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Time-resolved FTIR difference spectroscopy in combination with specific isotope labeling for the study of A1, the secondary electron acceptor in photosystem 1.

ABSTRACT: A phylloquinone molecule (2-methyl, 3-phytyl, 1, 4-naphthoquinone) occupies the A(1) binding site in photosystem 1 particles from Synechocystis sp. 6803. In menB mutant photosystem 1 particles from the same species, plastoquinone-9 occupies the A(1) binding site. By incubation of menB mutant photosystem 1 particles in the presence of phylloquinone, it was shown in another study that phylloquinone will displace plastoquinone-9 in the A(1) binding site. We describe the reconstitution of unlabeled ((16)O) and (18)O-labeled phylloquinone back into the A(1) binding site in menB photosystem 1 particles. We then produce time-resolved A(1)(-)/A(1) Fourier transform infrared (FTIR) difference spectra for these menB photosystem 1 particles that contain unlabeled and (18)O-labeled phylloquinone. By specifically labeling only the phylloquinone oxygen atoms we are able to identify bands in A(1)(-)/A(1) FTIR difference spectra that are due to the carbonyl (C=O) modes of neutral and reduced phylloquinone. A positive band at 1494 cm(-1) in the A(1)(-)/A(1) FTIR difference spectrum is found to downshift 14 cm(-1) and decreases in intensity on (18)O labeling. Vibrational mode frequency calculations predict that an antisymmetric vibration of both C=O groups of the phylloquinone anion should display exactly this behavior. In addition, phylloquinone that has asymmetrically hydrogen bonded carbonyl groups is also predicted to display this behavior. The positive band at 1494 cm(-1) in the A(1)(-)/A(1) FTIR difference spectrum is therefore due to the antisymmetric vibration of both C=O groups of one electron reduced phylloquinone. Part of a negative band at 1654 cm(-1) in the A(1)(-)/A(1) FTIR difference spectrum downshifts 28 cm(-1) on (18)O labeling. Again, vibrational mode frequency calculations predict this behavior for a C=O mode of neutral phylloquinone. The negative band at 1654 cm(-1) in the A(1)(-)/A(1) FTIR difference spectrum is therefore due to a C=O mode of neutral phylloquinone. More specifically, calculations on a phylloquinone model molecule with the C(4)=O group hydrogen bonded predict that the 1654 cm(-1) band is due to the non hydrogen bonded C(1)=O mode of neutral phylloquinone.

SUBMITTER: Hastings G

PROVIDER: S-EPMC2480661 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Time-resolved FTIR difference spectroscopy in combination with specific isotope labeling for the study of A1, the secondary electron acceptor in photosystem 1.

Hastings Gary G Bandaranayake K M Priyangika KM Carrion Enrique E

Biophysical journal 20080215 11

A phylloquinone molecule (2-methyl, 3-phytyl, 1, 4-naphthoquinone) occupies the A(1) binding site in photosystem 1 particles from Synechocystis sp. 6803. In menB mutant photosystem 1 particles from the same species, plastoquinone-9 occupies the A(1) binding site. By incubation of menB mutant photosystem 1 particles in the presence of phylloquinone, it was shown in another study that phylloquinone will displace plastoquinone-9 in the A(1) binding site. We describe the reconstitution of unlabeled ...[more]

PMID: 18281389

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Project description:Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site. As H/D exchange kinetics followed by FTIR spectroscopy can provide information on the conformational changes in proteins where solvent accessibility is affected, here the amide I region was used to measure the exchange rates of the different elements of the secondary structure for aromatase in the ligand-free form and in the presence of the substrate androstenedione and the inhibitor anastrozole. Biphasic exponential functions were found to fit the H/D exchange data collected as a function of time. Two exchange rates were assigned to two populations of protons present in different flexible regions of the protein. The addition of the substrate androstenedione and the inhibitor anastrozole lowers the H/D exchange rates of the α-helices of the enzyme when compared to the ligand-free form. Furthermore, the presence of the inhibitor anastrozole lowers exchange rate constant (k1) for β-sheets from 0.22±0.06 min(-1) for the inhibitor-bound enzyme to 0.12±0.02 min(-1) for the free protein. Dynamics effects localised in helix F were studied by time resolved fluorescence. The data demonstrate that the fluorescence lifetime component associated to Trp224 emission undergoes a shift toward longer lifetimes (from ≈5.0 to ≈5.5 ns) when the substrate or the inhibitor are present, suggesting slower dynamics in the presence of ligands. Together the results are consistent with different degrees of flexibility of the access channel and therefore different conformations adopted by the enzyme in the free, substrate- and inhibitor-bound forms.

Dataset Information

Time-resolved FTIR difference spectroscopy in combination with specific isotope labeling for the study of A1, the secondary electron acceptor in photosystem 1.

Publications

Time-resolved FTIR difference spectroscopy in combination with specific isotope labeling for the study of A1, the secondary electron acceptor in photosystem 1.

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