2D-hyperfine sublevel correlation spectroscopy of tyrosyl radicals.

Hyperfine sublevel correlation (HYSCORE) spectroscopy has been used to study the tyrosyl radicals in Photosystem II and bovine liver catalase. The HYSCORE data allow a complete resolution of all the 1H hyperfine tensors of these radicals. The present work shows that the proper analysis of the HYSCORE data allows the complete assignment of the 1H-hyperfine tensors in tyrosine radicals and this offers an alternative experimental tool relative to ENDOR.

Multifrequency high-field EPR study of the tryptophanyl and tyrosyl radical intermediates in wild-type and the W191G mutant of cytochrome c peroxidase.

Multifrequency (95, 190, and 285 GHz) high-field electron paramagnetic resonance (EPR) spectroscopy has been used to characterize radical intermediates in wild-type and Trp191Gly mutant cytochrome c peroxidase (CcP). The high-field EPR spectra of the exchange-coupled oxoferryl--trytophanyl radical pair that constitutes the CcP compound I intermediate [(Fe(IV)=O) Trp*(+)] were analyzed using a spin Hamiltonian that incorporated a general anisotropic spin-spin interaction term. Perturbation expressions of this Hamiltonian were derived, and their limitations under high-field conditions are discussed. Using numerical solutions of the completely anisotropic Hamiltonian, its was possible to simulate accurately the experimental data from 9 to 285 GHz using a single set of spin parameters. The results are also consistent with previous 9 GHz single-crystal studies. The inherent superior resolution of high-field EPR spectroscopy permitted the unequivocal detection of a transient tyrosyl radical that was formed 60 s after the addition of 1 equiv of hydrogen peroxide to the wild-type CcP at 0 degrees C and disappeared after 1 h. High-field EPR was also used to characterize the radical intermediate that was generated by hydrogen peroxide addition to the W191G CcP mutant. The g- values of this radical (g(x)= 2.00660, g(y) = 2.00425, and g(z)= 2.00208), as well as the wild-type transient tyrosyl radical, are essentially identical to those obtained from the high-field EPR spectra of the tyrosyl radical generated by gamma-irradiation of crystals of tyrosine hydrochloride (g(x)= 2.00658, g(y) = 2.00404, and g(z) = 2.00208). The low g(x)-value indicated that all three of the tyrosyl radicals were in electropositive environments. The broadening of the g(x) portion of the HF-EPR spectrum further indicated that the electrostatic environment was distributed. On the basis of these observations, possible sites for the tyrosyl radical(s) are discussed.

Comparative electron paramagnetic resonance study of radical intermediates in turnip peroxidase isozymes.

The occurrence of isozymes in plant peroxidases is poorly understood. Turnip roots contain seven season-dependent isoperoxidases with distinct physicochemical properties. In the work presented here, multifrequency electron paramagnetic resonance spectroscopy has been used to characterize the Compound I intermediate obtained by the reaction of turnip isoperoxidases 1, 3, and 7 with hydrogen peroxide. The broad (2500 G) Compound I EPR spectrum of all three peroxidases was consistent with the formation of an exchange-coupled oxoferryl-porphyrinyl radical species. A dramatic pH dependence of the exchange interaction of the [Fe(IV)=O por(*+)] intermediate was observed for all three isoperoxidases and for a pH range of 4.5-7.7. This result provides substantial experimental evidence for previous proposals concerning the protein effect on the ferro- or antiferromagnetic character of the exchange coupling of Compound I based on model complexes. Turnip isoperoxidase 7 exhibited an unexpected pH effect related to the nature of the Compound I radical. At basic pH, a narrow radical species ( approximately 50 G) was formed together with the porphyrinyl radical. The g anisotropy of the narrow radical Delta(g) = 0.0046, obtained from the high-field (190 and 285 GHz) EPR spectrum, was that expected for tyrosyl radicals. The broad g(x) edge of the Tyr* spectrum centered at a low g(x) value (2.00660) strongly argues for a hydrogen-bonded tyrosyl radical in a heterogeneous microenvironment. The relationship between tyrosyl radical formation and the higher redox potential of turnip isozyme 7, as compared to that of isozyme 1, is discussed.

Solid-phase synthesis of peptides containing the spin-labeled 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC).

2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino4-carboxylic acid (TOAC) is a nitroxide spin-labeled, achiral Calpha-tetrasubstituted amino acid recently shown to be not only an effective beta-turn and 3(10)/alpha-helix promoter in peptides, but also an excellent rigid electron paramagnetic resonance probe and fluorescence quencher. Here, we demonstrate that TOAC can be effectively incorporated into internal positions of peptide sequences using Fmoc chemistry and solid-phase synthesis in an automated apparatus.

Conformation of bacteriochlorophyll molecules in photosynthetic proteins from purple bacteria.

Fourier transform near-infrared resonance Raman spectroscopy can be used to obtain information on the bacteriochlorophyll a (BChl a) molecules responsible for the redmost absorption band in photosynthetic complexes from purple bacteria. This technique is able to distinguish distortions of the bacteriochlorin macrocycle as small as 0.02 A, and a systematic analysis of those vibrational modes sensitive to BChl a macrocycle conformational changes was recently published [Näveke et al. (1997) J. Raman Spectrosc. 28, 599-604]. The conformation of the two BChl a molecules constituting the primary electron donor in bacterial reaction centers, and of the 850 and 880 nm-absorbing BChl a molecules in the light-harvesting LH2 and LH1 proteins, has been investigated using this technique. From this study it can be concluded that both BChl a molecules of the primary electron donor in the photochemical reaction center are in a conformation close to the relaxed conformation observed for pentacoordinate BChl a in diethyl ether. In contrast, the BChl a molecules responsible for the long-wavelength absorption transition in both LH1 and LH2 antenna complexes are considerably distorted, and furthermore there are noticeable differences between the conformations of the BChl molecules bound to the alpha- and beta-apoproteins. The molecular conformations of the pigments are very similar in all the antenna complexes investigated.

Effects of hydrogen bonds on the redox potential and electronic structure of the bacterial primary electron donor.

The primary donor, P, of photosynthetic bacterial reaction centers (RCs) is a dimer of excitonically interacting bacteriochlorophyll (BChl) molecules. The two constituents are named PL and PM to designate their close association with the L- and M-subunits, respectively, of the RC protein. A series of site-directed mutants of RCs from Rhodobacter sphaeroides has been constructed in order to model the effects of hydrogen bonding on the redox midpoint potential and electronic structure of P. The leucine residue at position M160 was genetically replaced with eight other amino acid residues capable of donating a hydrogen bond to the C9 keto carbonyl group of the PM BChl a molecule of P. Fourier transform (FT) (pre)resonance Raman spectroscopy with 1064 nm excitation was used to (i) determine the formation and strengths of hydrogen bonds on this latter keto carbonyl group in the reduced, neutral state (PO), and (ii) determine the degree of localization of the positive charge on one of the two constituent BChl molecules of P in its oxidized, radical cation state (P*+). A correlation was observed between the strength of the hydrogen bond and the increase in PO/P*+ redox midpoint potential. This correlation is less pronounced than that observed for another series of RC mutants where hydrogen bonds to the four pi-conjugated carbonyl groups of P were broken or formed uniquely involving histidinyl residues [Mattioli, T. A., Lin, X., Allen, J. P. and Williams, J. C. (1995) Biochemistry 34, 6142-6152], indicating that histidinyl residues are more effective in raising the PO/P*+ redox midpoint potential via hydrogen bond formation than are other hydrogen bond-forming residues. In addition, an increase in positive charge localization is correlated with the strength of the hydrogen bond and with the PO/P*+ redox midpoint potential. This latter correlation was analyzed using an asymmetric bacteriochlorophyll dimer model based on Hückel-type molecular orbitals in order to obtain estimates of certain energetic parameters of the primary donor. Based on this model, the correlation is extrapolated to the case of complete localization of the positive charge on PL and gives a predicted value for the P/P+ redox midpoint potential similar to that experimentally determined for the Rb. sphaeroides HL(M202) heterodimer. The model yields parameters for the highest occupied molecular orbital energies of the two BChl a constituents of P which are typical for the oxidation potential of isolated BChl a in vitro, suggesting that the protein, as compared to many solvents, does not impart atypical redox properties to the BChl a constituents of P.

EPR investigation of compound I in Proteus mirabilis and bovine liver catalases: formation of porphyrin and tyrosyl radical intermediates.

Compound I of Proteus mirabilis and bovine liver catalases (PMC and BLC, respectively) were studied combining EPR spectroscopy and the rapid-mix freeze-quench techniques. Both enzymes, when treated with peroxyacetic acid, form a catalytic intermediate which consists of an oxoferryl porphyrin pi-cation radical. In PMC this intermediate is semistable, and an unexpected reversible equilibrium under pH influence takes place between two forms of compound I with different coupling between the oxoferryl and the porphyrin pi-cation radical. At acid pH, one form has a ferromagnetic character as in Micrococcus luteus compound I. At neutral pH, another form with a much smaller coupling, reminiscent of the horse radish peroxidase compound I, is detected. The approximate midpoint, estimated for these changes in the range 5.3 < pH < 6.0, approaches the pKa value of an histidyl residue. The residues possibly involved in the transformation are discussed in terms of the known structure of PMC compound I. The EPR spectrum of BLC compound I (pH 5.6), obtained in the millisecond time scale (40 ms), also showed a mixture of two forms which, most probably, correspond to two different magnetic exchange interactions, as in the case of PMC. Taken together, the low-temperature electronic absorption and the EPR spectra of BLC compound I formed in the 0.04-15 s range show that the porphyrin pi-cation radical disappears and, instead, a tyrosyl radical is formed. ENDOR experiments confirm our previously estimated hyperfine couplings to the C2,6 and C3,5 ring protons and the beta-methylene protons of the purported tyrosyl radical. Candidates for such a tyrosyl radical are discussed in connection with the possible electron transfer pathways between the heme active site and the NADPH cofactor.

Temperature-dependent behavior of bacteriochlorophyll and bacteriopheophytin in the photosynthetic reaction center from Rhodobacter sphaeroides.

We have reexamined the temperature dependence of resonance Raman (RR) spectra of the bacteriochlorin cofactors bound to reaction centers from Rhodobacter sphaeroides. Three types of resonant excitations were performed, namely, Soret band, bacteriopheophytin Qx-band, and near-infrared, Qy-band (pre)resonances. Sample temperature was varied from 300 to 10 K. In both Soret-resonant and Qy-preresonant Raman spectra, the ca. 1610-cm(-1) band corresponding to a bacteriochlorophyll CaCm methine bridge stretching mode is observed to increase in frequency by 4-6 cm(-1) as temperature is decreased from 300 to 15 K. This upshift is interpreted as arising from a change in conformation of the bacteriochlorophyll macrocycles. It may be nonspecific to the protein-bound cofactors, since a similar 4-cm(-1) upshift was observed in the same temperature range for BChl a in solution. Qx-resonant Raman spectra of either of the two bacteriopheophytin (BPhe) cofactors were obtained selectively using excitations at 537 and 546 nm. No significant frequency shift was observed for the CaCm stretching mode of BPheL between 200 and 15 K. We conclude, at variance with a previous report, that the macrocycle of the BPheL primary electron acceptor does not undergo any significant conformational change in the 200-15 K temperature range. Qy-preresonant excitation of RCs at 1064 nm provided selective Raman information on the primary electron donor (P primary). The stretching frequencies of the two conjugated keto and acetyl carbonyl groups of the M-branch primary donor BChl cofactor (P(M)) did not significantly change between 300 and 10 K. In contrast the keto carbonyl stretching frequency of cofactor P(L) was observed to upshift by 5 cm(-1), while its acetyl carbonyl frequency downshifted by 2 cm(-1). The latter shift indicated that the strong H-bond between the acetyl group of P(L) and His L168 may have slightly strengthened at 10 K. Excitation at 1064 nm of chemically oxidized RCs selectively provided RR spectra of the primary donor in its radical P.+ state. These spectra can be interpreted as a decrease of the localization of the positive charge on P(L) from 78% to 63% when the temperature decreased from 300 to 10 K resulting in a more electronically symmetric dimer. Possible origins of the temperature dependence of the positive charge delocalization in P.+ are discussed.

Influence of Asn/His L166 on the hydrogen-bonding pattern and redox potential of the primary donor of purple bacterial reaction centers.

The primary electron donor (P) of the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter (Rb.) sphaeroides is constituted of two bacteriochlorophyll molecules in excitonic interaction. The C2 acetyl carbonyl group of one of the two bacteriochlorophyll molecules (PL), the one more closely associated with the L polypeptide subunit, is engaged in a hydrogen bond with histidine L168, while the other pi-conjugated carbonyl groups of P are free from such hydrogen-bonding interactions. The three-dimensional X-ray crystal structures of the RC from several strains of Rb. sphaeroides reveal that asparagine L166 probably interacts indirectly with P through His L168. Such an interaction is expected to modulate the hydrogen bond between P and His L168, a residue which is highly conserved in purple bacteria. RC mutants of Rb. sphaeroides where asparagine L166 was genetically replaced by leucine [NL(L166)], histidine [NH(L166)], and aspartate [ND(L166)] were studied using Fourier transform (FT) Raman spectroscopy. All of these mutations resulted in an increase in the strength of the hydrogen bond between His L168 and the acetyl carbonyl group of P(L), as observed in the FT Raman spectrum, by the 2-4 cm(-1) decrease in vibrational frequency of the 1620 cm(-1) band which has been assigned to this specific acetyl carbonyl group [Mattioli, T. A., Lin, X., Allen, J. P., & Williams, J. C. (1995) Biochemistry 34, 6142-6152]. At pH 8, the NH(L166) mutation showed the greatest change in the P0/P.+ redox midpoint potential (515 mV), increasing it by ca. 30 mV compared to that of wild type (485 mV). A similar increase in P0/P.+ redox midpoint potential for NH(L166) compared to that of wild type is also observed at pH 5, 6, and 9.5. The p0/P.+ midpoint potential of the NL(L166) mutant was comparable to that of wild type at all pH values. In contrast, for the ND(L166) mutant, the midpoint potential shows a markedly different pH dependency, being 25 mV higher than wild type at pH 5 but 20 mV lower than wild type at pH 9.5. The hydrogen bond interactions of the primary electron donor from Rhodospirillum (Rsp.) centenum were determined from the FT Raman vibrational spectrum which exhibits a 1616 cm(-1) band similar to what is seen in the NH(L166) and ND(L166) Rb. sphaeroides mutants. Comparison of the sequence of the L subunit determined for the Rsp. centenum RC with that of other species indicates that positions L166 and L168 are occupied by His residues. The stronger hydrogen bond between the conserved His L168 and the acetyl carbonyl group of P(L), observed in the primary donor of Rsp. centenum and of several bacterial species which are known to possess a histidine residue at the analogous L166 position, is proposed to be due to interactions between these two histidine residues.

Hydrogen-bond interactions of the primary donor of the photosynthetic purple sulfur bacterium Chromatium tepidum.

We have used near-infrared Fourier transform (pre)resonance Raman spectroscopy to determine the protein interactions with the bacteriochlorophyll (BChl) dimer constituting the primary electron donor, P, in the reaction center (RC) from the thermophilic purple sulfur bacterium Chromatium tepidum. In addition, we report the alignment of partial sequences of the L and M protein subunits of C. tepidum RCs in the vicinity of the primary donor with those of Rhodobacter sphaeroides and Rhodopseudomonas viridis. Taken together, these results enable us to propose the hydrogen-bonding pattern and the H-bond donors to the conjugated carbonyl groups of P. Selective excitation (1064-nm laser radiation) of the FT (pre)-resonance Raman spectra of P in its neutral (P degree) and oxidized (P degree +) states were obtained via their electronic absorption bands at 876 and 1240 nm, respectively. The P degree spectrum exhibits vibrational frequencies at 1608, 1616, 1633, and 1697 cm-1 which bleach upon P oxidation. The P degree + spectrum exhibits new bands at 1600, 1639, and 1719 cm-1. The 1608-cm-1 band, which downshifts to 1600 cm-1 upon oxidation, is assigned to a CaCm methine bridge stretching mode of the P dimer, indicating that each BChl molecule possesses a single axial ligand (His L181 and His M201, from the sequence alignment). The 1616- and 1633-cm-1 bands correspond to two H-bonded pi-conjugated acetyl carbonyl groups of each BChl molecule. with different H-bond strengths: the 1616-cm-1 band is assigned to the PL C2 acetyl group which is H-bonded to a histidine residue (His L176), while the 1633-cm-1 band is assigned to the PM C2 acetyl carbonyl, H-bonded to a tyrosine residue (Tyr M196). Both PL and PM C9 keto carbonyls are free from interactions and vibrate at the same frequency (1697 cm-1). Thus, the H-bond pattern of the primary donor of C. tepidum differs from that of Rb. sphaeroides in the extra H-bond to the PM C2 acetyl carbonyl group; that of PL is H-bonded to a histidine residue in both primary donors (His L168 in Rb. sphaeroides and His L176 in C. tepidum). The P degree/P degree + redox midpoint potentials were measured to be +497 and +526 mV for isolated C. tepidum RCs with and without the associated tetraheme cytochrome c subunit, respectively, and +502 mV for intracytoplasmic membranes. The positive charge localization was estimated to be 69% in favor of PL, indicating a more delocalized situation over the primary donor of C. tepidum than that of Rb. sphaeroides (estimated to be 80% on PL). These differences in physicochemical properties are discussed with respect to the proposed structural model for the microenvironment of the primary donor of C. tepidum.

Structure and protein binding interactions of the primary donor of the Chloroflexus aurantiacus reaction center.

Soret resonance, QX resonance, and QY near-infrared Fourier transform (FT) (pre)resonance Raman spectroscopies were used to determine pigment-protein interactions of specific bacteriochlorin molecules in the reaction center from Chloroflexus aurantiacus. FT Raman spectroscopy, using 1064 nm excitation, was used to selectively obtain preresonance and resonance vibrational Raman spectra of the primary donor (P) of reaction centers (RCs) from Chloroflexus aurantiacus in the Po and P.+ states, respectively. The FT Raman spectrum of RCs in their neutral P (Po) state exhibits bands at 1605, 1632, 1648, and 1696 cm-1 which are attributable to P in its resting neutral state. Specifically, the latter three Raman bands can be assigned to the conjugated C2 acetyl and C9 keto carbonyl groups of the bacteriochlorophyll (BChl) molecules constituting P. The observation of at least three such bands is indicative of a non-monomeric nature of P, consistent with the proposal that it is a dimer of BChl molecules. The 1632 cm-1 band is consistent only with a hydrogen bonded BChl acetyl carbonyl, while the 1648 cm-1 band is assigned to a non-hydrogen bonded acetyl carbonyl. The 1696 cm-1 band is consistent only with a non-hydrogen bonded keto carbonyl group; from the unusually high intensity of this latter band compared to the others, we propose that the 1696 cm-1 band contains contributions from two keto carbonyl groups, both free of hydrogen bonds. From published protein sequence alignments of the L and M subunits of Rhodobacter (Rb.) sphaeroides and Chloroflexus aurantiacus we assign the 1632 cm-1 band as arising from the C2 acetyl carbonyl of the analogous PM constituent of P, which is hydrogen bonded to tyrosine M187 in the Chloroflexus RC, and propose a pigment-protein structural model for the primary donor of Chloroflexus aurantiacus. The FT Raman spectrum of RCs in the P degrees+ state indicates that one component of the 1696 cm-1 band has upshifted 21 cm-1 to 1717 cm-1. Compared to Rb. sphaeroides which showed a 26 cm-1 upshift for the corresponding band, the 21 cm-1 upshift indicates that the + charge is more delocalized over the P.+ species of Chloroflexus; we estimate that ca. 65% of the + charge is localized on one of the two BChl molecules of the Chloroflexus primary donor as compared to ca. 80% for Rb. sphaeroides. The consequences of the proposed structure of the Chloroflexus primary donor in terms of its Po/P.+ redox midpoint potential are discussed.

Effects of hydrogen bonding to a bacteriochlorophyll-bacteriopheophytin dimer in reaction centers from Rhodobacter sphaeroides.

The properties of the primary electron donor in reaction centers from Rhodobacter sphaeroides have been investigated in mutants containing a bacteriochlorophyll (BChl)--bacteriopheophytin (BPhe) dimer with and without hydrogen bonds to the conjugated carbonyl groups. The heterodimer mutation His M202 to Leu was combined with each of the following mutations: His L168 to Phe, which should remove an existing hydrogen bond to the BChl molecule; Leu L131 to His, which should add a hydrogen bond to the BChl molecule; and Leu M160 to His and Phe M197 to His, each of which should add a hydrogen bond to the BPhe molecule [Rautter, J., Lendzian, F., Schulz, C., Fetsch, A., Kuhn M., Lin, X., Williams, J. C., Allen J. P., & Lubitz, W. (1995) Biochemistry 34, 8130-8143]. Pigment extractions and Fourier transform Raman spectra confirm that all of the mutants contain a heterodimer. The bands in the resonance Raman spectra arising from the BPhe molecule, which is selectively enhanced, exhibit the shifts expected for the addition of a hydrogen bond to the 9-keto and 2-acetyl carbonyl groups. The oxidation--reduction midpoint potential of the donor is increased by approximately 85 mV by the addition of a hydrogen bond to the BChl molecule but is only increased by approximately 15 mV by the addition of a hydrogen bond to the BPhe molecule. An increase in the rate of charge recombination from the primary quinone is correlated with an increase in the midpoint potential. The yield of electron transfer to the primary quinone is 5-fold reduced for the mutants with a hydrogen bond to the BPhe molecule. Room- and low-temperature optical absorption spectra show small differences from the features that are typical for the heterodimer, except that a large increase in absorption is observed around 860-900 nm for the donor Qy band in the mutant that adds a hydrogen bond to the BChl molecule. The changes in the optical spectra and the yield of electron transfer are consistent with a model in which the addition of a hydrogen bond to the BChl molecule increases the energy of an internal charge transfer state while the addition to the BPhe molecule stabilizes this state. The results show that the properties of the heterodimer are different depending on which side is hydrogen-bonded and suggest that the hydrogen bonds alter the energy of the internal charge transfer state in a well-defined manner.

Pulsed EPR studies of the binuclear Mn(III)Mn(IV) center in catalase from Thermus thermophilus.

The nature of possible protein ligands to the binuclear metal core in manganese catalase from Thermus thermophilus has been addressed by EPR and ESEEM (pulsed EPR) spectroscopies. The three-pulse ESEEM spectrum of the superoxidized Mn(III)Mn(IV) enzyme obtained at 3429 G shows a frequency pattern with peaks at 0.60, 1.45, 2.06, and 5.03 MHz that is assigned to the magnetic coupling in the exact cancellation regime of one 14N atom that coordinates the Mn dimer, with magnetic parameters e2Qq = 2.34 MHz, eta = 0.51, and Aiso = 2.45 MHz. When the enzyme is chemically modified by reductive methylation, dramatic effects are detected both in the CW-EPR spectrum and in the ESEEM data. Spectral simulations of the CW-EPR signal suggest that the alterations in the spectra are related to the properties of the hyperfine coupling tensors of the Mn ions and of the g tensor, which changes from axial symmetry (gparallel - gperpendicular = 0.018) in the untreated catalase to a nearly isotropic symmetry (gparallel - gperpendicular = 0.002) in the modified enzyme. The three-pulse ESEEM spectrum of the catalase is also completely altered after the reductive methylation, with a rather different frequency pattern at 1.57, 2.35, 3.88, and 6.00 MHz. These data are interpreted as indicating that the hyperfine interaction from the coupled 14N donor is profoundly modified by the methylation treatment, changing from Aiso = 2.45 MHz to a larger value. The spectra are compared with ESEEM data obtained on two polynuclear Mn systems with 14N donors: the Mn cluster of Photosystem II inhibited by 14NH4Cl, and the model compound [Mn2(bipy)4(mu-O)2](ClO4)3. It is found that the ESEEM data measured on the untreated Mn(III)Mn(IV) catalase resemble those on the Photosystem II manganese site, suggesting that the coupled 14N coordinates the Mn dimer in an analogous fashion. By analogy to the mode of binding of ammonia in Photosystem II proposed by Britt et al. [Britt, R. D., Zimmermann, J. L., Sauer, K., & Klein, M. P. (1989) J. Am. Chem. Soc. 111, 3522-3532], it is proposed that a 14N atom bridges the two Mn ions in Mn(III)Mn(IV) catalase. By contrast, comparison of the data obtained on the methylated enzyme with those on the model compound suggests that the 14N couplings are similar in both systems; this is indicative of a terminal 14N ligand in the modified catalase.(ABSTRACT TRUNCATED AT 400 WORDS)

Spin label EPR study of lipid solvation of supramolecular photosynthetic protein complexes in thylakoids.

Lipid-protein association in the chloroplast membrane and its various thylakoid fractions from higher plants, namely pea and maize, rich in Photosystem I (PSI) and Photosystem II (PSII), respectively, were studied using EPR spectroscopy of spin-labelled lipid molecules. All the EPR spectra consisted of two spectral components corresponding to bulk fluid lipids and solvation lipids motionally restricted at the hydrophobic surface of membrane proteins. Spin-labelled stearic acid and phosphatidylglycerol exhibited marked selectivity towards the supramolecular protein complexes of both PSI and PSII although to different extent. In addition, lipid-protein titration experiments are described for partially delipidated PSII-enriched membrane fractions of pea chloroplasts, incorporating unlabelled egg phosphatidylcholine prior to or after the incorporation of spin-labelled lipids. Two sets of solvation sites were resolved by timed labelling experiments and a significant result of these studies was that a well-defined population of solvation sites (approx. 100 mol lipids/820 kDa protein) was rapidly exchanged by laterally diffusing membrane lipids, while other solvation sites (approx. 50 mol lipids/820 kDa protein) were exchanged much slower or not exchanged at all.