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Multiple Scattering X-Ray Absorption Studies of Zn²⁺ Binding Sites in Bacterial Photosynthetic Reaction Centers

Lisa Giachini ^* , Francesco Francia , Antonia Mallardi , Gerardo Palazzo ^¶, Emilio Carpenè ^||, Federico Boscherini ^*  and Giovanni Venturoli  

^* Dipartimento di Fisica, Università di Bologna, Bologna, Italy; Istituto Nazionale per la Fisica della Materia (INFM), UdR di Bologna, Bologna, Italy; Laboratorio di Biochimica e Biofisica, Dipartimento di Biologia, Università di Bologna, Bologna, Italy; Istituto per i Processi Chimico-Fisici, CNR, Bari, Italy; ^¶ Dipartimento di Chimica, Università di Bari, Bari; and ^|| Dipartimento di Biochimica, Sezione Biochimica Veterinaria, Università di Bologna, Ozzano Emilia, Bologna, Italy

Correspondence: Address reprint requests to Giovanni Venturoli, E-mail: ventur{at}alma.unibo.it.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Binding of transition metal ions to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides has been previously shown to slow light-induced electron and proton transfer to the secondary quinone acceptor molecule, Q_B. On the basis of x-ray diffraction at 2.5 Å resolution a site, formed by AspH124, HisH126, and HisH128, has been identified at the protein surface which binds Cd²⁺ or Zn²⁺. Using Zn K-edge x-ray absorption fine structure spectroscopy we report here on the local structure of Zn²⁺ ions bound to purified RC complexes embedded into polyvinyl alcohol films. X-ray absorption fine structure data were analyzed by combining ab initio simulations and multiparameter fitting; structural contributions up to the fourth coordination shell and multiple scattering paths (involving three atoms) have been included. Results for complexes characterized by a Zn to RC stoichiometry close to one indicate that Zn²⁺ binds two O and two N atoms in the first coordination shell. Higher shell contributions are consistent with a binding cluster formed by two His, one Asp residue, and a water molecule. Analysis of complexes characterized by 2 Zn ions per RC reveals a second structurally distinct binding site, involving one O and three N atoms, not belonging to a His residue. The local structure obtained for the higher affinity site nicely fits the coordination geometry proposed on the basis of x-ray diffraction data, but detects a significant contraction of the first shell. Two possible locations of the second new binding site at the cytoplasmic surface of the RC are proposed.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The bacterial photosynthetic reaction center (RC) is a membrane-bound pigment-protein complex that initiates the conversion of light into chemical energy. It catalyzes a series of light-driven electron and proton transfer reactions resulting in the formation across the bacterial membrane of a proton gradient utilized for ATP synthesis (Okamura et al., 2000). In the RC of Rhodobacter sphaeroides, after absorption of a photon, electron transfer proceeds from the first excited singlet state of the primary donor P (a bacterioclorophyll dimer) through a series of intermediate acceptors (a bacteriopheophytin and a primary quinone acceptor Q_A) to a loosely bound secondary quinone Q_B, yielding the membrane-spanning charge separated state Upon re-reduction of P⁺ by its physiological electron donor (a soluble cytochrome c₂), a new photoexcitation of P leads to the full reduction and protonation of to QH₂, which dissociates from the Q_B site and acts as a mobile electron and proton carrier from the RC to the bc₁ complex (Crofts and Wraight, 1983; Feher et al., 1989; Gunner, 1991; Okamura et al., 2000).

During the last decades the RC from Rb. sphaeroides has been the subject of an impressive number of spectroscopic investigations which provided a detailed thermodynamic and kinetic characterization of electron and proton transfer processes (Woodbury and Allen, 1995; Hoff and Deisenhofer, 1997; Okamura et al., 2000). The availability of site-directed mutants and of x-ray diffraction (XRD) data at atomic resolution (Allen et al., 1986, 1987a,b; Ermler et al., 1994) has made the RC from Rb. sphaeroides an ideal system for exploring the basic structure-function relationships that govern biological electron and proton transfer. Two challenging aspects are intensively investigated by exploiting the RC as a model: the nature of the conformational protein dynamics coupled to electron transfer; and the role of specific amino acids or local protein environments in influencing electron and proton transfer (see e.g., Paddock et al., 1990; Takahashi and Wraight, 1992; McMahon et al., 1998; Balabin and Onuchic, 2000; Poluektov et al., 2003; Baxter et al., 2004).

Recently, elucidation of these issues has taken advantage of the discovery that binding of metal ions (Zn²⁺ and Cd²⁺) to the RC affects the rate of specific electron and proton transfer processes. Utschig et al. (1998) have shown that Zn²⁺ binds to the RC in a site distinct from the Fe site, the only one constitutive transition metal ion site of the RC, located between the Q_A and Q_B acceptors. Upon binding of Zn²⁺ the transfer of the first electron from to Q_B is slowed and the room-temperature kinetics become distributed over the microsecond to millisecond time scale. This observation has suggested that Zn²⁺ acts by limiting protein local motions important for rapid electron transfer (Utschig et al., 1998). Paddock et al. (1999) confirmed and extended these results, demonstrating that Zn²⁺ and Cd²⁺ binding decreases by more than one order of magnitude the rate constant of the proton-assisted second electron transfer, i.e., the reaction In the presence of Zn²⁺ and Cd²⁺ the rate of proton transfer is slowed by a factor >10² and becomes the rate-limiting step (Paddock et al., 1999; Gerencsér and Maroti, 2001).

Axelrod et al. (2000) have determined the binding site of Zn²⁺ in the RC using XRD at 2.5 Å resolution. They found that the metal ion site is located 20 Å from the Q_B site at the cytoplasmic surface of the RC. Bound to the Zn are the imidazole side chains of two histidines (HisH126, HisH128) and the side chain of an aspartic acid (AspH124). The most significant structural difference between the RC with and without a bound metal ion is the positions of the side-chain ligands. Both histidines appear to undergo side-chain conformational changes that enable them to bind to the metal ions via the N atoms of the imidazole ring. One of the oxygens of the side chain of AspH124 is also coordinated to the metal ion. In addition to the three amino acidic residues a water molecule was also proposed to interact with the Zn²⁺ suggesting tetrahedral coordination, which is the predominant geometry found in Zn²⁺ containing proteins. XRD data indicate that Cd²⁺ binding involves the same cluster of residues and possibly three water molecules in an octahedral coordination geometry.

The location of this binding site has opened the way to a series of studies, which contributed significantly to identify the dominant proton pathway connecting the protein surface to Q_B. By combining site-directed mutagenesis and reversible binding of Zn²⁺ and Cd²⁺ it was shown that: a), HisH126 and HisH128 are involved in the pathway of the first proton to at the entry point (Paddock et al., 1999; Ädelroth et al., 2001); b), proton transfer proceeds through AspM17 and AspL210 (Paddock et al., 2000); and c), the second proton supplied to (Q_BH)^– by GluL212 and leading to the formation of quinol share the same entry point of the first proton, close to the metal binding cluster AspH124, HisH126, and HisH128 (Ädelroth et al., 2000). By examining the pH dependence of Cd²⁺ binding in native and in mutant RCs that lack the imidazole group of HisH126 or HisH128 Paddock et al. (2003) concluded that inhibition of proton transfer by the metal ion is predominantly a consequence of competition with protons for binding to HisH126 and HisH128, thus hampering the function of these residues as proton donor/acceptors along the proton pathway to the Q_B site. This study allowed determination of the proton affinities (i.e., pK_A values) of the histidines, a parameter of fundamental importance for the proton transfer processes.

The studies summarized above show that metal ion binding to the RC is a powerful tool in elucidating subtle aspects of electron and proton transfer. The local structure of the bound metal ions is expected to carry valuable information that can be profitably used to elucidate the details of the inhibition mechanisms. For these reasons we undertook x-ray absorption fine structure (XAFS) spectroscopy studies of Zn²⁺ binding site(s) in noncrystallized purified Zn-RC complexes.

XAFS (Koninsgsberger and Prins, 1988) is an ideal tool for selectively probing the local structure of a metal ion in a protein. XAFS obtains local structural information from the analysis of the modulations in the photoelectric absorption cross section, which occur above a core level absorption edge, yielding a quantitative measurement of the local structure in the first few coordination shells. Especially when the scattering atoms have a low atomic number, as in the field of biomolecules, the inclusion of multiple scattering (MS) contributions in the analysis of the XAFS can be required and can in fact provide information which is essential in order to allow the identification of the amino acidic residues coordinated to the metal, as indicated by Strange et al. (1987). The continuous development of advanced data simulation and analysis programs, which are able to treat MS contributions to the XAFS spectra (Filipponi et al., 1995; Ankudinov et al., 1998; Rehr and Albers, 2000), has significantly improved the reliability of the technique. Although the early studies concentrated on methodological aspects (existence and importance of MS), recent progress allows using MS as a structural tool (see e.g., Meneghini and Morante, 1998).

In the field of metalloproteins one important advantage of XAFS is that it is applicable to noncrystallized samples. Moreover, it can be usefully combined with x-ray diffraction to significantly improve the accuracy of the determination of the interatomic distances, which can be determined typically within a few hundredths of an Å for the first shell. Both these aspects are important for our investigation of Zn²⁺ binding site(s). We recall that the Fe site geometry in the RC has been previously studied by Eisenberger et al. (1982), Bunker et al. (1982), and recently by Chen et al. (2004). For recent reviews of the application of XAS to metalloproteins, see Samar Hasnain and Hodgson (1999) and Samar Hasnain and Strange (2003).

In this article, we report an XAFS analysis of Zn-RC complexes characterized by different Zn²⁺ to RC stoichiometries embedded in polyvinyl alcohol (PVA) films. Our data define the local structure of the high affinity site located by XRD and reveal a second structurally distinct binding site, involving three N atoms and one O atom.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Reaction centers were purified from Rb. Sphaeroides R26 according to Gray et al. (1990). The concentration of RCs was measured spectrophotometrically by the absorbance at 802 nm, using an extinction coefficient ₈₀₂ = 288 mM^–1 cm^–1 (Straley et al., 1973). The amounts of Zn and Fe bound to the RC were determined by atomic absorption spectroscopy using an Instrumentation Laboratories (Lexington, MA) IL S11 model spectrophotometer. Analysis for Fe content of the examined preparations yielded a Fe/RC ratio of 0.9 ± 0.1, consistent with one Fe site per RC. As a consequence essentially coincident Zn/RC stoichiometries were obtained when RC was evaluated spectrophotometrically or on the basis of Fe content. In agreement with Utschig et al. (1998) we found that purified RCs, without any incubation in Zn containing buffers, were characterized by a Zn/RC molar ratio of 0.15.

RC-Zn complexes were prepared using essentially the procedures described by Utschig et al. (1998), which consist in incubating the RC suspension with stoichiometric excess of Zn²⁺ followed by size exclusion chromatography to separate the free metal ion from the Zn-RC complexes. By incubating the RC with different equivalents of ZnSO₄ per RC followed by an appropriate gel filtration chromatography protocol (see below) we could systematically obtain RC-Zn complexes containing 1 or 2 Zn²⁺ per RC. To prepare samples characterized by a Zn²⁺/RC ratio close to 2, 1 mL suspension of 90 µM RC was incubated for 15 h at ice temperature with a four-time excess of ZnSO₄ in 10 mM Tris buffer, pH 7.9, containing 0.045% LDAO and 280 mM NaCl. At the end of the incubation time, the sample was sequentially eluted through two Sephadex G25 (Pharmacia, Uppsala, Sweden) columns (1cm diameter x 15 cm and 0.5 cm diameter x 20 cm, respectively) equilibrated with 0.045% LDAO, 50 mM NaCl, 10 mM Tris pH 7.9. EDTA was additionally present at 10 µM only in the first column. Metal analysis of three independent preparations yielded average values of 2.4 ± 0.2 and 1.8 ± 0.2 Zn/RC in the protein fractions collected after the first and the second column respectively, with 0.2 being the standard deviation between the different sample preparations. Good separation between free and RC-bound Zn²⁺ was indicated by column elution profiles, which exhibited two distinctly separated Zn²⁺ peaks, in agreement with Utschig et al. (1998).

The reversibility of Zn²⁺ binding was tested by dialysis of RC-Zn complexes prepared as described above against the metal chelating resin Chelex 100. When samples, characterized by Zn/RC 2 were dialyzed for 5 h against 100-volume of 0.045% LDAO, 10µM EDTA, 10 mM Tris pH 7.9 containing 5 g of Chelex 100 metal-chelating resin (Bio-Rad, Hercules, CA, 100-200 mesh Na form) and for 5 h against 200-volume of 0.045% LDAO, 50 mM NaCl, 10 mM Tris pH 7.9, Zn/RC stoichiometry was not reduced appreciably. However, when the first dialysis step was prolonged to 48 h, the Zn²⁺ content was reduced to levels lower than 0.2 Zn²⁺/RC, similarly to what observed by Utschig et al. (1998) for RC-Zn complexes characterized by 1.0 ± 0.2 Zn/RC.

These measurements strongly suggests that at least one additional Zn binding site is present in the RC besides the high affinity one documented in the literature. The existence of a second, lower affinity, Zn²⁺ binding site, is corroborated by the observation that a RC-Zn²⁺ complex characterized by 1.8 Zn/RC could be obtained following the above described procedure under different incubation conditions, i.e., at overall RC and ZnSO₄ concentrations in the incubation medium decreased by a factor of five. We note that these concentrations (18 µM RC and 72 µM ZnSO₄) are a factor of two lower than those used by Utschig et al. (1998) to prepare stoichiometric RC-Zn complexes. Samples characterized by Zn/RC ratios close to 1 were obtained essentially following the same procedure except that RCs were incubated in the presence of 1.2 equivalents of ZnSO₄.

Utschig et al. (1998) have shown that stoichiometric binding of Zn does not displace the buried Fe constitutively bound to the RC. We routinely determined the Fe content of our preparations by absorption spectroscopy and always found Fe/RC ratios very close to one, both in native RCs (containing low amounts of Zn (<0.2 Zn/RC)) and in Zn-RC complexes characterized by 2 Zn/RC. This confirms the conclusion of Utschig et al. (1998) and also indicates that binding of two Zn metal ions per RC does not interfere with Fe binding.

Two samples were analyzed by XAFS, characterized by 1.9 and 0.9 Zn/RC, hereafter named sample 1 and sample 2, respectively.

Zn-RC containing PVA films were prepared by supplementing the Zn-RC suspension described above with 2.5% w/v PVA (Fluka, Buchs, Switzerland, M_w 130000). The final RC concentration was typically 20–30 µM. To form the film, the suspension was dehydrated under dry N₂ flow for 5 h. Further dehydration was obtained under vacuum. Zinc 3,5-heptanedionate (Lancaster, Morecambe, UK), Zinc 5,10,15,20-tetra(4-pyridyl) –21H, 23H-porphine and zinc phthalocyanine (Aldrich, Milwaukee, WI) were used as reference compounds.

Zn K-edge XAFS measurements were performed at the BM 8 "GILDA" beam-line of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. An Si(311) double crystal monochromator employing dynamical sagittal focusing (Pascarelli et al., 1996) was used; the photon flux was of the order of 5 x 10¹⁰ photons per second and the spot size was 1 x 1 mm². Harmonics were rejected using a pair of grazing incidence Pd-coated mirrors. The energy dependence of the Zn absorption coefficient was monitored by measuring the intensity of the Zn K fluorescence, using a 13-element hyper-pure Ge detector equipped with fast digital electronics with a peaking time equal to 1 µs (Ciatto et al., 2004).

Samples were measured at both room and liquid nitrogen temperature, yielding virtually identical results. The data analyzed are the average of four spectra taken with an integration time of 30 s/point. The presence of time-dependent shifts in the absorption threshold energy, linked to radiation induced reduction, was explicitly checked and was ruled out.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Data analysis and model compounds
Together with the two samples described above, XAFS spectra of three Zn containing molecules of known structure, in which Zn is present in a local coordination similar to that expected in the RC, were measured. As mentioned above, XRD data at 2.5 Å resolution (Axelrod et al., 2000) have identified a Zn²⁺ binding site, indicating that the metal ion is coordinated to two imidazole nitrogens from HisH126 and HisH128 and one oxygen from AspH124. A water molecule has been proposed to interact with Zn²⁺, determining a tetrahedral coordination. Following these indications, we chose as reference compounds Zn-phthalocyanine, Zn-porphine, and Zn-heptanedionate. In Zn-phthalocyanine and Zn-porphine the metal ion coordinates four nitrogens belonging to pyrrolic rings. The structural unit of the Zn site in these compounds describes very well the bond of the metal ion with a histidine residue through the N atom of the imidazole ring. In Zn-heptanedionate the first coordination shell around the absorber is composed by four oxygens and the higher shells contain carbon atoms in a configuration similar to that of the aspartic acid residue. In Fig. 1 we report the local structure of Zn in the described configurations.

View larger version (18K): [in this window] [in a new window]   FIGURE 1  Sketch of the atomic structures considered in the analysis of the model compounds and of the amino acidic residues bound to Zn2+. (Top) on the left the pyrrolic ring, the considered structural unit of Zn-phthalocyanine and Zn-porphine; on the right the imidazole of histidine. (Bottom) on the left the considered group of atoms of Zn-heptanedionate; on the right the side chain of aspartic acid.

View larger version (28K): [in this window] [in a new window]   FIGURE 2  The magnitude of the Fourier transforms of the XAFS data for the two samples and three reference compounds; reference 1 is Zn-porphine, reference 2 is Zn-phthalocyanine, and reference 3 is Zn-heptanedionate. In the inset, the raw, background subtracted data for sample 2 are shown.

For all the reference compounds good fits were obtained. Best fitting structural parameters, which are in good agreement with the known values (Drabløs et al., 1999), are listed in Table 1. In particular the first shell Zn-O and Zn-N interatomic distances are within 0.04 Å of the published values for similar Zn coordination. In Fig. 3, we show, as an example, the fit for Zn-phthalocyanine together with all the significant contributions. The contributions are labeled by where i is a running index and n indicates the number of "legs" in the scattering path (n = 2 for a single scattering signal). The atomic configuration corresponding to each signal is listed in Table 1, using the atom labels indicated in Fig. 1. The fit of Zn-phthalocyanine was performed in the range k = 2.4 – 12 Å^–1 and R = 1.2 – 4 Å with a total of 14 fitting parameters: the many-body amplitude reduction factor a common shift in the energy origin E, and for each of the six paths a distance variation and a Debye Waller factor; the coordination numbers were kept fixed. We found that extending the analysis to the higher coordination shells was essential in order to improve the reliability of the local structural parameters of the first shell. In particular we found that, when analyzed in real space, some of the higher shell signals (both single scattering and MS) have a low distance tail which extends below the first shell peak.

View larger version (17K): [in this window] [in a new window]   FIGURE 3  Fit of the XAFS spectrum of Zn-phthalocyanine. The top curve shows the inverse Fourier transform (continuous line) and its fit (dots); also shown are the six contributions labeled as described in the text.

Zn-RC complexes
We now describe the analysis of the data relative to the two samples, starting with sample 2 (Zn/RC ratio equal to 0.9 ± 0.1, based on atomic absorption determination, see Materials and Methods). We recall that in Fig. 1 we depict the structure of the residues coordinating the metal ion in the RC (atoms are labeled according to the Protein Data Bank conventions) and we use the XRD determination by Axelrod et al. (2000) as a starting point of our analysis.

The contributions included in the fitting are described in the following in order of increasing distance from the Zn atom. We take the first coordination shell as composed by 4 atoms (2 O, 2 N). To reduce the number of fitting parameters, we grouped together the higher shell atoms in sets with similar distances from the absorber, in some cases making atom substitutions. In this way, we have identified three other coordination shells, and the corresponding single scattering signals. The second shell contains four C belonging to histidines (C₁ and C), a C and an O belonging to aspartic acid (C, O₁). This shell is approximated with six C atoms at a distance, which is expected to be close to the Zn-C second shell distance obtained for Zn-phthalocyanine or Zn-porphine. The third shell is composed by two C atoms which are the two C_ß of the histidines, and the corresponding distance is expected to be close to the Zn-C₆ in the pyrrolic ring. The fourth shell is composed by the two C and the two N atoms of the histidines (C₂, N₂) and by a C of the aspartic acid (C_ß), and was approximated by five C atoms. Finally, we have also included the two MS paths we have previously shown to be important in the study of Zn-porphine and Zn-phthalocyanine. In using the same ₃ signals as in the reference compounds we make some approximations by exchanging certain atoms, as can be deduced from a comparison of Table 1 and Table 2. In particular, four of the twelve paths approximated by a Zn-N-C signal are in fact Zn-O-C (two paths) or Zn-O-O (two paths) and of the eight paths approximated by a Zn-N-C signal four are in fact Zn-N-N. As theoretical signals we used the ones generated from the same cluster we built in the study of Zn-phthalocyanine after substituting the C₄ atom of pyrrole ring with the N₂ atom.

View larger version (17K): [in this window] [in a new window]   FIGURE 4  Fit of the XAFS spectrum of sample 2. The top curve shows the inverse Fourier transform (continuous line) and its fit (dots); also shown are the seven contributions labeled as described in the text.

We now come to the analysis of sample 1, characterized by a Zn/RC ratio equal to 1.9 ± 0.2. As detailed under Materials and Methods, a Zn/RC stoichiometry close to two could be obtained reproducibly by incubating the RC with a four-times excess ZnSO₄ at two RC concentrations. Analysis of the complete gel chromatography elution profile in these preparations evidences a good separation between free and RC-bound Zn²⁺, making it extremely unlikely that the stoichiometry found in sample 1 includes a contribution from free Zn ions. The reversibility of Zn²⁺ binding was demonstrated by equilibrium dialysis against a metal chelating resin (see Materials and Methods). The visible-NIR absorption spectrum of RC-Zn complexes characterized by 2 Zn/RC (not shown) is essentially coincident with that of RC not incubated with Zn ions. In particular the spectrum does not reveal any alteration of the Q_y band of the bacteriochlorophyll dimer at 865 nm, indicating that no anomalous aggregation of the RC complexes has occurred after incubation even at relatively high metal concentrations. This absorption band, in fact, has been shown to respond with a sizeable blue shift to RC aggregation (Palazzo et al., 2004).

In Fig. 2, we see that the Fourier transform of the XAFS signal for this sample is very different compared to that of sample 2 in the region corresponding to the higher coordination shells. Control measurements, in which the signal of a Zn-PVA matrix, in the absence of RC, was acquired (not shown), further confirm the absence of free Zn ions in sample 1. Therefore we can reasonably expect that, besides the site of high affinity discussed in sample 2, there is at least another site of minor affinity. Although the occurrence of two or more lower affinity sites (partially occupied in sample 1) cannot be excluded a priori, we made the simplifying assumption of a single lower affinity site and attempted to extract the XAFS signal of this unknown site. Using the measured values of the Zn/RC ratio we determine the XAFS signal of this second Zn²⁺ site as:

where _site1 and _site2 are the XAFS functions relative to the two sites, of major and minor affinity respectively, and _exp is the XAFS function extracted from our data of sample 1.

The fundamental problem in analyzing _site2 was that we had no preliminary indications about the local structure. However, it is reasonable to assume that Zn binds in the first shell to N and O, and, since we already had precise information about these distances, we tried to fit the first shell using Zn-O and Zn-N theoretical signals. In a first step we left as free parameter the number of N and O atoms, with the constrain that the total number of first shell bonds was four; we found the best fit for three N and one O atoms. Having determined the composition of the first coordination shell, we proceeded in studying the second shell.

We reasonably expect the second shell to be composed by C atoms, since this is the case in all the probable residues. We verified that the only path which fits our data in the second shell is a Zn-C one, similar to that found in Zn-heptanedionate, with a distance (3.26 Å) which is considerably longer than that found in Zn-porphine or Zn-phthalocyanine; this strongly suggests that we can exclude the presence of C from a histidine residue. An exact determination of the numbers of carbon atoms in second shell was found to be impossible because of the large errors associated with this parameter when it was left free. The number of C atoms is taken constant at the value of three. In Fig. 5 and in Table 3 we show the results obtained from the fit.

View larger version (19K): [in this window] [in a new window]   FIGURE 5  Fit of signal from the lower affinity site. The top curve shows the inverse Fourier transform (continuous line) and its fit (dots); also shown are the three contributions labeled as described in the text.

Since second shell analysis of the lower affinity binding site revealed by our investigation leads to exclude coordination to histidine residues, ligands providing the three N atoms of the first shell can be restricted to Gln, Asn, Lys, and Arg. Inspection of a database of Zn binding sites in proteins of known structure (http://tanna.bch.ed.ac.uk/cngroups.html), indicates a number of sites which include one or two Gln; Asn and Lys are less frequently present as donors of an N atom and only three binding sites contain one Arg residue. On this basis, we attempted to identify possible locations of the lower affinity binding site by searching for clusters of three N atoms belonging to Gln, Asn, Lys, or Arg residues lying on the hydrophilic surface of the RC protein. We discarded clusters in which the distance between two N atoms exceeds 7 Å as well as those containing two or more Arg residues. By interrogating the XRD structure of the RC (Protein Data Bank entry 1AIJ; Stowell et al., 1997) for clusters satisfying the above criterion, we found two groups (GlnH199-AsnH201-ArgH202 and AsnL199-ArgM267-LysH62) lying on the cytoplasmic hydrophilic portion and one on the periplasmic surface of the L subunit (GlnL264-LysL268-AsnL280). All three putative sites include an Arg and/or a Lys residue. Coordination of a metal ion by these amino acids is uncommon, requiring the deprotonation of the charged amino group of Lys and of guanidinium group of Arg, both characterized by typically high pK_a values. However it has been suggested that the coordinating ability of these groups can be strongly enhanced by the proximity of a His residue, which would act as a base, facilitating their deprotonation (Khangulov et al., 1998; Ferraroni et al., 2002). No His is present in the surroundings of the putative periplasmic site. At variance, a His residue is found in the proximity of ArgH202 and ArgM267-LysH62 of the two cytoplasmic sites (HisH204 and HisM145, respectively). In the latter, cluster coordination could be further facilitated by the presence of the carboxyl group of GluM263 (the O atoms of which being at 3 Å from the side chain N of LysH62). In the XRD structure of the RC determined in the presence of Zn²⁺ (Protein Data Bank entry 1DV6; Axelrod et al., 2000) a Cl^– is also detected at 3–4 Å from the N atoms of the guanidinium group of ArgM267. Interestingly a chlorine anion is present at one of the Arg containing Zn binding sites documented by XRD (Ferraroni et al., 2002).

Finally, we recall that two Zn²⁺ binding sites have been recently located by XRD in the cytochrome bc₁ complex (Berry et al., 2000). Also in this integral protein complex, Zn²⁺ has been shown to inhibit electron and proton transfer (Link and von Jagow, 1995; Klishin et al., 2002) and both Zn²⁺ binding sites identified in the crystallographic structure have been considered as potential inhibitory sites (Berry et al., 2000).

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
X-ray absorption spectroscopy of stoichiometric Zn-RC complexes allows a clear-cut determination of the local structure of the high affinity Zn²⁺ binding site previously identified by x-ray diffraction at 2.5 Å resolution (Axelrod et al., 2000). Accurate analysis of the XAFS regions of the spectrum in terms of single and multiple scattering paths nicely fits, up to the fourth coordination shell, a Zn²⁺ binding cluster which consists of HisH126, HisH128, AspH124, and a water molecule. The coordination geometry obtained in the noncrystalline sample characterized by a Zn/RC ratio close to one reproduces well the one proposed on the basis of XRD data in RC crystals soaked in the presence of the metal and determines the first shell distances with an error equal to 0.03 Å. The first shell distances resulting from our study are considerably contracted in comparison with those determined by XRD.

At variance with what reported by Utschig et al. (1998) we found that incubation of RCs in the presence of a moderate stoichiometric excess of Zn over the protein, followed by gel filtration chromatography, results systematically in Zn-RC complexes characterized by Zn/RC ratios close to two. This strongly suggests that the high affinity site located by XRD is not the only Zn²⁺ binding site of the RC. Both RC-bound Zn ions can be removed by equilibrium dialysis against a metal chelating resin. The XAFS spectrum of one of such samples (Zn/RC = 1.9 ± 0.2) is consistent with the existence of a new, lower affinity site which involves one O and three N, not belonging to a histidine residue. The possible contribution of the lower affinity binding site to inhibition of electron and/or proton transfer should be considered in functional studies performed in the presence of excess metal ion.

By interrogating the crystallographic structures of the RC we have proposed two possible locations of the second binding site at the cytoplasmic surface of the reaction center.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The authors thank P. Turina, B. A. Melandri, S. Ciurli, and C. Tomasini for useful discussions.

The financial support of MIUR of Italy is acknowledged by F.F. and G.V. (grant PRIN/2003 "Bioenergetica: genomica funzionale, meccanismi molecolari ed aspetti fisiopatologici"; grant FIRB/2001 "Meccanismi molecolari della fotosintesi"); and by G.P. (grant PRIN/2003 "Nuovi biosensori basati su neurorecettori immobilizzati"). G.P. was supported by the Consorzio Interuniversitario per lo sviluppo dei Sistemi a Grande Interfase (CSGI-Firenze). F.F. was also supported by A. M. Contigliozzi and A. Contigliozzi. Financial support for measurements at ESRF were provided by INFM.

Submitted on August 3, 2004; accepted for publication December 15, 2004.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
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