Electron nuclear double resonance (ENDOR) and special triple (ST) resonance spectroscopies have been used to study the cation radicals of the primary donor, P680, and two secondary donor chlorophylls (Chl) in photosystem 2 (PS2). Two different preparations were employed, Tris-washed PS2 membranes and PS2 reaction centers (D1-D2-I-Cytb559 complex). One secondary donor Chl a cation radical, Chl1.+, was generated in the Tris-washed preparation, while the P680.+ radical cation and a further Chl a cation radical, Chl2.+, were produced in the reaction center preparation. The ENDOR spectrum of the primary donor radical cation of photosystem 1 (P700.+) is also presented for comparison. Hyperfine coupling constants for methyl groups have been measured for all three PS2 radical species and assigned by comparison with previously published spectra of Chl a radicals in vitro. Electron spin densities were calculated from these hyperfine couplings. Comparison of ENDOR spectral features with those of Chla.+ in vitro indicates similar values for Chl1.+ and Chl2.+ radicals but an apparent reduction in unpaired electron spin density for P680.+. It has been proposed from the more detailed studies of purple bacterial reaction centers that such a reduction in spin density can be interpreted as a delocalization over two Chl a molecules. Our calculations therefore suggest that P680.+ is a weakly coupled chlorophyll pair with 82% of the unpaired electron spin located on one chlorophyll of the pair at 15 K. Environmental or geometrical changes to the chlorin ring structure to give a novel monomeric primary donor are also possible.(ABSTRACT TRUNCATED AT 250 WORDS)
The dark stable neutral tyrosine radical YD. of photosystem 2 (PS2) has been studied using electron nuclear double-resonance (ENDOR) and electron paramagnetic resonance (EPR) spectroscopies. The proton hyperfine coupling constants of all four ring protons and both beta-methylene protons have been determined for YD. in three species covering the range of oxygenic organisms; a higher plant (spinach), an alga (Chlamydomonas reinhardtii), and a cyanobacterium (Phormidium laminosum). It has generally been assumed that the properties of Yd. are the same in all oxygenic organisms, while in fact there are small but significant differences. The beta-proton coupling constants are shown to be species dependent while the ring proton coupling constants are not. Estimation of the electron spin density distribution of Yd. from all three organisms has been done. This shows that changes in beta-proton coupling constants in each organism arise from the slightly different orientation of the tyrosine ring, relative to the beta-protons. The electron spin density distribution within the tyrosine ring is organism independent. The variations in the beta-proton coupling constants are reflected in the corresponding EPR spectra, where small variations in line width have been detected. These data delineate the range of natural variation in the spectroscopic properties of YD., and by assigning the features of the ENDOR spectrum, provide a basis for both the unification of studies of YD. in different organisms and the study of YZ.. The results are discussed in relation to data in the recent study (Hoganson & Babcock, 1992) using YD. in the cyanobacterium, Synechocystis PCC 6803.
Hybrid density functional calculations (B3LYP) are performed on
the p-benzosemiquinone anion radical in
its free and hydrogen-bonded forms. Geometries and hyperfine
couplings are reported. A variety of basis
sets ranging from split valence to full triple-ζ are employed.
Converged results for hyperfine couplings are
observed at the double-ζ level. Hydrogen bonding principally
leads to increased spin density on the carbonyl
carbon leading to an increase in the 13C isotropic and
anisotropic hyperfine coupling of this atom.
Comparison
with experimentally determined isotropic and anisotropic hyperfine
couplings shows good quantitative
agreement between theoretical calculation and experiment.
A novel mechanism for the final stages of Nature's photosynthetic water oxidation to molecular oxygen is proposed. This is based on a comparison of experimental and broken symmetry density functional theory (BS-DFT) calculated geometries and magnetic resonance properties of water oxidising complex models in the final metastable oxidation state, S 3. We show that peroxo models of the S 3 state are in vastly superior agreement with the current experimental structural determinations compared with oxohydroxo models. Comparison of experimental and BS-DFT calculated 55 Mn hyperfine couplings for the EPR visible form shows better agreement for the oxo-hydroxo model. An equilibrium between oxo-hydroxo and peroxo models is proposed for the S 3 state and the major implications for the final steps in the water oxidation mechanism are analysed and discussed.
The
identity of a key intermediate in the S2 to S3 transition of nature’s water-oxidizing complex (WOC)
in Photosystem 2 is presented. Broken-symmetry density functional
theory (BS-DFT) calculations and Heisenberg–Dirac–van
Vleck (HDvV) spin ladder calculations show that an S2 state
open cubane model of the WOC containing a μ-hydroxo O4 changes
from an S = 5/2 form to an S = 7/2, form upon deprotonation of
W1. Combined with X-band electron paramagnetic resonance (EPR) spectral
analysis, this indicates that the g = 4.1 EPR signal
corresponds to an S = 5/2 form
of the WOC with W1 present as a water ligand to Mn4, while
the g = 4.8/4.9 form observed at high pH values corresponds
to an S = 7/2 form, with W1
as a hydroxo ligand. The latter is also likely to represent the form
needed to progress to S3 in the functioning enzyme.
Selective 15N isotope labeling of the cytochrome bo3 ubiquinol oxidase from E. coli with auxotrophs was used to characterize the hyperfine couplings with the side-chain nitrogens from R71, H98, and Q101 residues and peptide nitrogens from R71 and H98 residues around the semiquinone (SQ) at the high-affinity QH site. The 2D ESEEM (HYSCORE) data have directly identified the Nε of R71 as an H-bond donor carrying the largest amount of the unpaired spin density. In addition, weaker hyperfine couplings with the side-chain nitrogens from all residues around the SQ were determined. These hyperfine couplings reflect a distribution of the unpaired spin density over the protein in the SQ state of the QH site and strength of interaction with different residues. The approach was extended to the virtually inactive D75H mutant, where the intermediate SQ is also stabilized. We found that the Nε from a histidine residue, presumably H75, carries most of the unpaired spin density instead of the Nε of R71, as in the wild-type bo3. However, the detailed characterization of the weakly coupled 15Ns from selective labeling of R71 and Q101 in D75H was precluded by overlap of the 15N lines with the much stronger ~1.6 MHz line from quadrupole triplet of the strongly coupled 14Nε from H75. Therefore, a reverse labeling approach, in which the enzyme was uniformly labeled except for selected amino acid types, was applied in order to probe the contribution of R71 and Q101 to the 15N signals. Such labeling has shown only weak coupling with all nitrogens of R71 and Q101. We utilize density functional theory based calculations to model the available information about 1H, 15N and 13C hyperfine couplings for the QH site and to describe the protein-substrate interactions in both enzymes. In particular, we identify the factors responsible for the asymmetric distribution of the unpaired spin density and ponder the significance of this asymmetry to the quinone’s electron transfer function.
A new
paradigm for the high- and low-spin forms of the S2 state
of nature’s water-oxidizing complex in Photosystem
II is found. Broken symmetry density functional theory calculations
combined with Heisenberg–Dirac–van Vleck spin ladder
calculations show that an open cubane form of the water-oxidizing
complex changes from a low-spin, S = 1/2, to a high-spin, S = 5/2, form on protonation of the bridging O4 oxo. We
show that such models are fully compatible with structural determinations
of the S2 state by X-ray free-electron laser crystallography
and extended X-ray absorption fine structure and provide a clear rationale
for the effect of various treatments on the relative populations of
each form observed experimentally in electron paramagnetic resonance
studies.
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