Light-induced generation of superoxide radicals and hydrogen peroxide in isolated thylakoids has been studied with a lipophilic spin probe, cyclic hydroxylamine 1-hydroxy-4-isobutyramido-2,2,6,6-tetramethylpiperidinium (TMT-H) to detect superoxide radicals, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitron (4-POBN) to detect hydrogen peroxide-derived hydroxyl radicals. Accumulation of the radical products of the above reactions has been followed using electron paramagnetic resonance. It is found that the increased production of superoxide radicals and hydrogen peroxide in higher light is due to the enhanced production of these species within the thylakoid membrane, rather than outside the membrane. Fluorescent probe Amplex red, which forms fluorescent product, resorufin, in the reaction with hydrogen peroxide, has been used to detect hydrogen peroxide outside isolated chloroplasts using confocal microscopy. Resorufin fluorescence outside the chloroplasts is found to be suppressed by 60% in the presence of the inhibitor of aquaporins, acetazolamide (AZA), indicating that hydrogen peroxide can diffuse through the chloroplast envelope aquaporins. It is demonstrated that AZA also inhibits carbonic anhydrase activity of the isolated envelope. We put forward a hypothesis that carbonic anhydrase presumably can be attached to the envelope aquaporins. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.
Recent electron paramagnetic resonance (EPR)
experiments (Proskuryakov, I. I.; Klenina, I. B.; Hore, P.
J.;
Bosch, M. K.; Gast, P.; Hoff, A. J. Chem. Phys.
Lett. 1996, 257, 333) on the primary
radical pair in bacterial
reaction centers are interpreted in detail. The spin-correlated
radical pair model is extended to include the
time dependence of the EPR intensities, differential lifetime
broadening of the basic four-line spectrum, and
averaging over the inhomogeneous distributions of hyperfine
interactions in the two radicals. Values of the
singlet and triplet recombination rate constants and the magnitude and
sign of the exchange interaction of the
radical pair are obtained from the shape and linewidth of the
spin-polarized EPR spectra and the kinetics of
formation of the triplet state of the primary electron donor. For
Rhodobacter sphaeroides at 70 K, k
S
= (1.2
± 0.3) × 107 s-1,
k
T = (5.7 − 8.8) × 108
s-1, −J = 8 − 6 G; and for
Rhodopseudomonas viridis at 190 K,
k
S = (8 ± 2) × 107
s-1, k
T = (6.5 − 10.0) ×
108 s-1, −J = 17 − 16 G.
The reliability of these values is
discussed in the light of the energetic heterogeneity of the radical
pair energies (Ogrodnik, A.; Keupp, W.;
Volk, M.; Aumeier, G.; Michel-Beyerle, M. E. J.
Phys. Chem. 1994, 98,
3432−3439) and of the sensitivity
and selectivity of the EPR measurements toward the three
parameters.
The first time-resolved magnetophotoselection study of the primary donor triplet state ( 3 P) in bacterial reaction centers from Rhodobacter sphaeroides R26 and Rhodopseudomonas Viridis is reported. With direct excitation of the primary donor, this approach provides the orientation (spherical coordinates δ, colatitude, and γ, longitude) of the excited optical transition moment in the principal triplet axis system. It is essentially free from the effects of spin-lattice relaxation of 3 P, enabling magnetophotoselection measurements over a wide temperature range. Two independently measured triplet-state spectra, excited with light polarized parallel and perpendicular to the EPR magnetic field, are simulated with the same set of parameters. This procedure results in a high precision (ca. (5°with sufficient signal/noise ratio) of the obtained spherical coordinates of the optical transition moment vector. We find δ ) 80 ( 5°, γ ) 70 ( 5°and δ ) 75 ( 5°, γ ) 70 ( 5°for Rb. sphaeroides R26 and Rps. Viridis, respectively. We demonstrate that excitation of the sample with nonpolarized light is essentially nonisotropic. Neglect of this effect in spectral simulations of light-induced signals may lead to considerable error in the parameters determined.
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