%0 Journal Article %J The Journal of Physical Chemistry %D 1996 %T Calcium and Magnesium Binding in Native and Structurally Perturbed Purple Membrane %A Griffiths, Jennifer A. %A King, John %A Yang, Difei %A Browner, Richard %A El-Sayed, Mostafa A %X The number and identity of the metal cations bound to wild-type bacteriorhodopsin (bR) are determined by using inductively coupled plasma mass spectrometry (ICP-MS) and ICP emission techniques. The results indicate that there at ≈2 total Ca2+ and Mg2+ per bR molecule with a ratio of ≈3:1 Ca2+ to Mg2+. This observed ratio is found to agree with the calculated ratio using previously determined binding constants for the two high affinity sites of Ca2+ to deionized bR (Zhang; et al. Biophys. J. 1992, 61, 1201). This suggests that the high-affinity binding sites in deionized bR are similar to those in native bR. Structural perturbation of the native membrane by cleavage of the C-terminus decreases the number of ions per bR to 1.4. The observed ratio of total ions in this sample to total ions in bR is found to agree with that calculated using known binding constants for each. The results on the number of metal cations/bR and their ratio in bacterio-opsin agrees with the calculated number using previously observed binding constants in deionized bO (Yang; et al. Biophys J., in press) only if one assumes that the second high-affinity site (not the first) is removed by retinal removal. Removal of 75% of the lipids from the purple membrane is found to greatly reduce the number of metal cations from 2 to 0.16. This suggest that if metal cations are in the two high-affinity sites (which are the only type of binding sites evident in our native bR sample), the removal of lipids, known to change the protein tertiary structure, changes also the metal ion binding sites.The number and identity of the metal cations bound to wild-type bacteriorhodopsin (bR) are determined by using inductively coupled plasma mass spectrometry (ICP-MS) and ICP emission techniques. The results indicate that there at ≈2 total Ca2+ and Mg2+ per bR molecule with a ratio of ≈3:1 Ca2+ to Mg2+. This observed ratio is found to agree with the calculated ratio using previously determined binding constants for the two high affinity sites of Ca2+ to deionized bR (Zhang; et al. Biophys. J. 1992, 61, 1201). This suggests that the high-affinity binding sites in deionized bR are similar to those in native bR. Structural perturbation of the native membrane by cleavage of the C-terminus decreases the number of ions per bR to 1.4. The observed ratio of total ions in this sample to total ions in bR is found to agree with that calculated using known binding constants for each. The results on the number of metal cations/bR and their ratio in bacterio-opsin agrees with the calculated number using previously observed binding constants in deionized bO (Yang; et al. Biophys J., in press) only if one assumes that the second high-affinity site (not the first) is removed by retinal removal. Removal of 75% of the lipids from the purple membrane is found to greatly reduce the number of metal cations from 2 to 0.16. This suggest that if metal cations are in the two high-affinity sites (which are the only type of binding sites evident in our native bR sample), the removal of lipids, known to change the protein tertiary structure, changes also the metal ion binding sites. %B The Journal of Physical Chemistry %I American Chemical Society %V 100 %P 929 - 933 %8 1996 %@ 0022-3654 %G eng %U http://dx.doi.org/10.1021/jp952951i %N 3 %! J. Phys. Chem. %R doi: 10.1021/jp952951i %0 Journal Article %J Proceedings of the National Academy of Sciences of the United States of America %D 1996 %T Detection of a Yb3+ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups. %A Roselli, Cecile %A Boussac, A %A Mattioli, T A %A Griffiths, Jennifer A. %A El-Sayed, Mostafa A %K Bacteriorhodopsins %K Binding Sites %K Metals, Rare Earth %K Phospholipids %K Retinaldehyde %K Spectroscopy, Near-Infrared %X Near infrared Yb3+ vibronic sideband spectroscopy was used to characterize specific lanthanide binding sites in bacteriorhodopsin (bR) and retinal free bacteriorhodopsin (bO). The VSB spectra for deionized bO regenerated with a ratio of 1:1 and 2:1 ion to bO are identical. Application of a two-dimensional anti-correlation technique suggests that only a single Yb3+ site is observed. The Yb3+ binding site in bO is observed to consist of PO2- groups and carboxylic acid groups, both of which are bound in a bidentate manner. An additional contribution most likely arising from a phenolic group is also observed. This implies that the ligands for the observed single binding site are the lipid head groups and amino acid residues. The vibronic sidebands of Yb3+ in deionized bR regenerated at a ratio of 2:1 ion to bR are essentially identical to those in bO. The other high-affinity binding site is thus either not evident or its fluorescence is quenched. A discussion is given on the difference in binding of Ca2+ (or Mg2+) and lanthanides in phospholipid membrane proteins. %B Proceedings of the National Academy of Sciences of the United States of America %V 93 %P 14333-7 %8 1996 Dec 10 %G eng %N 25 %1 http://www.ncbi.nlm.nih.gov/pubmed/8962051?dopt=Abstract %0 Journal Article %J The Journal of Physical Chemistry %D 1996 %T Effect of Binding of Lanthanide Ions on the Bacteriorhodopsin Hexagonal Structure:  An X-ray Study %A Griffiths, Jennifer A. %A El-Sayed, Mostafa A %A Capel, Malcom %X The effect of the binding of trivalent lanthanide metal cations (Eu3+, Ho3+, and Dy3+) on the hexagonal structure of bacteriorhodopsin (bR) is investigated at different pH using x-Ray diffraction to examine films made by slow evaporation of the corresponding regenerated bR. It is observed that the lanthanide-regenerated bR (at a ratio of 2:1 metal ion to bR) does not form a 2D structure isomorphous to that of native bR or Ca2+-regenerated samples at low sample pH. The native bR hexagonal structure is recovered by titration of the sample with sodium hydroxide. The pH at which the hexagonal structure is recovered depends on the charge density of the lanthanide ion used for the regeneration. The higher the charge density of the ion, the higher the pH at which an isomorphous lattice is formed. A model is proposed in which at normal or low pH a complex bidentate and monodentate type binding (which disrupts the lattice hexagonal structure) exists between a lanthanide ion, the O- of PO2- groups, and/or the amino acid residues. At high pH, complexation with OH- takes place, which converts this binding to a simple monodentate type complex that leads to the recovery of the lattice structure. An equation is derived for the pH at which this conversion takes place and is found to be proportional to the binding constant of the lanthanide ions to the O- of the PO2- groups or the amino acid residues and inversely proportional to the binding constant of the lanthanide ion to the OH- groups. This predicts an increase of conversion pH with the charge density of the lanthanide ion, as observed. %B The Journal of Physical Chemistry %I American Chemical Society %V 100 %P 12002 - 12007 %8 1996 %@ 0022-3654 %G eng %U http://dx.doi.org/10.1021/jp960741f %N 29 %! J. Phys. Chem. %R doi: 10.1021/jp960741f %0 Journal Article %J The Journal of Physical Chemistry %D 1996 %T Fluorine Substitution Effects on the Photodissociation Dynamics of Iodobenzene at 304 nm %A Griffiths, Jennifer A. %A Jung, Kwang-Woo %A El-Sayed, Mostafa A %X The photodissociation dynamics of pentafluoroiodobenzene are investigated by state-selective one-dimensional translation spectroscopy at 304 nm. We have determined the one-dimensional recoil distribution and the spatial distribution in the form of the anisotropy parameter, ?, as well as the photodissociation relative yields of both ground-state I(3P3/2) and excited-state I*(2P1/2) iodine photofragments. The results are compared to those observed for iodobenzene at 304 nm. As in iodobenzene, two velocity distributions were observed for the dissociation channel which gives ground-state iodine:? a sharp, high recoil velocity peak assigned previously to n,σ* excitation and a slow recoil velocity distribution peak assigned previously to π,π* excitation. Unlike in C6H5I, the I* distribution is relatively strong and its spatial anisotropy can be measured. The fluorine perturbation has led to a number of different observations that can be summarized as follows:? (1) The high velocity distribution has a lower average value and much broader width, suggesting more rapid energy redistribution to the fluorinated phenyl ring prior to and during the dissociation process, resulting from stronger coupling between the n,σ* and π,π* states and/or a longer excited-state lifetime; (2) the slow distribution is weaker and has an almost isotropic spatial distribution (the anisotropy parameter ? ≈ 1.0), while in the iodobenzene spectrum ? is correlated with the recoil velocity; (3) the I* quantum yield for C6F5I is 14 times larger than that for iodobenzene; and (4) ? is correlated with the velocity in the I* spectrum found for C6F5I which is not observed for iodobenzene. These observed fluorine perturbations are attributed to an increased mixing between the charge-transfer state (resulting from electron transfer from the iodine nonbonding electrons to the π* orbitals of the fluorinated benzene ring) and both the n,σ* and the ring π,π* states. This leads to two effects:? (1) a decrease in the nonbonding electron density on the iodine, which decreases the spin-orbit interaction between the n,σ* states themselves, resulting in a decrease in the curve-crossing probability (thus increasing the I* yield) and (2) an increase in the coupling between the repulsive n,σ* states and the fluorinated phenyl π,π* states, leading to an increase in the rate of energy redistribution.The photodissociation dynamics of pentafluoroiodobenzene are investigated by state-selective one-dimensional translation spectroscopy at 304 nm. We have determined the one-dimensional recoil distribution and the spatial distribution in the form of the anisotropy parameter, ?, as well as the photodissociation relative yields of both ground-state I(3P3/2) and excited-state I*(2P1/2) iodine photofragments. The results are compared to those observed for iodobenzene at 304 nm. As in iodobenzene, two velocity distributions were observed for the dissociation channel which gives ground-state iodine:? a sharp, high recoil velocity peak assigned previously to n,σ* excitation and a slow recoil velocity distribution peak assigned previously to π,π* excitation. Unlike in C6H5I, the I* distribution is relatively strong and its spatial anisotropy can be measured. The fluorine perturbation has led to a number of different observations that can be summarized as follows:? (1) The high velocity distribution has a lower average value and much broader width, suggesting more rapid energy redistribution to the fluorinated phenyl ring prior to and during the dissociation process, resulting from stronger coupling between the n,σ* and π,π* states and/or a longer excited-state lifetime; (2) the slow distribution is weaker and has an almost isotropic spatial distribution (the anisotropy parameter ? ≈ 1.0), while in the iodobenzene spectrum ? is correlated with the recoil velocity; (3) the I* quantum yield for C6F5I is 14 times larger than that for iodobenzene; and (4) ? is correlated with the velocity in the I* spectrum found for C6F5I which is not observed for iodobenzene. These observed fluorine perturbations are attributed to an increased mixing between the charge-transfer state (resulting from electron transfer from the iodine nonbonding electrons to the π* orbitals of the fluorinated benzene ring) and both the n,σ* and the ring π,π* states. This leads to two effects:? (1) a decrease in the nonbonding electron density on the iodine, which decreases the spin-orbit interaction between the n,σ* states themselves, resulting in a decrease in the curve-crossing probability (thus increasing the I* yield) and (2) an increase in the coupling between the repulsive n,σ* states and the fluorinated phenyl π,π* states, leading to an increase in the rate of energy redistribution. %B The Journal of Physical Chemistry %I American Chemical Society %V 100 %P 7989 - 7996 %8 1996 %@ 0022-3654 %G eng %U http://dx.doi.org/10.1021/jp952662u %N 19 %! J. Phys. Chem. %0 Journal Article %J The Journal of Physical Chemistry %D 1996 %T Monodentate vs Bidentate Binding of Lanthanide Cations to PO2- in Bacteriorhodopsin %A Griffiths, Jennifer A. %A Masciangioli, Tina M. %A Roselli, Cecile %A El-Sayed, Mostafa A %X The frequency difference between the symmetric and antisymmetric stretching vibration of PO2- in phosphatidylglycerol phospate (PGP) is used to differentiate between monodentate and bidentate binding of these groups to metal cations in the membrane of bacteriorhodopsin (bR) and phosphatidylglycerol phospate. The binding of Ca2+ to PGP is found to have a frequency difference corresponding to monodentate binding. The symmetric and antisymmetric PO2- bands in bR show similar frequency shifts upon Ca2+ binding, which is independent of pH. This suggests that Ca2+ has a monodentate type binding with the PO2- in bR. In contrast, the PO2- symmetric and antisymmetric frequencies of PGP complexes with trivalent lanthanide cations with higher charge density (Ho3+ and Dy3+) are observed to have smaller separations and to increase their separation with increasing pH toward the value observed for Ca2+ binding. Lanthanide cations (Ho3+, Dy3+, Eu3+, Nd3+, and La3+) binding in bR at pH 4 show small frequency separations that are observed to have similar frequency shifts with pH, the magnitude of which is dependent on the cation. It is proposed that at low pH the lanthanide cations with higher charge density have bidentate binding to bR, while at high pH, complexation with the OH- competes with one of the oxygens of the PO2- for the binding of the lanthanide ion thus changing the bidentate to monodentate type binding.The frequency difference between the symmetric and antisymmetric stretching vibration of PO2- in phosphatidylglycerol phospate (PGP) is used to differentiate between monodentate and bidentate binding of these groups to metal cations in the membrane of bacteriorhodopsin (bR) and phosphatidylglycerol phospate. The binding of Ca2+ to PGP is found to have a frequency difference corresponding to monodentate binding. The symmetric and antisymmetric PO2- bands in bR show similar frequency shifts upon Ca2+ binding, which is independent of pH. This suggests that Ca2+ has a monodentate type binding with the PO2- in bR. In contrast, the PO2- symmetric and antisymmetric frequencies of PGP complexes with trivalent lanthanide cations with higher charge density (Ho3+ and Dy3+) are observed to have smaller separations and to increase their separation with increasing pH toward the value observed for Ca2+ binding. Lanthanide cations (Ho3+, Dy3+, Eu3+, Nd3+, and La3+) binding in bR at pH 4 show small frequency separations that are observed to have similar frequency shifts with pH, the magnitude of which is dependent on the cation. It is proposed that at low pH the lanthanide cations with higher charge density have bidentate binding to bR, while at high pH, complexation with the OH- competes with one of the oxygens of the PO2- for the binding of the lanthanide ion thus changing the bidentate to monodentate type binding. %B The Journal of Physical Chemistry %I American Chemical Society %V 100 %P 6863 - 6866 %8 1996 %@ 0022-3654 %G eng %U http://dx.doi.org/10.1021/jp9533279 %N 16 %! J. Phys. Chem. %R doi: 10.1021/jp9533279 %0 Journal Article %J Pure and applied chemistry %D 1995 %T On the molecular mechanisms of the rapid and slow solar-to-electric energy storage processes by the other natural photosynthetic system, bacteriorhodopsin %A El-Sayed, Mostafa A %A Griffiths, Jennifer A. %A Song, Li %A Zhang, N. %X Upon the absorption of solar energy by retinal in bacterioi..>dopsin highly specific photoisomerization of the retinal around the C13 -C14 bond takes place. This is followed by the formation of a number of intermediates resulting from conformational changes of the protein around the retinal which leads to the deprotonation of the protonated Schiff base of the retinylidene system. Thisis the switch of the proton pump which leads to the last step in the storage of solar energy in the form of electric energy by this photosynthetic system. The removal of metal cations from bR is found to inhibit the deprotonation process. In the present paper we summarize the results of our studies and the others regarding two important questions in the conversion process: 1) what is(are) the molecular mechanism(s) of the protein catalysis of the photoisomerizationprocess and 2) what is the role of metal cations in the deprotonation process of the protonated Schiff base (the switch of the proton pump)? In order to answer the first question, the results of the subpicosecondphotoisomerization rate of retinal in bR and in a number of its relevant mutants are discussed in terms of the steric and electronic factors. In an effort to answer the second question,we discussed the results of the binding studies of Ca*+to bR, to its mutants and to bR after its C- terminus is cleaved. From these results and the results of Roux et al. on the 31P NMR of Nd3+ regenerated bR, we concluded that one or two metal cations strongly bound to the protein but not on the surface, are functionally important. The model in which these metal cation@)control the pK values of Aspartic acids in the 85 and 212 positions and that of the protonated Schiff base (PSB) during the photocycle is discussed. %B Pure and applied chemistry %I BLACKWELL SCIENTIFIC PUBLICATIONS %V 67 %P 149-149 %@ 0033-4545 %G eng %0 Journal Article %J Journal of Chemical Physics %D 1995 %T Photofragment translational spectroscopy of Ibr at 304 nm: Polarization dependence and dissociation dynamics %A Jung, Kwang-Woo %A Griffiths, Jennifer A. %A El-Sayed, Mostafa A %X The photodissociation dynamics of IBr has been studied at 304 nm by state-selective photofragment translational spectroscopy. Velocity distributions, anisotropy parameters, and relative quantum yields are obtained for the ground I(P-2(3/2)) and spin-orbit excited state I*(P-2(1/2)) iodine atoms, which are produced from photodissociation of IBr at this wavelength. Two sharp velocity distributions observed for the I channel suggest the two dissociation pathways that correlate with ground-state iodine formation. Based on the expected translational energy release and the energy separation between the peaks, the two distributions have been assigned to dissociation of IBr to form I(P-2(3/2))+Br(P-2(3/2)) and I(P-2(3/2))+Br*(P-2(1/2)) with the former channel appearing at higher translational energy. The I* distribution shows one strong peak indicating that there is one dominant channel for formation of I* atoms at this wavelength which has been assigned to dissociation of IBr to form I*(P-2(1/2))+Br(P-2(3/2)) with a quantum yield of 0.1. The I* signal formed from the I*(P-2(1/2))+Br*(P-2(1/2)) dissociation channel is observed very weakly. The observed anisotropy parameter indicates that the I+Br* product (beta=-0.7) is formed mainly from the perpendicular (1) Pi(1)(2341)<--X transition while the I*+Br channel (beta=1.8) is formed predominantly from the parallel 3 Pi(0+)(2341)<--X transition followed by curve crossing to the (3) Sigma(0+)(-)(2422) State. The recoil energy dependence of the anisotropy parameter in the I atom produced in the I+Br channel shows a positive beta value above maximum of the peak recoil energy and a negative value below the peak maximum of the recoil energy distribution. These results are interpreted in terms of the presence of more than one path for the formation of I+Br photoproduct with opposite polarization for their absorbing transitions, most likely the (3) Pi(0+)(2341)<--X and the (3) Pi(1)(2341)<--X transitions. The possible excited state dynamics which give the observed results are discussed in terms of the previously proposed potential energy diagrams for IBr and ICl. (C) 1995 American Institute of Physics. %B Journal of Chemical Physics %V 103 %P 6999-7005 %8 Oct 22 %@ 0021-9606 %G eng %M WOS:A1995TA44100018 %R 10.1063/1.470326