9720
J. Phys. Chem. B 2000, 104, 9720-9727
Isotope-Based Discrimination between the Infrared Modes of Plastosemiquinone Anion Radicals and Neutral Tyrosyl Radicals in Photosystem II‡,§ Sunyoung Kim,† Jason S. Patzlaff, Thomas Krick, Idelisa Ayala, Roseann K. Sachs, and Bridgette A. Barry* UniVersity of Minnesota, Department of Biochemistry, Molecular Biology, & Biophysics, St. Paul, Minnesota 55108 ReceiVed: February 2, 2000; In Final Form: August 3, 2000
Photosystem II (PSII) conducts the light-driven oxidation of water and reduction of plastoquinone. Difference Fourier transform infrared (FT-IR) spectroscopy can be used to obtain information about structural changes which occur in protein and cofactors when charge separation occurs. The focus of this work was the assignment of vibrational lines to two different species in PSII: the tyrosyl radical, Z•, and the plastosemiquinone anion radical, QA-. Difference FT-IR experiments were conducted with cyanobacterial PSII samples, in which the tyrosine ring was uniformly 13C-labeled, in which tyrosine was 13C-labeled at carbon 4, and in which plastoquinone was methyl-deuterated. At 80 K, difference FT-IR spectra reflect the oxidation of chlorophyll/ carotenoid and the one-electron reduction of QA; no significant D or Z contribution to the spectrum is observed under these conditions. At 264 K, difference FT-IR spectra reflect the oxidation of redox-active tyrosines Z and D; no significant QA- contribution is observed under these conditions. At 80 K, isotope-induced shifts were observed in spectral features at 1482 and 1469 cm-1 upon deuteration of plastoquinone. At 264 K, isotope-induced shifts were observed in a 1478 cm-1 line upon 13C- labeling of tyrosine, but little change was observed upon plastoquinone deuteration. These data support the assignment of a positive 1478 cm-1 line to a tyrosyl radical vibrational mode and positive 1482 and 1469 cm-1 lines to plastosemiquinone anion vibrational modes. Hybrid Hartree-Fock/density functional calculations of p-cresyl radical’s vibrational frequencies and isotopic frequency shifts support this assignment.
Introduction Photosystem II (PSII), a multisubunit complex that oxidizes water and reduces PQ, is required for oxygenic photosynthesis in plants, algae, and cyanobacteria. Water oxidation involves a tetranuclear manganese cluster and a redox active tyrosine, called Z.1,2 Four charge separations in PSII create the oxidizing equivalents necessary for the oxidation of two water molecules to molecular oxygen. Several redox-active amino acids and cofactors are known to be involved in oxygen evolution and charge separation:3 tyrosine Z, primary chlorophyll donor (P680), pheophytin, quinones, and nonheme iron. After photoexcitation, P680 transfers an electron to the quinone, QA. QA functions as a one-electron acceptor, cycling between the neutral (QA) and oneelectron reduced state (QA-) in which the semiquinone anion radical is formed. A second PQ acceptor, QB, acts as a twoelectron gate, accepting two electrons and two protons.4 P680+ is reduced by tyrosine Z, which in turn forms a radical.5 Z•, the neutral radical, is then reduced by the manganese cluster on a ms-µs time scale.6 Site-directed mutagenesis and isotopic * Author to whom correspondence should be addressed at University of Minnesota, 1479 Gortner Ave., St. Paul, MN 55108. Phone: 612-624-6732. Fax: 612-625-5780. E-mail:
[email protected]. † Present address: Virginia Tech, Department of Biochemistry, 124 Engel Hall, Blacksburg, VA 24061. ‡ This work was supported by NIH GM43273 (B.A.B.), NSF MCB 9973324 (R.K.S.), and NIH GM 19541 (I.A.). § Abbreviations: chl, chlorophyll; D, tyrosine 160 in the PSII D2 polypeptide; DCBQ, 2,6-dichloro-p-benzoquinone; EPR, electron paramagnetic resonance spectroscopy; FT-IR, Fourier transform infrared spectroscopy; MS, mass spectrometry; PSII, photosystem II; PQ, plastoquinone; PQ-9, plastoquinone-9; Z, tyrosine 161 in the PSII D1 polypeptide.
labeling experiments show that Z is Y161 in the D1 polypeptide of PSII7-10 and that Z is required for oxygen evolution.7-9,11 A second redox-active tyrosine in PSII is tyrosine D. Site-directed mutagenesis and isotopic labeling experiments define D as Y160D212-14 and demonstrate that D is not required for oxygen evolution.13-15 The function of D is unknown, but tyrosine D is oxidized by P680+, generating a neutral radical.12,16 Difference FT-IR spectroscopy identifies vibrational bands of amino acid residues and prosthetic groups that are perturbed upon the transition between two protein functional states. Because the vibrational spectrum reflects changes in structure, hydrogen bonding, and protonation state, this form of lightminus-dark spectroscopy offers a method with which dynamic information about PSII can be obtained. Here, our objective was the identification of tyrosyl radical and semiquinone anion radical vibrational modes through specific isotopic labeling of tyrosine and PQ in situ (Figure 1). The assignment of a vibrational mode at 1478 cm-1 has been discussed extensively in the literature. This line has been assigned to the C-O vibrational mode of QA- 17-21 and to the vibrational modes of Z• and D•22-28 in PSII. This is the first report in which the overlapping vibrational contributions of QA- and Z• have been spectrally distinguished. Materials and Methods Isotopic Labeling of Cultures. Using cyanobacterial cultures, isotopic labeling of tyrosine in situ was performed, as described.12,26,27 Cultures contained 0.5 mM phenylalanine, 0.25 mM tryptophan, and either 0.25 mM 13C(6)-tyrosine {(L-4-
10.1021/jp000410+ CCC: $19.00 © 2000 American Chemical Society Published on Web 09/16/2000
QA and Tyr Vibrational Modes in Photosystem II
J. Phys. Chem. B, Vol. 104, No. 41, 2000 9721
Figure 1. Structures of the (A) tyrosyl radical and (B) PQ-9 semiquinone anion radical. Asterisks denote the position of the isotopic label: 13C for the tyrosine radical and 2H for the plastosemiquinone anion radical.
hydroxyphenyl-13C6)-alanine, 99% labeled, Isotec, Miamisburg, OH} or 13C(1)-tyrosine, which is 13C-labeled at ring carbon 4, adjacent to the phenolic oxygen {(L-hydroxyphenylalanine-413C )-alanine, 99% labeled, Cambridge Isotope, Woburn, MA, 1 and 95-99% labeled, Isotec}. Using a methionine-tolerant cyanobacterial culture, isotopic labeling of PQ-9 in situ was performed12,29 through the use of 0.2 M 2H3-methionine {C2H3SC1H2C1H2C1H(N1H2)COO1H, 98% 2H, Isotec}. Mass Spectral Analyses. Incorporation of label into the amino acids from photosynthetic complexes was examined using mass spectrometry.30 Protein samples were purified from cyanobacterial thylakoid preparations11 and acid hydrolyzed by incubation with 10 N HCl for 24 h under anaerobic conditions. tert-Butyldimethylsilyl derivatives of the amino acids were generated.30 Quantitation of amino acid isotopic labeling was measured using electron impact GC-MS at 70 eV (KRATOS MS25, Ramsey, NJ). Incorporation of label into pigments and PQ in photosynthetic complexes was examined using mass spectral techniques. Pigment and PQ purification and identification were performed by reverse phase HPLC.31 Quantitation of pigment and quinone isotopic labeling was performed with electrospray mass spectrometry (LCQ, Finnigan MAT, San Jose, CA). Protein Purification and Manganese Depletion. PSII particles were purified from cyanobacterial cultures.1,11 Chlorophyll quantitation in cyanobacterial preparations was conducted in 100% methanol.32 Oxygen evolution rates for representative PSII samples were the following: wildtype, 2700; methionine, 1100; 2H3-methionine, 1000 µmol O2 mg-1 chl h-1. For experiments at 264 K, manganese was depleted from Synechocystis PSII samples using hydroxylamine.23 The manganese-depleted sample, at 1 mg chl/mL, was buffered in 5 mM HEPES-NaOH, pH 7.5. Difference FT-IR Spectroscopy. Infrared data associated with Z•-minus-Z in manganese-depleted cyanobacterial PSII were recorded on a Nicolet Magna 550 II spectrometer with a MCT-A detector.26 Samples were partially dehydrated33 and contained 3 mM ferricyanide and 3 mM ferrocyanide. Spectral conditions were the following: resolution, 4 cm-1; mirror velocity, 2.5 cm/s; apodization function, Happ-Genzel; levels of zero filling, 1; data acquisition time, 4.0 min; and temperature, 264 K. Infrared data associated with quinone reduction at 80 K were obtained from manganese-containing cyanobacterial PSII using continuous illumination and a Nicolet 60SXR spectrometer (Madison, WI) with a MCT-B detector and a Hansen liquid
Figure 2. Mass spectral analysis performed on tyrosine. The amino acid was derived from photosynthetic complexes isolated from control cyanobacteria (A) and from cyanobacteria cultured in the presence of 13 C(6)-tyrosine (B). In (A) and (B), electron impact ionization data were obtained following anaerobic protein hydrolysis, tert-butyldimethylsilylderivatization, and gas chromatography. In (B), the sample was also partially enriched for 15N (24). Non-15N-enriched samples gave a similar result (data not shown).
nitrogen cryostat (Santa Barbara, CA).34,35 PSII samples contained one molar equivalent of potassium ferricyanide per mole of PSII reaction center. Spectral conditions were the following: resolution, 4 cm-1; mirror velocity, 2.5 cm/s; apodization function, Happ-Genzel; levels of zero filling, 3; data acquisition time, 7.5 min; and temperature, 80 K. For both infrared measurements, the sample absorbance at 1655 cm-1 (amide I band) was less than 0.9, and the continuous illumination source was red- and heat-filtered. Data recorded in the light were ratioed directly to data recorded in the dark. Difference spectra were normalized to an amide II absorbance of 0.35, correcting for any small deviations in sample concentration or path length. Computational Methods. The authors are grateful to Prof. R. Wheeler and S. Boesch at the University of Oklahoma, who calculated vibrational frequencies for the p-cresyl radical, a model for the tyrosyl radical.36,37 Isotopic substitution calculations were performed for the neutral p-cresyl radical in which all six of the ring carbons were replaced with 13C and in which carbon 4 was replaced with 13C. These calculations were performed by methods previously described29 for the neutral plastosemiquinone-1 radical, a model for QA-. Results Isotopic Labeling of Tyrosine and PQ in PSII. In this work, our goal was to distinguish vibrational lines from the tyrosyl radical and the plastosemiquinone anion radical in the 15201420 cm-1 region. Tyrosine was 13C-labeled (Figure 1A) by culturing cyanobacteria in the presence of phenylalanine, 13Ctyrosine, and tryptophan. In the first labeling experiment, tyrosine was 13C(6)-labeled. Based on mass spectral analysis of derivatized amino acids,30 there is little scrambling of label into other amino acid residues (data not shown). Moreover,
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Kim et al.
Figure 3. Mass spectral analysis performed on PQ-9 from control cyanobacterial PSII (A) and from PSII, which was isolated from cyanobacteria cultured in the presence of 13C(1)-tyrosine (B) and 13C(6)-tyrosine (C). In (A)-(C), electrospray ionization data were obtained on samples dissolved in methanol and following extraction and HPLC purification. Under these conditions, the most abundant molecular ion is at 749 (12), corresponding to [M + H]-. Spectra shown are representative examples. The average amount of 13C(1)-PQ labeling was 20 ( 10%; the average amount of 13C(6)-PQ labeling was 64 ( 2%. The larger standard error for the 13C(1) case was due to variation in the protonation state of PQ-9; this variation is evident upon comparison of (A)-(C).
tyrosine, derived by acid hydrolysis, exhibited the expected 6 amu increase in 90% of the sample (Figure 2). This high level of isotope incorporation into tyrosine is the expected result, because under these growth conditions, cyanobacteria are functional auxotrophs for tyrosine and are dependent on the import of labeled tyrosine for growth.12 Mass spectral analysis of PQ-9 (Figure 3A) shows that culturing cyanobacteria on 13C(6)-tyrosine led to a 6 amu increase in 64% of PQ (Figure 3C). Culturing cyanobacteria on 13C(1)-tyrosine led to a 1 amu increase in 20% of a PQ sample (Figure 3B). Therefore, comparison of data acquired with these two isotopomers will allow us to distinguish spectral contributions from tyrosine, which is extensively labeled in each case, from PQ contributions, which are less quantitatively and more variably labeled. As previously reported, deuteration of tyrosine did not lead to significant PQ labeling12 (e7%, data not shown), and 13C-labeling of tyrosine did not lead to significant isotope incorporation into chlorophyll24 (data not shown). These results have important ramifications for understanding the biosynthesis of PQ in cyanobacteria; this aspect is under further investigation. In addition to tyrosine labeling, PQ labeling was performed. The methyl groups of PQ were deuterated (Figure 1B) from 2H -methionine. Mass spectral analysis of methionine shows that 3 62% of the sample, derived by acid hydrolysis, is 2H3-labeled (Table 1). This result is consistent with culturing methods for the methionine-tolerant strain. From the subset of five amino acids examined, we conclude that label scrambling into other amino acid residues was not significant (Table 1). In particular, tyrosine shows no significant labeling (Table 1). However, 87% of the PQ in the cyanobacterial PSII sample was found to be deuterated (Table 2). In addition to 2H3-labeling of PQ, partial chlorophyll labeling occurs.29 Electrospray mass spectrometry of chlorophyll shows that approximately 68% of the sample is 2H -labeled (Table 2). The correlation of 2H enrichment, when 3 3 methionine and chlorophyll are compared (Tables 1 and 2), is consistent with studies showing that methionine is the C1 donor of the 134 methyl group.38 This is the only methyl group of chlorophyll a derived from methionine.38 Our previous work has shown that the phytol tail of chlorophyll is not deuterated, suggesting that carotenoids are not labeled in these samples.29
TABLE 1: Quantitation of Isotope Labeling in Amino Acids, Derived from Acid Hydrolysis of Cyanobacterial Photosynthetic Complexes. Proteins Were Isolated from Methionine-Tolerant Cyanobacterial Cultures Grown Either in the Presence of Methionine or 2H3-Methionine percent labela
residue
mass change
Tyr
