(I1) have been characterized by Abstract published
in Advance ACS
Abstracts. November 15, 1993.
0022-3654/93/2097-13216$04.00/0
magnetic susceptibility: CW EPR,lO and time-resolved EPR11 measurements. One can easily prepare the bacterial reaction centers such that there are only two paramagnetic species per protein molecule, the oxidized primary electrondonor, (BChla)z+, and the non-heme Fe(I1). Since the dimensions of the reaction center ensure a relatively large interenzyme distance between paramagnetic sites,12we can consider the intraenzyme (BChla)Z+Fe(I1) interaction to be pairwise. Our interest in the bacterial reaction center also stems from earlier studies of electron spin-lattice relaxation in the bacterial reaction center and the related protein complex in plants, photosystem 11. In a study by Norris et al.,l3 it was demonstrated that the spin-lattice relaxation rate of (BChla)2+was enhanced by the non-heme Fe(I1). However, while our analysis predicts that the spin-lattice relaxation of (BChla)*+should be distinctly nonexponential,vide post, this was not reported by Norris et al.13 In previous work by our group, it was demonstrated that the spin-lattice relaxation of a stable tyrosine radical in photosystem 11, YD.,was enhanced by another paramagnetic site.' While there are a number of paramagnetic sites within Mn-depleted photosystem 11, the most likely source of relaxation enhancement is the non-heme Fe(II), which is homologous to the non-heme Fe(I1) in the bacterial reaction center. The temperature dependence for the relaxation enhancement of (BChla)*+should be identical to that of YD',if the same fast relaxer, i.e., the nonheme Fe(II), is responsible for the relaxation enhancement. In addition, the magnitude of the relaxation enhancement should be inversely proportional to the distance between the two sites. Examination of the spin-lattice relaxation of (BChla)2+ allows us to test these predictions. In higher plants, photosynthesis takes place within an organelle called the chloroplast. Photosyntheticbacteria lack this organelle. Instead, they localize their photosynthetic machinery within invaginations of the cell membrane. When the cells are lysed, these invaginations become closed vesicles called chromatophores. (BChla)z+ can be generated by freezing chromatophores or purified reaction centers in liquid nitrogen under illumination. 0 1993 American Chemical Society
Bacterial Photosynthetic Reaction Center
The Journal of Physical Chemistry, Vol. 97,No. 50, 1993 13217
In frozen solutions of bacterial reaction centers, it was observed that (BChla)2+decayed overnight at 77 K. If the samples were thawed and frozen under illumination again, only about half of the original concentration of (BChla)2+was produced. Kleinfeld et al.“ have made similar observations. In contrast, when (BChla)2+ was generated by the same treatment in chromatophore-bound reaction centers, (BChla)2+was stable over a period of days. When chromatophore samples were thawed and frozen under illumination again, the same concentration of (BChla)z+ was produced, even after four or five cycles of freezing and thawing. Since (BChl)2+ could be fully regenerated in chromatophores,they were used for the bulkof data collection. Unlike purified reaction centers, there seemed little likelihood that freezing under illumination caused photochemical damage to the reaction centers. In this work, we have measured the spin-lattice relaxation of (BChla)2+ in Fe(I1)-containing reaction centers from Rb. sphaeroidesover the temperature range 3.8 K IT I 22 K. These data have been analyzed by application of the dipolar model developed in earlier studies.3~4Isotropic (scalar) and orientation dependent (dipolar) rate constants are extracted by fitting the dipolar model to the saturation-recovery EPR transients of (BChla)z+. The magnitude and temperature dependence of the scalar rate constants of (BChla)2+ are compared to the 1/ TI’Sof several porphyrin radicals which could potentially serve as models of the relaxation behavior of (BChla)z+ in the absence of the non-heme Fe(I1). The magnitude and temperature dependence of the dipolar rate constants of (BChla)2+are compared to those of YD’in Mn-depleted photosystem I1 in order to establish the source of the relaxation enhancement of YD*and its distance from the non-heme Fe(I1).
The form of the equation decribing the intrinsic spin-lattice relaxation rate depends on the mechanism(s) of relaxation. Organic radicals in solid matrices often show an approximately quadratic temperature dependence. Possible mechanisms for the spin-lattice relaxation of organic radicals are considered in the discussion. The spin-lattice relaxation rate due to scalar exchangedepends on the properties of both the fast and slow relaxing spins and is given by3J5
where Jexis the exchange coupling between the fast (non-heme Fe(I1)) and slow ((BChla)2+) relaxers, pf and g+ are the magnetic moment and g factor, respectively, of the fast relaxing spin, w, and wfarethe resonant frequencies for theslow and fast relaxers, respectively, 0 is the Bohr magneton, and T2r is the transverse (spin-spin) relaxation time of the fast relaxing spin. The orientation-dependent rate constant, kle, arises from the dipole-dipole interaction between the slow and fast relaxing spins. One expects the dipole-dipole interaction to be modulated by the anisotropy of the g values, the hyperfine interaction, and the zero-field splittings of the paramagnetic centers. However, for this analysis, we assume that both spins are isotropic. In this limit, the appropriate equation for kle has been derived by Kulikov and Likhtenstein16and by Goodman and Leigh17
Equations In order to describe the expected spin-lattice relaxation of (BChla)z+ in the bacterial reaction center, we assume that relaxation enhancement arises from the pairwise interaction of the “slow”relaxing spin ((BChla)2+)with the “fast” relaxing spin (the non-heme Fe(I1)). Since the protein is in a frozen glass, for each pair of slow and fast relaxers the interspin (spin-to-spin) vector is in a fixed but random orientation with respect to the static magnetic field. The dipole-dipole induced relaxation enhancement is a function of the angle 8 between the interspin vector and the static magnetic field and all orientations of the interspin vector between 0 and T are possible. The saturationrecovery EPR transient, which reflects the spin-lattice relaxation of all of the slow relaxing spins, can then be described by the following equation’
~ ( r =) 1 - N
sin e ( e + l w + k l ) J t ) de
(1)
where kllahr is the contribution of isotropic relaxation processes and kie is the contribution of the orientation-dependent interactions. For each orientation, the recovery is single-exponential with an overall rate constant, kllalrr + kls. However, since the observed recovery is the sum of many singleexponential recoveries, each with a different overall rate constant, it is nonexponential. We refer to eq 1 as the “dipolar model” for the spin-lattice relaxation of the slow relaxing spin. We assume that their are two possible contributions to the scalar rate constant, i.e.
where kli is the relaxation rate of the “slow” relaxing spin in the absence of the “fast” relaxing spin, i.e., the “intrinsic” relaxation rate, and kloxis the relaxation rate due to the isotropic Heisenberg exchange interaction between the two spins. We refer to klexas the “scalar exchange” rate.
(k) y{++ 2
k,, =
2
=
3C+
where ys is the magnetogyric ratio for the “slow” spin and r is the interspin distance. The terms B, C, and E are defined as follows: m
m
C=
E=
1 If
sin2 0 cos2 e
1
+ w:Tlr
1
e + (os+ w , ) ~ T , ,sin‘ ~
.l2f
(7)
Tlf is the longitudinal (spin-lattice) relaxation time of the fast relaxing spin, and 8 is the angle between the magnetic field and the interspin vector. For a very fast relaxing spin, Le.,
eq 4 becomes 2
2
k,, = 3 i T 2 f (1 - 3 cos2 0)’
+
3TIf sin2 8 cos28 + In this limit, one predicts that kle will be inversely proportional to temperature since Tlf is inversely proportional to temperature and Tvis inversely proportional to, or independent of, temperature. In the opposite limit where
13218 The Journal of Physical Chemistry, Vol. 97, No. 50, 1993 q 4 becomes
3 sin4 e 2(w, + 4 T 2 f In this limit, one predicts that kle will be directly proportional to temperature since l / T l f is proportional to temperature and 1/ T2f is proportional to, or independent of, temperature. On the basis of the results of Norris et al.13 and the results presented in this paper, eq 9 gives the correct limit for the interaction of the non-heme Fe(I1) with (BChla)2+over the temperature range we have studied. We identify the first, second, and third terms in brackets on the right-hand side of eq 9 as the B, C, and E terms, respectively. If eq 9 applies, then whether the B or C term dominates depends on the relaxation times, Tlr and T2f, and the energy, wf, of the appropriate transition(s) of the fast relaxer, the non-heme Fe(11). We favor the CtermanalysislssincethevalueofTlfpredicted from the observed dipolar relaxation enhancement is consistent with the limit imposed by the Massbauer data. However, since precise values of Tlr, Tzf, and wf are not known, we cannot rule out the possibility that the B term dominates the dipolar-induced spin-lattice relaxation enhancement. We have analyzed the saturation-recovery data twice, once assuming kle is dominated by the C term and once by assuming that it is dominated by the B term. (See discussion.) The appropriate equations for when the B term dominates the dipolar-induced relaxation have been developed in earlier work.’ For clarity, we only consider here the limit where klbisdominated by the Cterm. Equation 9 simplifies to
= kfd sin2 e cos2 e
(10)
where
We will refer to kfd as the “dipolar rate constant”. If the fast relaxer is the same for two slow relaxing spins, eq 11 predicts that the temperature dependence of their dipolar rate constantswill also be the same since only thosevariables associated with the fast relaxer, Le., pf and Tlf, are potentially temperature dependent. Therefore, eq 11 predicts that a plot of kfd vs T for YD’in Mn-depleted photosystem I1 should have the same slope as a plot of kfdvs Tfor (BChla)2+in the bacterial reaction center if, in both cases, the non-heme Fe(I1) is the primary source of relaxation enhancement. Furthermore, sincethedistance between the (BChla)z+ radical and the non-heme Fe(I1) in the bacterial reaction center is known, eq 11can beusedto calculate thedistance between YD’and the non-heme Fe(I1) in photosystem I1 without explicit knowledge of p? and Tlf, i.e.
where rsF is the distance between the slow and fast relaxer and kfd(S)is the dipolar rate constant for the slow relaxer. Note that we have assumed that the magnetogyric ratio of the two radicals is the same. Experimental Section Sample Preparation. Preparation of Chromatophores and Reaction Center Samples. Rb. sphaeroides R26.1 cells were grown anaerobically in modified Hutners medium.19 Rb.
Hirsh and Brudvig sphaeroides R26.1 is a mutant strain of Rb. sphaeroides which is unable to synthesize carotenoids. Hence, the reaction center of Rb. sphaeroides R26.1 differs from the wild type in that it lacks a carotenoid pigment. Chromatophores were obtained by French pressure disruption of whole cells at 20 000 psi followed by ultracentrifugation at 250 000 X g for 90 min. Adventitious metal ions, particularly Mn(II), were removed from the chromatophores by the following procedure. To a suspension of chromatophores, ODsm = 27, calcium ionophore (A23187), 5 mM in dimethyl sulfoxide (DMSO), was added to give a final concentration of 0.7 pM calcium ionophore. The chromatophore suspension was diluted by a third of its original volume with a buffer containing 15 mM tris(hydroxymethy1)aminomethane (Tris), 10 mM (ethylenedinitri1o)tetraacetic acid (EDTA), pH 8.0. The suspension was stirred on ice for 15 min before ultracentrifugation at 250 000 X g for 1 h. The chromatophoreswere resuspended in 15 mM Tris, pH 8.0, and pelleted by ultracentrifugation at 250 000 X g for 1 h. This last step was repeated. The pelleted chromatophores were finally suspended in a minimal volume of 33% (v/v) ethylene glycol in 15 mM Tris buffer. A 300-pL volume of this suspension was then placed into an EPR tube. (BChla)2+was generated by illuminating the EPR sample with light from a microscope lamp and submerging the sample in liquid nitrogen. Preparation of Bacteriochlorophyll a Samples. Bacteriochlorophyll a (BChla) came partially purified from Sigma (61.9 wt % BChla). A UV/vis spectrum of this mixture was identical to that observed for pure bacteriochlorophylla. Several absorption maxima common to bacteriochlorophylla and bacteriopheophytin a are shifted to significantly shorter wavelengths in bacteriophe phytin a. However, they were not observed in the UV/vis spectrum of the mixture from Sigma. Since there was no evidence for the presence of other chromophores, the partially purified bacteriochlorophyll a was used as delivered. Partially purified BChla, 1 mg, was stirred overnight in methanol to givea saturated solution of approximately 190 r M BChla. A 350-pL aliquot of this solution was mixed 1:l with glycerol. A 300-p1 aliquot of BChla in methano1:glycerol was placed in an EPR tube and made anaerobic by three freezepumpthaw cycles on a vacuum line. Then the BChla was oxidized to the cation radical by using iodine. Iodine was transferred to the EPR tube containing the frozen solution of BChla by sublimation and the two were mixed upon thawing the BChla solution. Preparation of Ni(Il) octaethylporphyrin EPR Samples. Scheidt et al.20 have shown that when Ni(I1) octaethylporphyrin (OEP) is oxidized with half an equivalent of [(4-BrPh)~N]SbClb, it spontaneously forms a singly oxidized Ni(I1) porphyrin dimer ( [Ni(II)(OEP/2)]2). Ni(I1) octaethylporphyrin and [(4BrPh)3N]SbCl6 were used as received from Aldrich. [Ni(II)(OEP’/2)I2 was produced in methylene chloride by the method of Scheidt et a1.20 The reaction mixture was diluted 1:2 with 2-methyltetrahydrofuran. Aliquots of the resulting solution were transferred to EPR tubes and frozen in liquid nitrogen. The frozen solution formed a transparent glass. EPR Measurements. Saturation-recovery EPR spectroscopy and conventionalcontinuous-wave(CW) EPR spectroscopywere performed on a home-built X-band pulsed EPR spectrometer.21 Saturation-recovery EPR transients were obtained with direct CW detection (without magnetic-field or microwave-frequency modulation) in the absorption mode as described previously.3.21 Sample temperatures were controlled by an Oxford ESR-900 cryostat system. Sample temperatures were checked by using a Si diode temperature sensor held at the sample position. The X-band EPR lineshapes of the (BChla)2+, BChla+, and [Ni(II)(OEP’/2)]2radicalsshowsimpleGaussianlineshapes with no resolvableg-anisotropy or hyperfine couplings. The measured
The Journal of Physical Chemistry, Vol. 97, No. 50, 1993 13219
Bacterial Photosynthetic Reaction Center
I
.
0
1
.
100
I
,
200
4
.
300
)
.
400
6
.
500
1
600
l . . . I . . . I . . . ) . . . I . . . I
ir"
0
I . . . . , ., .a ., . . . I . . . . I . . . . I 0 200 400 600 800 1000 Time
(mr)
Figure 1. Saturation-recoveryEPR transients obtained at 10 K with the best single-exponentialfib superimposed: (a) (BChla)2+: the observing microwave power level was 720 nW, the saturating microwave pulse (1 60 mW) waa of 6 ms duration; (b) [Ni(OEP)*/2]2:the observing microwave power level was 29 nW and the saturating microwave pulse (50 mW) was of 100 ms duration; (c) BChla+: the observing microwave power level was 29 nW and the saturating microwave pulse (50 mW) was of 217 nu
duration
20
40
60
80
100
Time (ms) Figure 2. (a) Saturation-recoveryEPR transient obtained for (BChla)z+ of the chromatophore-boundbacterial reaction center at 4 K with the fit by the C term dipolar model (eq 14) superimposed. The observing microwave power level was 450 nW and the saturating microwave pulse ( 5 0 0 mW) was of 22 ms duration. The rcsiduals (thedifferencebetween the saturation-recoverytransient and the fitted curve) for (b) the single exponential, (c) the dipolar model using the C term.
of cos 8. Equation 13 becomes
I
line widths for (BChla)2+ (AHm= 9.3 G)and BChla+ (AHpp= 13.3 G)agree with the published line widths.22 [Ni(II)(OEP'12)]2 showed a first-derivative peak-to-peak line width of 5.0 G. All saturation-recovery EPR transients were recorded at the zerocrossing point of the CW EPR first-derivative line shape, Le., at the absorption maximum. To see if the iron-semiquinone (Fe(II)-QA- or Fe(II)-QB-) species were generated in the chromatophore-bound reaction centers, low-temperature (7 K) CW EPR spectra were recorded at high microwave power (720 pW) over the g = 2 t o g = 1.6 field range. Fe(I1) is weakly exchange coupled with either Q ~ - o QBr and the iron-semiquinone pair produces a lifetime-broadened EPR signal centered at g = 1.8.1° No Fe(II)-semiquinone signal was detected in the illuminated chromatophore-bound reaction centers. The procedure used to generate (BChla)2+ in the chromatophores (see above) apparently allows the electron to be transferred from Qa or QBto an exogenous electron acceptor, potentially a quinone or 0 2 . We found that the yield of (BChla)Z+ was significantly reduced in anaerobic samples, suggesting that 0 2 may be the terminal electron acceptor. Since no Fe(I1)semiquinonesignal was detected in our samples, the analysis used here assumes that dipolar relaxation enhancement arises simply from interactions of (BChla)z+ with the non-heme Fe(I1). Analysis. Substitution of eq 10 for kid into eq 1 yields
qt)= 1 - Ne+&
s," sin g, (Skfbsin2
-9
8 t) d e
The summation in eq 14 converages slowly, possibly because sin 8 is reaching its maximum in eq 13, at a/2, as sin2 8 cos2 8 approaches zero. When eq 14 was fit to the saturation-recovery EPR data, values of differed by as much as 48% between fits with n = 20 and n = 1000 and values of kfd differed by as much as 13%between fits with n = 20 and n = 1000. By fitting a number of randomly selected saturation-recovery EPR transients, it was found that there was a consistenttrend; as n increased, klwlar and kfd decreased. Since the changes in klrakr and kfd were small upon increasing n from 200 to 1000,13 and II%, respectively, the values of klwlprand kfd at n = 1000 were taken as the "convergent" values of these two rate constants. Practical limits on how much time could be spent fitting the saturationrecovery EPR data prevented the use of large values of n. For n = 60, klwlar and kfd were within approximately 15 and 5% of the convergent values, respectively, and this value of n was used in fitting all the saturation-recovery EPR data presented in this work. Fits to eq 14 were obtained by a nonlinear regression program that employed the Marquardt algorithm.23 The methodology for determining scalar (klahr), dipolar (kld), and singleexponential (1/ T I )rate constants has been described previo~sly.~ In essence, it requires that k l d r and 1/Tl be measured as a function of observe power at each temperature since the observing microwave power will contribute to the observed isotropic relaxation rate. The true values of k l a h r and 1/ TIare found by extrapolation to zero power.
(13)
There is noanalytical solution to the integral in eq 13. However, it can be approximated by a summation over evenly spaced values
Results Spin-Lattice Relaxation of (Bchlp),+, BChlr+, and [Ni(II)(OEPP)]z. Saturation-recovery EPR transients of (BChla)z+
13220 The Journal of Physical Chemistry, Vol. 97, No. 50, 1993
I
Hirsh and Brudvig 8
8
r
8 I
10
Temperature
lo2
80 (K)
Figure 3. 1/T1of [Ni(OEP)*12]2(m), BChla+ (n),and (BChla)z+in
Fe(I1)-depleted reaction centers (a), and & l d l of (BChla)2+ in chromatophore-boundreaction centers (O),vs T. The values of l/T1 for (BChla)2+in Fe(I1)-depleted reaction centen were taken from Norris et a1.13
inchromatophore-boundreactioncenters, [Ni(II)(OEP'/2)]2,and BChla+ at 10 K are shown in Figure 1 with single-exponential fits superimposed. As parts b and c of Figure 1 show, the saturation-recovery traces of the model radicals, [Ni(II)(OEPI2)I2 and BChla+, were fit well by a single-exponentialwhile those of (BChla)z+, Figure la, were not. This was the case throughout the temperature ranged studied. For BChla+, the saturationrecovery EPR trace deviates somewhat from single-exponential behavior at the earliest time points. Possible reasons for this area taken up in the discussion. Figure 2a shows a saturation-recoveryEPR trace for (BChla)2+ in chromatophore-bound reaction centers at 4 K with the dipolarmodel fit incorporating the C term (sin2 B cos2 B angular dependence) superimposed. Parts b and c of Figures 2 show the residuals for the single-exponential fit and the dipolar-model fit incorporating the C term angular dependence, respectively, to the saturation-recovery EPR trace in Figure 2a. The residuals are displayed on an expanded vertical scale. Comparison of Isotropic Relaxation Rates for (BChla)j+, Bchlr+,and [Ni(II)(OEP'/2)b Figure 3 compares the temperature dependenceof the isotropicspin-lattice relaxation constants of [Ni(II)(OEP/2)]2, BChla+, (BChla)2+ in Fe(I1)-depleted reaction centers, and (BChla)z+in chromatophore-boundreaction centers. The data for the spin-lattice relaxation of (BChla)2+in Fe(I1)-depleted reaction centers are taken from the workof Norris et al.13 For BChla+, [Ni(II)(OEP/Z)]2, and (BChla)2+ in Fe(11)-depleted reaction centers, 1/TI is plotted vs temperature. Note that for Fe(I1)-depleted reaction centers, the spin-lattice relaxation of (BChla)2+is expected to be single-exponential.For (BChla)2+ in chromatophore-bound reaction centers, klrrhr is plotted vs temperature. The magnitude and temperature dependenceof l/T1 are thesame for BChla+and [Ni(II)(OEP/Z)]~. Both data sets show the roughly T2 temperature dependence that has been observed for other organic radicals which lack significant g or hyperfine a n i s o t r ~ p y . Comparing ~~ the l/T1 values for (BChla)2+in the Fe(I1)-depleted reaction centers with the klrrlar values for (BChla)z+ in the Fe(I1)-containing chromatophorebound reaction centers, one sees that the magnitude and temperature dependenceof these rate constants are also the same to within the uncertainty. However, they are very different from those of the BChla+ and [Ni(II)(OEP'/2)]2. The isotropic rate constants for (BChla)2+differ from those of BChla+ by an order of magnitude at 3.8 K and show a T temperature dependence. Comparison of Dipolar Relaxation Rates. The temperature dependence of the dipolar rate constants, k;, of (BChla)z+ in chromatophore-bound reaction centers of Rb. sphaeroides and
'
I
* * - . I
10
Temperature
80
(K)
Figure 4. kfd vs ?'for (BChla)2+( 0 )in chromatophore-bound reaction centers of Rb. sphaeroides and YD' (m) in Mn-depleted photosystem 11. The lines show the best power law fit (&fd = A T ) for each set of rate and YO' (&fd = (20 i constants: (BChla)2+ (&fd = (72 15)F,7M.1), 5 )P6fO.1).
*
the stable tyrosine radical YD*in Mn-depleted photosystem I1 are compared in Figure 4. The data for YD' in Mn-depleted photosystem I1 were presented in earlier work? but here they have been reanalyzed by using eq 14, the C term fit. The solid lines show the best power law fits for each set of rate constants.
Discussion Spin-Lattice Relaxation of (BChlah+, BChla+, and [Ni(II)( 0 E P P ) b . Figures 1and 2 show that, whilea single-exponential provides an adequate description of the spin-lattice relaxation of the model radicals, BChla+, and [Ni(II)(OEP/2)]2, it does not describe the spin-lattice relaxation of (BChla)2+ in chromatophore-bound reaction centers well. The nonexponentialityof the saturation-recovery EPR traces of (BChla)2+ are predicted by the dipolar model. The dipoledipole interaction between (BChla)z+ and the non-heme Fe(I1) is modulated by the orientationof theintenpinaxis with respect to thestaticmagnetic field. Since all orientations are present in the EPR sample, the observed saturation-recovery EPR trace is the superposition of recoveries with different spin-lattice relaxation rate constants. Since the distance between the primary electron donor, (BChla)2, and the non-heme Fe(I1) is 28 A, it is worth considering whether intermolecular paramagnetic interactions are contributing to thespin-lattice relaxation of (BChla)2+. Thedimensions of the reaction center protein complex make this unlikely.12 Both the (BChla)~and the non-heme Fe(I1) lie along the central axis of the reaction center and are 20-30 A from the surfaces of the reactioncenter in contact with themembrane bilayer. Therefore, it is unlikely that intermolecular paramagnetic interactions with other reaction centers or proteins in contact with the hydrophobic surfaceof the reaction center contribute to relaxation enhancement of (BChla)2+. The nearest protein/watcr interface is approximately 10 A from the center of the (BChla)2 and 25 A from the non-heme Fe(I1). Thesedistances make significant paramagnetic interactions with other reaction centers or proteins in contact with the hydrophilic surface of the reaction center also unlikely. Adventitious paramagnetic ions could approach to within 10 A of (BChla)2+,but the CW EPR spectra of the chromatophore samples indicate that the ionophore/EDTA treatment was effective in removing paramagnetic ions. No Mn(I1) was visible and the concentrations of high-spin iron(II1) and Cu(I1) were much less than that of the reaction centers (data not shown). We also found in preliminaryexperimentson purified reaction centers that the relaxation rate constants for (BChla)Z+ were the same as those for (BChla)z+ in the chromatophore-bound reaction
-
Bacterial Photosynthetic Reaction Center
The Journal of Physical Chemistry, Vol. 97, No. 50, 1993 13221
centers to within the uncertainty in the measurements (data not shown). These constraints on paramagnetic interactions with exogenous metal ions suggest that the nonexponential saturationrecovery transients of (BChla)2+do, in fact, arise from a pairwise interaction of (BChla)2+ with the non-heme Fe(I1). While the saturation-recovery EPR transients of [Ni(OEP)*Q were almost perfectly described by a single exponential, the saturation-recovery EPR transients of BChla+ show some deviation from single-exponential behavior, Figure l , b and c. However, the deviation is small compared to the saturationrecovery EPR transients of (BChla)2+,Figures l a and 2b. As was noted in the Experimental Section, the BChla came only partially purified and one explanation for the non-singleexponential recoveries is that there are two or more radical species present in the EPR sample. However, this seems unlikely since the UV/vis spectrum shows only BChla and the EPR line width, 13.3 G, agrets with the published line width for BChla+.22 It may be that there are two populations of BChla+, monomeric BChla+ and dimeric BChla+. The chlorophylls in general are known to dimerize in solution even at moderate concentrations. It is also possible that the deviation of the saturation-recovery EPR transients from single-exponentialbehavior is due to spectral diffusion or cross relaxation. If the rates of spectral diffusion or cross relaxation are comparable to the spin-lattice relaxation rate, then it is not possible to saturate these pathways completely by using long saturating pulses2sand the saturation-recoveryEPR transients deviate from a single exponentiaL2l Because the line width of BChla+ is much broader then that of [Ni(OEP)*12]2,a smaller fraction of the line width is saturated by the microwave pulse and more spins are 'available" to contribute to spectral diffusion or cross-relaxation processes. Comparison of Isotropic Relaxation Rates for (BCllla)z+, BCbla+, and [Ni(II)(OEP'12)]2.A quadratic temperature dependence for the temperature regime of these experiments ( T < 77 K) has been observed by Dalton et al.Z4 for the spin-lattice relaxation of a number of organic radicals produced by X-ray and UV irradiation in crystals and inclusion compounds. The authors present twopossiblemechanisms for the F temperature dependence: (1) a distribution of low-lying librational states, and (2) a distribution of exchange interactions or singlet-triplet splittings associated with exchange-coupled pairs of radicals. Dalton et al.Z4 were not able to distinguish between these two mechanisms on the basis of their experimental data. For the organic radicals that we have studied to date, the explanationthat low-lying librational states give rise to a (nearly) quadratic temperature dependence appears to be more likely. In earlier work we compared the spin-lattice relaxation behavior of the stable tyrosine radical, YD',in Mn-depleted photosystem I1 with the tyrosine radical generated by illuminating a frozen solution of L-tyrosine with UV light.' We found that the intrinsic spin-lattice relaxation rate (kli) of YD*has the same temperature dependence as 1/ T I of the UV-generated tyrosine radical. We have also compared the spin-lattice relaxation behavior of the stable tyrosyl radical of ribonucleotidereductase with the UVgenerated tyrosine r a d i ~ a l . At ~ low temperatures, where the dinuclear iron center of ribonucleotidereductase is diamagnetic, 1/ T Iis the same for the tyrosyl radical of ribonucleotide reductase and the UV-generated tyrosineradical. While it is plausible that the quadratic temperature dependence of an unprotectedtyrosine radical arises from exchange-coupled pairs, this is an unlikely mechanism for relaxation in the protein-bound tyrosine radicals of photosystem I1 and ribonucleotide reductase. Since the temperature dependence of the unprotected and protein-bound radicals is very similar, it seems likely that the same relaxation mechanism is responsible for the quadratic temperature dependence in both types of systems. We initially chose to look at BChla+as a model for the intrinsic spin-lattice relaxation rate of (BChla)2+. However, as Figure 3
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shows, the temperature dependence and magnitude of l/TI for BChla+ are significantly different from those of k l d r for (BChla)2+in the temperature range examined here. As stated in eq 2, klscrkris the sum of contributions from the intrinsic relaxation rate, kli, and the relaxation ratedue to scalar exchange, kl,. Therefore, one explanation for the difference is that there is a significant amount of scalar exchange coupling between the non-heme Fe(I1) and (BChla)2+. However, at a center-to-center distance of 28 A, scalar exchange is not expected to make a significant contribution to the spin-lattice relaxation. An alternate explanation is that klscrhrof (BChla)2+represents the intrinsic relaxation rate, kli, but that BChla+ is a poor model for the intrinsic relaxation behavior of (BChla)2+. This explanation is supported by comparison of k l d r of (BChla)z+ in the Fe(11)-containingchromatophorebound reaction centers with 1/ T I of (BChla)2+in the Fe(I1)-depleted reaction centers of Norris et al." Thetwosetsofrateconstantsareidenticalinbothmagnitude and temperaturedependence, within the scatter of thedata. Note that in the Fe(I1)-depleted reaction centers, the spin-lattice relaxation of (BChla)z+represents the intrinsic relaxation rate (kli),since there is no other source of relaxation enhancement in the bacterial reaction center. Therefore, we conclude that in the Fe(I1)-containingchromatophore bound reaction centers k l b = kli. The close agreement between our values for the intrinsic spin-lattice relaxation rate, kli, derived from the dipolar model and Norris et al.3 values for the intrinsic spin-lattice relaxation rate, 1/ T I ,measured directly in Fe(I1)-depleted reaction centers, indicates that the rate constants derived from the dipolar model are physically meaningful. Why is BChla+a poor model for the spin-lattice relaxation of (BChla)2+? One obvious difference between BChla+ and (BChla)2+is that BChla+ is a monomer of bacteriochlorophyll a and (BChla)2+is a dimer. Scheidt et al.20have proposed that the nickel and copper porphyrin dimer cations, [M(OEP)'/2]2, where M = Cu(I1) or Ni(II), can serve as spectroscopic models of the oxidized primary donor ((BChla)2+)in the bacterial reaction center. Both show an absorption band in the near-IR ((BChla)2+ 1300 nm, [M(OEP)*/2]2 1500 nm) which is not observed in the monomeric cations. The narrow, featureless EPR absorption signal of [Ni(II)(OEP)*/2]2is also reminiscent of (BChla)2+. However, Figure 3 clearly shows that the spin-lattice relaxation of the dimer [Ni(II)(OEP)'/2l2 is not significantly different from that of the monomeric BChla+. Since the relaxation properties of [Ni(II)(OEP)'/2]2 are the same as those of other organic radicals, apparently it is the environment of (BChla)2+which is different. Two features of the klscrhr data for (BChla)2+ stand out in Figure 3, the temperature dependence, which is roughly P,and the fast (compared to BChla+) spin-lattice relaxation at the lowest temperatures examined. The approximately P temperature dependence suggests that direct spin-phonon processes provide the most efficient pathway for spin-lattice relaxation of (BChla)z+ at these temperatures. The fast relaxation at low temperatures suggests that coupling to the lattice is strong. Interestingly, the packing and DebyeWaller factors of the X-ray crystal structure of the bacterial reaction indicate that the interior of the bacterial reaction center is very rigid. A I3C magic angle spinning NMR study of the bacterial reaction center2* supports these findings and indicates that the environment around (BChla)2+ is especially rigid. These structural factors could produce an environment in which direct spin-phonon relaxation processes dominate the spin-lattice relaxation of (BChla)Z+ at low temperatures. Estimate of the YD*-Fe(II) Distancein Photosystem II. Figure 4 shows the dipolar rate constants kfd of (BChla)2+ in the chromatophore-bound bacterial reaction center and k k of YD' in Mn-depletedphotosystem 11,plotted against temperature with power law fits superimposed. The fact that the temperature
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Hirsh and Brudvig
13222 The Journal of Physical Chemistry, Vol. 97, No. 50, 1993
dependenceof kfd is very similar for (BChla)2+and YD' suggests that the fast relaxer in photosystem I1 has the same spin-lattice relaxation properties as the fast relaxer in the bacterial reaction center, i.e., the non-heme Fe(I1). No direct measurement of the YD*-Fe(II) distance has been made. However if one assumes that there is a high degree of structural homology between the Land M subunitsof the bacterial reaction center and the D1 and D2 polypeptides of photosystem 11,29 then one predicts a distance of 37 A. Using the kfd of (BChla)z+ in the chromatophore-bound reaction center as a "yardstick" it should be possible to measure the distance between YD' and the non-heme Fe(I1) in photosystem I1 using eq 12. When the exponent is fixed at 1.65, the average of the two freely fit values, the best fit power law equations for kfd(Y/) and kfd((BCh1a):) are kfd(Yi) = (20 f 5)F'.65and &fd( (BChla):) = (72 f 15)F.65.Substituting for the rate constants and the known (BChla)z+-Fe(II) distance (28 A) in eq 12 yields a YD'-Fe(I1) distance of 35 3 A. This agrees well with the distance predicted from the structure of the bacterial reaction center. In earlier work, we had estimated this distance to be 1 3 8 A.3 However, this estimate relied on assumptions about the Fe(11)'s correlation time and resonant frequency which we no longer believe to be correct. Angular Dependence of The Dipolar Relaxation Enhancement. The results presented so far have been analyzed with the assumption that the C term dominates the dipolar-induced spinlattice relaxation (eq 9). *However, as we pointed out in the Introduction, the non-heme Fe(I1) is not sufficiently well characterized to say a priori whether the E or the C term will dominate eq 9. The saturation-recovery EPR transients from (BChla)z+ in chromatophore-bound reaction centers and from YD' in Mn-depleted photosystem I1 membranes were also analyzed using the dipolar model with the B term which has a (1 - 3 cosz 0)z angular dependence.3 The dipolar model incorporating the E term actually gives a slightlybetter fit to the observed recoveries at the earliest time points (data not shown). However, these "fast phase" time points may be influenced by incomplete saturation of the spectral diffusion channels for (BChla)2+. The values for kllalar determined by the E term fit differed by 15% or less from those of the C term fit. The temperature dependence of the dipolar rate constants was fit to a power law equation A p ) which yielded for YD', kfd = (2.0 f 0.5)F.7'".1and (kfd for (BChla)z+, dk: = (13 f 3)p,*f0.1. This is essentially the same temperature dependencefound using the Cterm fit. When the exponent n was fixed at the average value, 1.78, the value of A in the two power law fits was unchanged. Solving for the YD'-Fe(II) distance using an equation analogous to eq 12, one finds that the distance predicted from the E term fit is 38 f 4 A. This is the same distance, to within the uncertainty, as that determined using the C term angular dependence. Taking the average of the C term and E term distance predictions and their total spread as the uncertainty in this measurement, we predict that the YD*-Fe(II) distance is 37 f 5 A. In our analysis, we have assumed a particular angular dependencefor thedipoldipole interactionwhich ignorespossible complicationsfrom anisotropy or multiple correlation time(s) of the non-heme Fe(I1). We are currently investigatingthe angular dependence of the dipolar rate constant using oriented samples in order to explicitly determine its form. However, it is apparent
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from this analysis that even when the precise form of the angular dependence is unknown, the dipolar model can be used to extract intrinsic relaxation rates and, in the presence of a suitable model, distances between paramagnetic centers in proteins. Achwledgment. We thank Professor Harry Frank and the members of his research group, Roya Farhoosh, Mila Aldema, and Jennifer Innes, of the University of Connecticut for providing the cells of Rhodobacter sphaeroides R26.1 and assisting in the preparation of chromatophoresand reaction centers and Professor George Feher for access to unpublished saturation-recovery EPR data. This work was supported by the National Institutes of Health (GM36442). References and Notes (1) Moscr, C. C.; Keske, J. M.; Warncke, K.; Farid, R. S.;Dutton, P. L. Nature 1992. 355. 796802. (2) Beratan, D. N.; Onuchic, J. N.; Winlder, J. R.; Gray, H. B. Science 1992,258, 1740-1741. (3) Hirsh, D. J.; Beck, W. F.; Innw, J. B.; Brudvig, G.W. Biochemistry 1992,31, 532-541. (4) Hirsh, D. J.; Beck, W. F.;Lynch, J. B.; Que, L., Jr.; Brudvig, G. W. J . Am. Chem. Soc. 1992. 114. 7475-7481. ( 5 ) Koulougliotis, D:; Hikh, D. J.; Brudvig, G. W. J. Am. Chem. Soc. 1992.114.~322-a323. ~- , ---- - - - (6) Deisenhofer, J.; Epp, 0.;Miki, K.; Huber, R.; Michel, H. J. Mol. Biol. 1984, 180,385-398. (7) Allen, J. P.;Feher, G.; Yeatw, T. 0.;Komiya, H.;Recs, D. C. Proc. Narl. Acad. Sci. U.S.A. 1988.85. 8487-8491. (8) Chang, C.; Tide, D.; Tang, J.; Smith, U.; Norris, J.; Schiffer, M. FEES &ti. 1986. 205. 82-86. (9) Butler,-W.F.;Johnston,D.C.;Shore,H.B.;Frdkin,D.R.;Okamura, M. Y.; Feher, G. Biophys. J. 1980,32,967-992. (10) Butler, W. F.; Calvo, R.; Fredkin, D. R.; Isaacson, R. A.; Okamura, M. Y.; Feher, G. Biophys. J. 1984,45,947-973. (1 1) Calvo, R.; Butler, W. F.;Isaacson, R. A.; Okamura, M. Y.; Fredkin, D. R.; Feher, G. Biophys. J. 1982, 37, llla. Komiya, H.; Rccs, D. C.; Allen, J. P.; Feher, G. Proc. (12) Yeates, T. 0.; Natl. Acad. Sci. U.S.A. 1981,84,6438-6442. (13) Norris, J. R.;Thurnauer, M. C.; Bowman, M. K. Adv. Biol. Med. Phys. 1980,17, 365-416. (14) Kleinfeld, D.;Okamura, M. Y . ;Feher, G. Biochemistry 1984, 23, 5780-5786. (15) Abragam, A. The Principles of Nuclear Magnetism; Clarendon Press: Oxford, 1961. (16) Kulikw, A. V.; Likhtenstein,G.I. Ado. Mol. Relax. Interact. Processes 1911, IO, 41-79. (17) Goodman, G.; Leigh, J. S.,Jr. Biochemistry 1985,24,2310-2317. (18) Hirsh, D. J. Ph.D. Thesis, Yale University, 1993. (19) Cohen-Bazire, G.; Sistrom, W.R.; S t a n k , R. Y. J. Cell. Comp. Physiol. 1957,49,25-68. (20) Scheidt, W.R.; Cheng, B.; Haller, K. J.; Mislankar, A.; Rae, A. D.; Reddy, K. V.; Song, H.; Orosz, R. D.; R d , C. A,; Cukiernik, F.;Marchon, J.-C. J. Am. Chem. Soc. 1993,115, 1181-1183. (21) Beck, W. F.;Innes, J. B.; Lynch, J. B.; Brudvig, G. W. J. Magn. Reson. 1991, 91, 12-29. (22) McElroy, J. D.; Feher, G.; Mauzerall, D. C. Biochim. Biophys. Acra 1912,267,363-314. (23) Press, W. H.; Flannery, B. P.; Teukolhy, S.A.; Vetterling, W . T. NumericalRecipes in Pascal; CambridgeUniversityPress: Cambridge, 1989. (24) Dalton, L. R.;Kwiram, A. L.; Cowen, J. A. Chem. Phys. Lert. 1972, 17,495499. (25) Hyde, J. S.In Time-Domain Electron Spin Resonance; Kevan, L., Schwartz, R. N., Ed.; John Wiley & Sons: New York, 1979; pp 1-30. (26) Treutlein, H.; Schulten, K.; Deisenhofer, J.; Michel, H.; Brtlnger, A.; Karplus, M. In Structure of the Phorosyntheric Bacrerial Reacrion Center: X-ray Crystallography and OpticalSpectroscopy with PolartzedUght; Plenum Press: CEN Cadarache, Saint Paul lez Durance, France, 1987;pp 139-150. (27) Chang, C.-H.; El-Kabbani, 0.;Tide, D.; Norris, J.; Schiffer, M. Biochemistry 1991.30,5352-5360. (28) Fischer, M. R.;de Groot, H.J. M.; Raap, J.; Winkel, C.; Hoff, A. J.; Lugtenburg, J. Biochemistry 1992, 31, 11038-1 1049. (29) Michel, H.; Deisenhofer, J. Biochemistry 1988,27, 1-7. ~
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