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CHAPTER THREE

Ligation of the C-terminus of the D1 polypeptide of photosystem II to the oxygen evolving complex: A Dft-Qm/Mm study

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José A. Gascón1, Eduardo M. Sproviero, James P. McEvoy2, Gary W. Brudvig, and Victor S. Batista

D1-Ala344, comparable to FTIR data, even when the C-terminus is coordinated to calcium as suggested by X-ray diffraction models. These results suggest that ligation of the C-terminal carboxylate to calcium is consistent with both FTIR and X-ray diffraction experiments.

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Abstract The X-ray diffraction (XRD) models of the oxygen-evolving complex (OEC) of photosystem II (PSII) position the α-COO− group of D1-Ala344 near calcium. In contrast, the analysis of S2-minus-S1 FTIR difference spectra of PSII particles show a red-shift of the symmetric stretch of the α-COO− of D1-Ala344, but no change when Sr2+ is substituted for Ca2+ and have been interpreted to rule out calcium ligation. This paper addresses the apparent contradiction between FTIR and XRD models, regarding ligation of the C-terminal carboxylate of the mature D1 polypeptide in terms of the computational analysis of structural, vibrational and electronic properties of models of the OEC of PSII in the S1 and S2 states. The models are constructed and analyzed by using state-of-theart quantum mechanics/molecular mechanics (QM/ MM) hybrid methods. Consistently with FTIR data, it is found that the S1→S2 oxidation of the OEC induces a red-shift in the symmetric-stretch vibrational frequency of the α-COO− group of

1

University of Connecticut, Chemistry Department, Storrs, CT 06269, USA 2 Regis University, Chemistry Department, Denver, CO 80221, USA

Keywords Oxygen-evolving complex, photosystem II, FTIR, DFT, QM/MM

Introduction

The oxygen-evolving complex (OEC) of photosystem II (PSII) is a manganese- and calciumcontaining cofactor that catalyzes the oxidation of water to dioxygen according to the so-called ‘S-state’ catalytic cycle proposed by Joliot and Kok (Joliot et al. 1969; Kok et al. 1970). Prolonged research over many years of studies has provided considerable structural information regarding the Mn cluster and its local environment as well as fundamental insight into the OEC functionality and the underlying catalytic mechanism of photosynthetic water oxidation (Barber 2003; Diner and Babcock 1996; Renger 2001; Vrettos et al. 2001;

J.F. Allen, E. Gantt, J.H. Golbeck & B. Osmond (eds.), Photosynthesis. Energy from the Sun: 14th International Congress on Photosynthesis, 363–368. © 2008 Springer.

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Sr2+ although the ionic radius increases from 0.99 to 1.12 Å and numerous unassigned vibrational modes changed in the υsym(COO−) and υasym(COO−) regions of the spectrum (1,450–1,280 cm−1 and 1,650–1,510 cm−1, respectively). These observations seem to be in contradiction with XRD models. Recent theoretical studies have introduced structural models of the OEC of PSII in the S1 and S2 states, with complete ligation of the ‘3 + 1 Mn tetramer’ by amino-acid residues, water, hydroxide, chloride and calcium ions (Sproviero et al. 2006, 2007a, b). The computational models were developed from the empirical XRD models by quantum mechanics/molecular mechanics (QM/MM) structural refinement, exploring oxidation states compatible with EPR measurements, in an attempt to circumvent the effect of radiation damage caused by reduction of the Mn centers. The resulting models were found to be compatible with XRD and EXAFS measurements of PSII, offering a unique opportunity to investigate the vibrational spectroscopy of carboxylate residues directly coordinated to the OEC. In particular, this paper is focused on the DFT QM/MM analysis of vibrational frequency shifts ∆υsym(COO−) of the C-terminus of D1-Ala344 induced by oxidation of the OEC, or substitution of Sr2+ for Ca2+. The structural and electronic origins of vibrational frequency shifts, induced by oxidation, are also compared to those in simple hydrated models with common structural features.

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Yachandra et al. 1996). In addition, several groups have published X-ray diffraction (XRD) structures of PSII at 3–3.8 Å resolution (Ferreira et al. 2004; Zouni et al. 2001) in recent years (Biesiadka et al. 2004; Ferreira et al. 2004; Kamiya and Shen 2003; Zouni et al. 2001), resolving almost all amino acid residues and cofactors in the PSII transmembrane protein complex. However, the precise positions of the individual Mn ions and ligation of the OEC could not be resolved due to structural disorder and radiation damage caused by photoreduction of Mn ions (Yano et al. 2005). Therefore, an outstanding challenge is the elucidation of the configuration of the OEC metal cluster, including complete coordination by proteinaceous ligands and substrate water molecules. The proteinaceous ligation suggested by the XRD models of the OEC of PSII have been judged to be contradictory to FTIR data, especially with regards to ligation of several amino acid residues including D1-A344, D1-D170, D1-D342 and D1E189 (Strickler et al. 2005, 2006). This paper addresses such a disagreement between XRD and FTIR models with regards to ligation of the C-terminal carboxylate of the mature D1 polypeptide, a ligand that has long been thought to bind to one of the metal ions in the OEC (Diner 2001; Nixon et al. 1992). Even the most recent X-ray structures at 3–3.5 Å resolution suggests that the α-COO− group of D1-Ala344 is very close (or directly ligated) to Ca2+ in the OEC (Ferreira et al. 2004; Loll et al. 2005). In contrast, results from several FTIR studies have been considered as indicative of ligation of the C-terminus of D1-Ala344 to a manganese ion, likely oxidized during the S1→ S2 transition, but not to Ca2+ (Chu et al. 2004a). In fact, the most recent studies of the S2-minusS1 FTIR difference spectra of the purified PSII particles show that, upon S1→S2 oxidation, the frequency of the symmetric stretching mode of the D1-Ala344 carboxylate changes from ~1,356 cm−1 to either ~1,337 cm−1 (∆υsym(COO−) = −19 cm−1) or ~1,320 cm−1 (∆υsym(COO−) = −36 cm−1) (Strickler et al. 2005), likely due to a Mn3+ to Mn4+ transition (Penner-Hahn 1998). Such a red shift ∆υsym(COO−) remains unchanged upon substitution of Ca2+ by

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Methods DFT-QM/MM Models. The structural models are based on the recently developed DFT-QM/MM structures of the OEC of PSII in the S1 and S2 states (Sproviero et al. 2006). The models are prepared by hydration, completion and DFT QM/ MM refinement of the X-ray crystal structure of cyanobacterium Thermosynechococcus elongatus (Ferreira et al. 2004), explicitly considering 1,987 atoms of PSII. This includes the inorganic Mn3CaO4Mn complex and all amino-acid residues with α-carbon atoms within 15 Å from any atom

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Fig. 1 DFT QM/MM structural model of the OEC of PSII. Note that D1-Ala344 is unidentally ligated to Ca2+. The symmetric stretching mode of the D1-Ala344 carboxylate is indicated with arrows

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in the OEC metal ion center. Unrestricted DFT QM/MM computations are based on the two-layer ONIOM electronic-embedding (EE) link-hydrogen atom approach as implemented in Gaussian 03 (Frisch et al. 2004). The QM layer includes the Mn3CaO4Mn complex, directly ligated by the carboxylate groups of D1-E189, CP43-E354, D1-A344, D1-E333, D1D170, D1-D342, the imidazole ring of D1-H332 as well as bound water molecules, hydroxide and chloride ions (see Fig. 1). The molecular structure beyond the QM layer is described by the Amber MM force field. Fully relaxed QM/MM molecular structures are obtained at the ONIOM-EE (UHF B3LYP/ lacvp,6-31G(2df),6-31G:AMBER) level of theory by geometry optimization of the complete structural models in the presence of a buffer shell of amino acid residues with α-carbons within 15–20 Å from any atom in the OEC ion cluster. These are subject to harmonic constraints in order to preserve the natural shape of the system. DFTQM computations of vibrational frequencies of D1-Ala344 ligated to Ca2+ are performed on the fully optimized DFT QM/MM structures for both the S1 and S2 states. Reduced QM Models. Reduced models of hydrated high-valent manganese ions are investigated in order analyze the effect of Mn3+ → Mn4+ oxidation on the vibrational frequencies of a carboxylate ligand in close contact with the metal center. Model I (Fig. 2) involves unidentate ligation of the alanine carboxylate group to a hydrated Ca2+ ion that is in contact with the hydrated manganese center. For comparison, model II (Fig. 3) involves the alanine carboxylate directly ligated to the manganese center undergoing oxidation. Model I was constructed in the oxidized Mn4+ state, resembling the S2 state of PSII with net charge q = +1 |e| (Fig. 2, right panel). Water molecules and hydroxides (OH)− complete the preferred hexacoordination of Mn4+. The coordination sphere of calcium was completed with three water molecules and monodentate ligation of alanine, resulting in a total of six ligands. The coordination arrangements, shown in Fig. 2, stabilize the proper spin populations S = 2 and S = 3/2 state (i.e., Mn3+

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Fig. 2 Model I, in which Ala is coordinated to Ca2+ in close contact with a hydrated manganese ion undergoing Mn3+ → Mn4+

Fig. 3 Model II, where Ala is coordinated to a hydrated manganese ion undergoing Mn3+ to Mn4+ oxidation

and Mn4+) analogues of the S1 and S2 states, respectively. The S1 state analogue is prepared by geometry optimization at the DFT B3LYP level with the basis set 6-31G(d) for O, H, C, and N, and the lanl2dz basis set for Mn and Ca after one-electron reduction. Model II is constructed analogously by first preparing the oxidized state (Fig. 3 right), where Mn4+

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Results

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The DFT-QM/MM structural models predict that the symmetric stretch vibrational frequency of the α-COO− group of D1-Ala344 changes from 1,381 cm−1 in the S1 state to 1,369 cm−1 in the S2 state (∆υsym(COO−) = −12 cm−1), upon S1→S2 oxidation of the OEC of PSII (Table 1). These results indicate that a vibrational frequency-shift, comparable to the experimental shift (Strickler et al. 2005), can be caused by oxidation of the OEC even when the carboxylate ligand is not directly coordinated to the oxidized Mn ion. The molecular origin of the resulting vibrational frequency shift can be traced to the underlying redistribution of charge induced by the S1→S2 transition. According to the proposed structural models, the S1→S2 oxidation changes the overall charge of the OEC from neutral (i.e., net charge q = 0 |e|) in the S1 resting state to be positively charged (i.e., net charge q = + 1 |e|) in S2. The resulting charge accumulation induces charge transfer interactions between the α-COO− group of D1-Ala344 and the positively charged metal complex. These chargetransfer interactions induce delocalization of negative charge from the Ala carboxylate into the metal cluster, partially neutralizing the increase of positive charge due to oxidation of the Mn complex. As a

result of charge injection, there is a net decrease in bond orders associated with the α-COO− group of D1-Ala344. This is quantified in Table 1 (last column) in terms of ∆BO, a measure defined as the net change in the sum of bond orders associated with the α-COO− bonds C-O1, C-O2, and C-Ca (i.e., the bonds that exhibit the largest displacement along the symmetric stretch, as shown by the arrows in Fig. 1). The quantum mechanical analysis of reduced models of hydrated high-valent manganese ions also indicates that similar vibrational frequencies shifts are induced on carboxylate ligands, upon Mn3+ → Mn4+ oxidation, even when such ligands are not directly ligated to the oxidized metal center. Oxidation of the manganese center, in model I, induces a ∆υsym(COO−) = −40 cm−1 red-shift in the symmetric-stretch vibrational frequency of the Ala carboxylate, comparable to the experimental frequency shift (∆υsym(COO−) = −19 cm−1, or −36 cm−1). As indicated by the charge population analysis, the resulting red-shift induced by Mn3+ → Mn4+ oxidation in model I is also due to chargetransfer between the negatively charged carboxylate group of Ala and the positively charged metal complex in its oxidized state. Consequently, a reduction of bond orders of the Ala bonds exhibiting the larger displacement along the symmetric stretch vibrational mode is observed. Furthermore, it is found that substitution of Sr2+ for Ca2+ in model I produces only minor changes in the vibrational frequency of the symmetric stretch mode of Ala, shifting it from 1,390 to 1,391 cm−1 in the Mn3+ state and from 1,350 to 1,351 cm−1 in the Mn4+ state. These results are consistent with the observation that the substitution of Ca2+ by Sr2+ in PSII produces negligible changes in the vibrational frequencies of S1-Ala344 as manifested by the S2-minus-S1 FTIR spectra. The vibrational analysis of model II allowed for the comparative study of the effect of oxidation of a hydrated manganese ion on the vibrational frequencies of a COO− group of Ala directly ligated to the oxidized metal center (see Fig. 3). Table 1 shows that Mn3+ → Mn4+ oxidation in model II also induces a red-shift ∆υsym(COO−) = −51 cm−1 in the vibrational frequency of the symmetricstretch of the Ala carboxylate. While the computed

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is ligated by two water molecules, two hydroxides and the carboxylate group of alanine, and then the reduced state (Fig. 3, left) by geometry optimization after reduction.

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Table 1 Vibrational frequency (in cm−1) of the symmetric stretching mode of the Ala carboxylate in PSII, as described by FTIR (first row), and as computed for the computational structural models. ∆BO is a measure of the change in the bond order for all bonds relevant to the symmetric stretching mode Oxidation state Model

Mn3+

Mn4+

Freq. shift

∆BO

Exp. QM/MM I II

1,356 1,381 1,390 1,346

1,320, 1,337 1,369 1,350 1,297

−19,−36 −12 −40 −51

−0.017 −0.024 −0.050

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Conclusions

Barber J (2003) Photosystem II: The engine of life. Quart Rev Biophys 36:71–89. Biesiadka J, Loll B, Kern J, Irrgang KD, Zouni A (2004) Crystal structure of cyanobacterial photosystem II at 3.2 angstrom resolution: A closer look at the Mn cluster. Phys Chem Chem Phys 6:4733–4736. Chu H, Hillier W, Debus RJ (2004a) Evidence that the Cterminus of the D1 polypeptide of photosystem II is ligated to the manganese ion that undergoes oxidation during the S1 to S2 transition: An isotope-edited FTIR study. Biochemistry 43:3152–3166. Chu HA, Hillier W, Debus RJ (2004b) Evidence that the C-terminus of the D1 polypeptide of photosystem II is ligated to the manganese ion that undergoes oxidation during the S_1 to S_2 transition: An isotope-edited FTIR study. Biochemistry 43:3152–3166. Diner BA (2001) Amino acid residues involved in the coordination and assembly of the manganese cluster of photosystem II. Proton-coupled electron transport of the redox-active tyrosines and its relationship to water oxidation. Biochim Biophys Acta 1503:147–163 Diner BA, Babcock GT (1996) Structure, dynamics, and energy conversion efficiency in photosystem II. In: Ort DR, Yocum CF (eds) Oxygenic Photosynthesis: The Light Reactions. Kluwer, Dordrecht, The Netherlands, pp 213–247. Ferreira KN, Iverson TM, Maghlaoui K, Barber J, Iwata S (2004) Architecture of the photosynthetic oxygenevolving center. Science 303:1831–1838. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Montgomery JJA, Vreven T, Kudin KN, Burant JC, Millam JM, Iyengar SS, Tomasi J, Barone V, Mennucci B, Cossi M, Scalmani G, Rega N, Petersson GA, Nakatsuji H, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao O, Nakai H, Klene M, Li X, Knox JE, Hratchian HP, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski JW, Ayala PY, Morokuma K, Voth GA, Salvador P, Dannenberg JJ, Zakrzewski VG, Dapprich S, Daniels AD, Strain MC, Farkas O, Malick DK, Rabuck AD, Raghavachari K, Foresman JB, Ortiz JV, Cui Q, Baboul AG, Clifford S, Cioslowski J, Stefanov BB, Liu G, Liashenko A, Piskorz P, Komaromi I, Martin RL, Fox DJ, Keith T, Al-Laham MA, Peng CY, Nanayakkara A, Challacombe M, Gill PMW, Johnson B, Chen W, Wong MW, Gonzalez C, Pople JA (2004) Gaussian 03, Revision B.04. Gaussian, Wallingford, CT. Joliot P, Barbieri G, Chabaud R (1969) Un nouveau modele des centres photochimiques du systeme II. Photochem Photobiol 10:309–329. Kamiya N, Shen JR (2003) Crystal structure of oxygenevolving photosystem II from Thermosynechococcus vulcanus at 3.7-angstrom resolution. Proc Natl Acad Sci USA 100:98–103.

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We have shown that quantum mechanical calculations of vibrational frequency shifts due to S1→S2 oxidation of the OEC of PSII are consistent with FTIR measurements, even when the C-terminus of the D1 polypeptide of PSII is directly ligated to calcium as suggested by X-ray diffraction models. While the reported calculations can not rule out other possible ligation schemes, such as unidentate ligation of the carboxylate group of alanine to manganese, or the bridging of this group between manganese and calcium, we have shown that a red shift ∆υsym(COO−) = −36 cm−1 in the symmetric stretch vibrational frequency can still be consistent with unidentate ligation of the carboxylate group of alanine to Ca2+, when such a metal center is linked is linked to the redox-active Mn ion via µ-oxo bridges. Furthermore, we have shown that in silico substitution of Sr2+ for Ca2+ does not significantly affect the vibrational frequency of the symmetric stretch mode of a carboxylate ligand directly bound to calcium. These results, which are in agreement with FTIR experiments, suggest that D1-Ala344 bound to calcium in the OEC of PSII might be consistent with both X-ray diffraction models and FTIR data.

References

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frequency shift is larger than for model I, it is still of the same sign and order of magnitude, making it difficult to rule out one ligation scheme versus the other. Considering these results, in conjunction with the vibrational frequency shifts due to S1→S2 oxidation in the DFT QM/MM structural models, we conclude that the observed FTIR red-shift in the symmetric-stretch vibrational frequency of the α-COO− group of D1-Ala344 can not conclusively rule out the ligation scheme suggested by X-ray diffraction models, in which D1-Ala344 is very close or directly ligated to calcium.

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Acknowledgements. VSB acknowledges supercomputer time from NERSC and financial support from the Camille Dreyfus Teacher Scholar Award, the Sloan Fellowship and grants NSF CHE 0345987, NSF ECCS-0725118, NIH 2R01-GM043278-14 and DOE DE-FG02-07ER15909. GWB acknowledges support from the National Institutes of Health grant GM32715.

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Author Queries:

evolving complex of photosystem II. Curr Opin Struct Biol 17:173–180. Strickler MA, Walker LM, Hillier W, Debus RJ (2005) Evidence from biosynthetically incorporated strontium and FTIR difference spectroscopy that the C-terminus of the D1 polypeptide of photosystem II does not ligate calcium. Biochemistry 44:8571– 8577. Strickler MA, Hillier W, Debus RJ (2006) No evidence from FTIR difference spectroscopy that glutamate-189 of the D1 polypeptide ligates a Mn ion that undergoes oxidation during the S-0 to S-1, S-1 to S-2, or S-2 to S-3 transitions in photosystem II. Biochemistry 45:8801–8811. Vrettos JS, Limburg J, Brudvig GW (2001) Mechanism of photosynthetic water oxidation: Combining biophysical studies of photosystem II with inorganic model chemistry. Biochim Biophys Acta 1503:229– 245. Yachandra VK, Sauer K, Klein MP (1996) Manganese cluster in photosynthesis: Where plants oxidize water to dioxygen. Chem Rev 96:2927–2950. Yano J, Kern J, Irrgang KD, Latimer MJ, Bergmann U, Glatzel P, Pushkar Y, Biesiadka J, Loll B, Sauer K, Messinger J, Zouni A, Yachandra VK (2005) X-ray damage to the Mn4Ca complex in single crystals of photosystem II: A case study for metalloprotein crystallography. Proc Natl Acad Sci USA 102: 12047–12052. Zouni A, Witt HT, Kern J, Fromme P, Krauss N, Saenger W, Orth P (2001) Crystal structure of photosystem II from Synechococcus elongatus at 3.8 angstrom resolution. Nature 409:739–743.

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Kimura Y, Mizusawa N, Yamanari T, Ishii A, Ono T (2005) Structural changes of D1 C-terminal alpha-carboxylate during S-state cycling in photosynthetic oxygen evolution. J Biol Chem 280:2078–2083. Kok B, Forbush B, McGloin M (1970) Cooperation of charges in photosynthetic O2 evolution. 1. A linear four step mechanism. Photochem Photobiol 11:457–475. Loll B, Kern J, Saenger W, Zouni A, Biesiadka J (2005) Towards complete cofactor arrangement in the 3.0 angstrom resolution structure of photosystem II. Nature 438:1040–1044. Nixon PJ, Trost JT, Diner BA (1992) Role of the carboxy terminus of polypeptide D1 in the assembly of a functional water-oxidizing manganese cluster in photosystem II of the cyanobacterium Synechocystis sp. PCC 6803 – assembly requires a free carboxyl group at Cterminal position 344. Biochemistry 31:10859–10871. Penner-Hahn JE (1998) Structural characterization of the Mn site in the photosynthetic oxygen-evolving complex. Struct Bond 90:1–36. Renger G (2001) Photosynthetic water oxidation to molecular oxygen: Apparatus and mechanism. Biochim Biophys Acta 1503:210–228. Sproviero EM, Gascon JA, McEvoy JP, Brudvig GW, Batista VS (2006) QM/MM models of the O2-evolving complex of photosystem II. J Chem Theor Comput 2:1119–1134. Sproviero EM, Gascon JA, McEvoy JP, Brudvig GW, Batista VS (2007a) QM/MM computational studies of substrate water binding to the oxygen evolving complex of Photosystem II. Phil Trans Roy Soc Lond Series B, Biol Sci (in press). Sproviero EM, Gascon JA, McEvoy JP, Brudvig GW, Batista VS (2007b) Structural models of the oxygen-

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[Au1]: Chu et al. 2004b not cited. [Au2]: Ref is not cited. [Au3]: Please update Sproviero et al. 2007a.

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