Asako Ishii

Changes in Structural and Functional Properties of Oxygen-evolving Complex Induced by Replacement of D1-Glutamate 189 with Glutamine in Photosystem II LIGATION OF GLUTAMATE 189 CARBOXYLATE TO THE MANGANESE CLUSTER

A carboxylate group of D1-Glu-189 in photosystem II has been proposed to serve as a direct ligand for the manganese cluster. Here we constructed a mutant that eliminates the carboxylate by replacing D1-Glu-189 with Gln in the cyanobacterium Synechocystis sp. PCC 6803, and we examined the resulting effects on the structural and functional properties of the oxygen-evolving complex (OEC) in photosystem II. The E189Q mutant grew photoautotrophically, and isolated photosystem II core particles evolved oxygen at ∼70% of the rate of control wild-type particles. The E189Q OEC showed typical S2 state electron spin resonance signals, and the spin center distance between the S2 state manganese cluster and the YD (D2-Tyr-160), detected by electron-electron double resonance spectroscopy, was not affected by this mutation. However, the redox potential of the E189Q OEC was considerably lower than that of the control OEC, as revealed by the elevated peak temperature of the S2 state thermoluminescence bands. The mutation resulted in specific changes to bands ascribed to the putative carboxylate ligands for the manganese cluster and to a few carbonyl bands in mid-frequency (1800 to 1100 cm-1) S2/S1 Fourier transform infrared difference spectrum. Notably, the low frequency (650 to 350 cm-1) S2/S1 Fourier transform infrared difference spectrum was also uniquely changed by this mutation in the frequencies for the manganese cluster core vibrations. These results suggested that the carboxylate group of D1-Glu-189 ligates the manganese ion, which is influenced by the redox change of the oxidizable manganese ion upon the S1 to S2 transition.

Temperature-Sensitive Reaction of a Photosensor Protein YcgF: Possibility of a Role of Temperature Sensor

The spectrally silent photoreaction of a blue light sensor protein YcgF, composed of the N-terminal BLUF domain and the C-terminal EAL domain, was investigated by the time-resolved transient grating method. Comparing photoinduced reactions of full-length YcgF with that of the BLUF-linker construct, it was found that a major conformation change after photoinduced dimerization is predominantly localized on the EAL domain. Furthermore, the photoinduced conformational change displayed significant temperature dependence. This result is explained by an equilibrium of reactive and nonreactive YcgF species, with the population of photoreactive species decreasing as the temperature is lowered in the dark state. We consider that the dimer form is the nonreactive species and it is the dominant species at lower temperatures. The temperature sensitivity of the photoreaction of YcgF suggests that this protein could have a biological function as a temperature sensor as well as behaving as a light sensor.

Nonlineal relationship between g=2 doublet and multiline signals in Ca2+-depleted Photosystem II

Illuminating of the Ca(2+)-depleted PS II in the S(2) state for a short period induced the doublet signal at g=2 with concomitant diminution of the multiline signal, both in the presence and absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). In the absence of DCMU, the doublet signal decayed (t(1/2) approximately 7 min) during subsequent dark incubation at 273 K and the multiline signal was regenerated to the original amplitude with the same kinetics of the doublet decay. In the presence of DCMU, the doublet signal decayed much faster (t(1/2) approximately 1 min) by charge recombination with Q(A)(-), while the time course of the multiline recovery was inherently identical with that observed in the absence of DCMU. A simple theoretical consideration indicates the direct conversion from the doublet-signal state to the multiline state with no intermediate state between them. Lengthy dark storage at 77 K led to disappearance of the DCMU-affected doublet signal and a Fe(2+)/Q(A)(-) electron spin resonance (ESR) signal, but no recovery of the multiline signal. Notably, the multiline signal was restored by subsequent dark incubation at 273 K. The charge recombination between Q(A)(-) and the doublet signal species led to a thermoluminescence band at 7 degrees C in a medium at pH 5.5. The peak position shifted to 17 degrees C at pH 7.0, presumably due to a pH-dependent change in the redox property of a donor-side radical species responsible for the doublet signal. Based on these results, redox events in the Ca(2+)-depleted PS II are discussed in contradistinction with the normal processes in oxygen-evolving PS II.

Functional analysis of site-directed mutants on the C-terminal Leu-343 and Ala-344 of D1 protein

Previous studies suggested that mutagenesis around the C-terminal processing site on D1 protein could have major effects on the formation and/or conformation of the oxygen-evolving complex. Some of the mutants had a very slow processing rate (Hatano-Iwasaki et al. PMB (2000) 42, 353-363) and the other mutant carried two functionally distinct Mn complexes (Hatano-Iwasaki et al. BBA (2001) 1504, 299-310). In this study, we further mutagenized Leu343 and Ala344 of D1 protein in C. reinhardtii and performed biophysical characterizations. Thermoluminescence measurements revealed that 1) the L343P(LP) and A344P(AP) mutants have modified PSII centers which have higher redox potential of the Mn complex, and 2) only the LP mutant evolves oxygen at a lower rate. Measurements of fluorescence kinetics demonstrated that 1) the LP mutant has a lower charge recombination rate which may account for the lower oxygen evolving activity, and 2) AP mutant has a very slow re-reduction rate of the oxidized P680, inhibiting the normal electron transfer reaction around the donor-side. These mutants are further being analyzed by EPR to gain detailed information about the modification(s) on the Mn complex.