Lipid-protein interactions in thylakoid membranes of chilling-resistant and -sensitive plants studied by spin label electron spin resonance spectroscopy

Lipid-protein interactions in thylakoid membranes from lettuce, pea, tomato, and cucumber have been studied using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyl diglyceride and phosphatidylglycerol. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted lipids interacting with the integral membrane proteins. Comparison of the spectra from the same spin label in thylakoid membranes from different plants shows that the overall lipid fluidity in the membranes decreases with chilling sensitivity. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with integral membrane proteins. Thylakoid membranes of cucumber, a typical chilling-sensitive plant, have been found to have a higher proportion of motionally restricted lipids and a different lipid selectivity for lipid-protein interaction, as compared with those of pea, a typical chilling-resistant plant. This correlation with chilling sensitivity holds generally for the different plants studied. It seems likely that the chilling sensitivity in thylakoid membranes is not determined by lipid fluidity alone, but also by the lipid-protein interactions which could affect protein function in a more direct manner.     

Kinetics of carboxymyoglobin and oxymyoglobin studied by picosecond spectroscopy.

Picosecond studies of carboxymyoglobin (MbCO) and oxymyoglobin (MbO2) reveal that excitation at 530 nm induces photodissociation at less than 8 ps. The kinetic and structural changes were monitored by following absorbance changes at selected wave-lengths in the Soret (B) band and in the Q band. Within the 10 ps-0.45 ns period of time over which our experiments were conducted, the absorbance changes in the Soret and Q bands for MbCO and MbO2 correspond to the conventional long-term, steady-state deoxymyoglobin difference spectra (Mb-MbCO and Mb-MbO2), as determined by comparison of isosbestic, maximum, and minimum points. In addition, MbCO exhibits a decay to a steady state in the Soret band (monitored at 440 nm). The onset of the decay immediately follows photodissociation and has a rate of (8 +/- 3) X 10(9) s-1 (tau = 125 +/- 50 ps). During the 10 ps-0.45 ns observation window, relaxation is not seen for MbO2 in the Soret band, nor is relaxation observed in the Q band for either MbCO or MbO2. We conclude from these results that the steady state that we observed for MbCO and MbO2 is most likely the stable form of deoxymyoglobin, and the relaxational differences between MbCO and MbO2 observed in the Soret band indicate that the electronic destabilization after ligand detachment is very different for these molecules. We believe that these relaxational differences may be related to differences in tertiary structural changes, or due to the fact that the MbCO (S = 0) molecule passes through an intermediate spin Mb (S = 1) state before relaxing the the Mb (S = 2) state.     

Domain formation in sphingomyelin/cholesterol mixed membranes studied by spin-label electron spin resonance spectroscopy

Interactions of palmitoylsphingomyelin with cholesterol in multilamellar vesicles have been studied over a wide range of compositions and temperatures in excess water by using electron spin resonance (ESR) spectroscopy. Spin labels bearing the nitroxide free radical group on the 5 or 14 C-atom in either the sn-2 stearoyl chain of phosphatidylcholine (predominantly 1-palmitoyl) or the N-stearoyl chain of sphingomyelin were used to determine the mobility and ordering of the lipids in the different phases. Two-component ESR spectra of the 14-position spin labels demonstrate the coexistence first of gel (L(beta)) and liquid-ordered (L(o)) phases and then of liquid-ordered and liquid-disordered (L(alpha)) phases, with progressively increasing temperature. These phase coexistences are detected over a limited range of cholesterol contents. ESR spectra of the 5-position spin labels register an abrupt increase in ordering at the L(alpha)-L(o) transition and a biphasic response at the L(beta)-L(o) transition. Differences in outer splitting between the C14-labeled sphingomyelin and phosphatidylcholine probes are attributed to partial interdigitation of the sphingomyelin N-acyl chains across the bilayer plane in the L(o) state. In the region where the two fluid phases, L(alpha) and L(o), coexist, the rate at which lipids exchange between phases (<7 x 10(7) s(-)(1)) is much slower than translational rates in the L(alpha) phase, which facilitates resolution of two-component spectra.     

Spin-density distribution in the [FeO2]-complex. Electron spin resonance of myoglobin single crystals

X-irradiated oxymyoglobin (MbO2) exhibits electron spin resonance (ESR) spectra at 77 K due to two distinct [FeO2]-centers formed by electron addition to the dioxygen. In single crystals with 17O and 57Fe isotope enrichment in the heme-ligand complex, the full set of spectral parameters is analyzed for one of the centers (gmax = 2.23, gint = 2.13, gmin = 1.97; HOmax = 2.66 mT, HOint = 1.61 mT, HOmin = 0.57 mT; HFe max = 1.62 mT, HFe int = 0.57 mT, HFe min = 0.49 mT) and the iron-dioxygen spin-density distribution and bonding geometry is derived. The g-tensor is evaluated for the second species at 77 K (gmax = 2.25, gint = 2.11, gmin = 1.95). Both centers transform into secondary species at 180 K for which the g-tensor elements are analyzed (gmax = 2.31, gint = 2.18, gmin = 1.93; gmax = 2.35, gint = 2.21, gmin = 1.91).     

Ascorbic acid induced degradation of beta-glucan: Hydroxyl radicals as intermediates studied by spin trapping and electron spin resonance spectroscopy

The formation of hydroxyl radicals in beta-glucan solutions treated with ascorbic acid and iron(II) was demonstrated by ESR spin trapping based methods. Two different spin traps were tested, namely DMPO which is commonly used to detect hydroxyl radicals, and POBN often used to detect carbon centered radicals. The experiments performed showed that the presence of iron(II) with DMPO led to low DMPO-OH adduct stability and further to DMPO dimerization. The level of hydroxyl radicals formed during the beta-glucan radical mediated degradation was evaluated using two ESR spin trapping methods based on the use POBN together with either 2% (v/v) EtOH or DMSO. The addition of ascorbic acid together with iron(II) in beta-glucan solution led to an immediate maximal production of hydroxyl radicals while the presence of ascorbic acid alone led to a progressive production of radical. Further hydroxyl radicals were found to be formed when iron(II) was added alone in beta-glucan solutions. The viscosity loss observed in the three last mentioned beta-glucan solutions were found to relate with the formation of hydroxyl radicals. These data confirm the involvement of hydroxyl radical in the beta-glucan degradation.     

The electron spin relaxation of the electron acceptors of photosystem I reaction centre studied by microwave power saturation.

Photosystem I particles from spinach were reduced by illumination at 77 K. Under these conditions the one-electrom transfer from P-700 resulted in a reduction of only one acceptor molecule of the reaction centre. The EPR signals at g=2.05, 1.94 and 1.86 were attributed to reduced centre A and the smaller signals at g=2.07, 1.92 and 1.89 to reduced centre B. Reduction of both centres by dithionite in the dark lead to signals at g=2.05, 1.99, 1.96, 1.94, 1.92 and 1.89. Thus, the features at g=2.07 and 1.86 disappeared and new signals at g=1.99 and 1.96 were observed. From the spectral changes it followed that the iron-sulphur centres A and B interact magnetically. Temperature dependent EPR spectra demonstrated a faster electron spin relaxation of centre A than of centre B. These conclusions were corroborated using microwave power saturation of the respective EPR signals. The saturation data of the fully reduced centres A and B could not be fitted using the saturation equation for a one-electron spin system. The magnetic interaction between the (4Fe-4S) CENTRes of the electron acceptors A and B resulted in saturation properties which are simular to those of the 2(4Fe-4S) ferredoxin from Clostridium pasteurianum. For centre X a high proportion of homogeneous broadening of the EPR lines was inferred from the inhomogeneity parameter (b=1.83). It was, therefore, concluded that centre X is most probably an anion radical of chlorophyll. From the low temperature necessary for observing the EPR signal of centre X followed that the drastic relaxation enhancement has to be attributed to a magnetic interaction of the anion radical with iron.     

Exchange rates at the lipid-protein interface of myelin proteolipid protein studied by spin-label electron spin resonance

The electron spin resonance (ESR) spectra from spin-labeled phospholipids in recombinants of myelin proteolipid apoprotein with dimyristoylphosphatidylcholine have been simulated with the exchanged-coupled Bloch equations to obtain values for both the fraction of motionally restricted lipids and the exchange rate between the fluid and motionally restricted lipid populations. The rate of exchange between the two spin-labeled lipid components is found to lie in the slow exchange regime of nitroxide ESR spectroscopy. The values obtained for the fraction of motionally restricted component in the exchanged-coupled spectra are found to be in good agreement with those obtained previously by spectral subtraction for the same system [Brophy, P. J., Horváth, L. I., & Marsh, D. (1984) Biochemistry 23, 860-865]. The rate of lipid exchange off the protein is independent of lipid/protein ratio for a given spin-labeled phospholipid, as expected, and decreases with increasing selectivity of the various phospholipids for the protein. At 30 degrees C and for ionic strength 0.1 and pH 7.4, the off-rate constants are 4.6 X 10(6) s-1 for phosphatidic acid, 1.1 X 10(7) s-1 for phosphatidylserine, 1.6 X 10(7) s-1 for phosphatidylcholine, and 2.2 X 10(7) s-1 for phosphatidylethanolamine. These values are in the inverse ratio of the relative association constants of the various lipids for the protein (Brophy et al., 1984) and are appreciably slower than the rate of lipid lateral diffusion in dimyristoylphosphatidylcholine bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate binding to DNA photolyase studied by electron paramagnetic resonance spectroscopy.

Structural changes in Escherichia coli DNA photolyase induced by binding of a (cis,syn)-cyclobutane pyrimidine dimer (CPD) are studied by continuous-wave electron paramagnetic resonance and electron-nuclear double resonance spectroscopies, using the flavin adenine dinucleotide (FAD) cofactor in its neutral radical form as a naturally occurring electron spin probe. The electron paramagnetic resonance/electron-nuclear double resonance spectral changes are consistent with a large distance (> or =0.6 nm) between the CPD lesion and the 7,8-dimethyl isoalloxazine ring of FAD, as was predicted by recent model calculations on photolyase enzyme-substrate complexes. Small shifts of the isotropic proton hyperfine coupling constants within the FAD's isoalloxazine moiety can be understood in terms of the cofactor binding site becoming more nonpolar because of the displacement of water molecules upon CPD docking to the enzyme. Molecular orbital calculations of hyperfine couplings using density functional theory, in conjunction with an isodensity polarized continuum model, are presented to rationalize these shifts in terms of the changed polarity of the medium surrounding the FAD cofactor.     

Protein-tyrosyl radical interactions in photosystem II studied by electron spin resonance and electron nuclear double resonance spectroscopy: comparison with ribonucleotide reductase and in vitro tyrosine.

The stable tyrosine radical in photosystem II, YD*, has been studied by ESR and ENDOR spectroscopies to obtain proton hyperfine coupling constants from which the electron spin density distribution can be deduced. Simulations of six previously published ESR spectra of PSII (one at Q band; five at X band, of which two were after specific deuteration and two others were of oriented membranes) can be achieved by using a single set of magnetic parameters that includes anisotropic proton hyperfine tensors, an anisotropic g tensor, and noncoincident axis systems for the g and A tensors. From the spectral simulation of the oriented samples, the orientation of the phenol head group of YD* with respect to the membrane plane has been determined. A similar orientation for YZ*, the redox-active tyrosine in PSII that mediates electron transfer between P680 and the oxygen-evolving complex, is expected. ENDOR spectra of YD* in PSII preparations from spinach and Synechocystis support the set of hyperfine coupling constants but indicate that small differences between the two species exist. Comparison with the results of spectral simulations for tyrosyl radicals in ribonucleotide reductase from prokaryotes or eukaryotes and with in vitro radicals indicates that the spin density distribution remains that of an odd-alternant radical but that interactions with the protein can shift spin density within this basic pattern. The largest changes in spin density occur at the tyrosine phenol oxygen and at the ring carbon para to the oxygen, which indicates that mechanisms exist in the protein environment for fine-tuning the chemical and redox properties of the radical species.     

Lipid-protein interactions with cardiac phospholamban studied by spin-label electron spin resonance

Phospholamban is a cardiac regulatory protein that, in its monomeric form, inhibits the Ca(2+)-ATPase. Lipid-protein interactions with a synthetic variant of phospholamban, in which all cysteine residues are replaced with alanine, have been studied by spin-label electron spin resonance (ESR) in different lipid host membranes. Both the stoichiometry and selectivity of lipid interactions were determined from the two-component ESR spectra of phospholipid species spin-labeled on the 14 C atom of the sn-2 chain. The lipid stoichiometry is determined by the oligomeric state of the protein and the selectivity by the membrane disposition of the positively charged residues in the N-terminal section of the protein. In dimyristoylphosphatidylcholine (DMPC) membranes, the stoichiometry (N(b)) is 7 lipids/monomer for the full-length protein and 4 for the transmembrane section (residues 26-52). These stoichiometries correspond to the dimeric and pentameric forms, respectively. In palmitoyloleoylphosphatidylcholine, N(b) = 4 for both the whole protein and the transmembrane peptide. In negatively charged membranes of dimyristoylphosphatidylglycerol (DMPG), the lipid stoichiometry is N(b) = 10-11 per monomer for both the full-length protein and the transmembrane peptide. This stoichiometry corresponds to monomeric dispersion of the protein in the negatively charged lipid. The sequence of lipid selectivity is as follows: stearic acid > phosphatidic acid > phosphatidylserine = phosphatidylglycerol = phosphatidylcholine > phosphatidylethanolamine for both the full-length protein and the transmembrane peptide in DMPC. Absolute selectivities are, however, lower for the transmembrane peptide. A similar pattern of lipid selectivity is obtained in DMPG, but the absolute selectivities are reduced considerably. The results are discussed in terms of the integration of the regulatory species in the lipid membrane.     

The electron spin relaxation of the electron acceptors of Photosystem I reaction centre studied by microwave power saturation

Photosystem I particles from spinach were reduced by illumination at 77 K. Under these conditions the one-electron transfer from P-700 resulted in a reduction of only one acceptor molecule of the reaction centre. The EPR signals at g = 2.05, 1.94 and 1.86 were attributed to reduced centre A and the smaller signals at g = 2.07, 1.92 and 1.89 to reduced centre B. Reduction of both centres by dithionite in the dark lead to signals at g = 2.05, 1.99, 1.96, 1.94, 1.92 and 1.89. Thus, the features at g = 2.07 and 1.86 disappeared and new signals at g = 1.99 and 1.96 were observed. From the spectral changes it followed that the iron-sulphur centres A and B interact magnetically. Temperature dependent EPR spectra demonstrated a faster electron spin relaxation of centre A than of centre B.

These conclusions were corroborated using microwave power saturation of the respective EPR signals. The saturation data of the fully reduced centres A and B could not be fitted using the saturation equation for a one-electron spin system. The magnetic interaction between the [4Fe-4S] centres of the electron acceptors A and B resulted in saturation properties which are similar to those of the 2[4Fe-4S] ferredoxin from Clostridium pasteurianum.

For centre X a high proportion of homogeneous broadening of the EPR lines was inferred from the inhomogeneity parameter (b = 1.83). It was, therefore, concluded that centre X is most probably an anion radical of chlorophyll. From the low temperature necessary for observing the EPR signal of centre X followed that the drastic relaxation enhancement has to be attributed to a magnetic interaction of the anion radical with iron.     

New sensitive agents for detecting singlet oxygen by electron spin resonance spectroscopy.

Free radicals are well-established transient intermediates in chemical and biological processes. Singlet oxygen, though not a free radical, is also a fairly common reactive chemical species. It is rare that singlet oxygen is studied with the electron spin resonance (ESR) technique in biological systems, because there are few suitable detecting agents. We have recently researched some semiquinone radicals. Specifically, our focus has been on bipyrazole derivatives, which slowly convert to semiquinone radicals in DMSO solution in the presence of potassium tert-butoxide and oxygen. These bipyrazole derivatives are dimers of 3-methyl-1-phenyl-2-pyrazolin-5-one and have anti-ischemic activities and free radical scavenging properties. In this work, we synthesized a new bipyrazole derivative, 4,4'-bis(1p-carboxyphenyl-3-methyl-5-hydroxyl)-pyrazole, DRD156. The resulting semiquinone radical, formed by reaction with singlet oxygen, was characterized by ESR spectroscopy. DRD156 gave no ESR signals from hydroxyl radical, superoxide, and hydrogen peroxide. DRD156, though, gives an ESR response with hypochlorite. This agent, nevertheless, has a much higher ability to detect singlet oxygen than traditional agents with the ESR technique.