X-ray absorption near-edge structure spectra of the manganese (Mn) cluster in physiologically native intermediate states of photosynthetic water oxidation induced by short laser flash were measured with a compact heat-insulated chamber equipped with an x-ray detector near the sample surface. The half-height energy of the Mn Kedge showed a period-four oscillation dependent on cycling of the Joliot-Kok's oxygen clock. The flash number-dependent shift in the Mn K-edge suggests that the Mn cluster is oxidized by one electron upon the S(0)-to-S(1), S(1)-to-S(2), and S(2)-to-S(3) transitions and then reduced upon the S(3)-to-S(0) transition that releases molecular oxygen.

The D1 protein of the photosystem II (PSII) reaction center has a rapid turnover and is specifically degraded under illumination in vivo. When isolated PSII membranes were treated in darkness with 10 mM hydrogen peroxide (H2O2), an active form of oxygen that is generated at the acceptor side of PSII under illumination, proteins of the PSII reaction center were specifically damaged in almost the same way as observed under illumination with strong light. The D1 protein and, to a lesser extent, the D2 protein were degraded to specific fragments, and cross-linked products (the covalently linked adduct of the D1 protein and the alpha subunit of cytochrome b559 and the heterodimer of the D1 and D2 proteins) were generated concomitantly. The site of cleavage of the D1 protein that gave rise to a major fragment of 22 kDa was located in the loop that connects membrane-spanning helixes IV and V. Treatment with H2O2 caused the same damage to proteins in isolated thylakoids and in core complexes that contained the non-heme iron at the acceptor side, but not in isolated reaction centers depleted of the iron. From these observations and the effects of reagents that are known to interact with the non-heme iron, it is suggested that the damage to proteins is caused by oxygen radicals generated by the non-heme iron in the Fe(II) state in a reaction with H2O2. It is proposed, moreover, that a similar mechanism is operative during the selective and specific degradation of the D1 protein under illumination.

. In order to assign LHCI proteins in the thylakoid membranes, the PSI-LHCI supercomplex that retains all of the major LHCI proteins was purified. Seven distinct LHCI proteins were resolved from the purified supercomplex by a high-resolution SDS polyacrylamide gel electrophoresis, and their N-terminal amino acid sequences were determined. One LHCI protein (band e) was newly found, although the other six LHCI proteins corresponded to those previously reported. Genomic clones encoding these seven LHCI proteins were newly isolated and the nucleotide sequences were determined. A comprehensive characterization of all members of Lhc gene family in this alga revealed that LHCI proteins are more highly diverged than LHCII, suggesting functional differentiation of the protein components in LHCI. Neighbor joining trees were constructed for LHC proteins from C. reinhardtii and those of Arabidopsis thaliana or Galdieria sulphuraria to assess evolutionary relationships. Phylogenetic analysis revealed that (1) green algal LHCI and LHCII proteins are more closely related to one another than to LHCI proteins in red algae, (2) green algae and higher plants possess seven common lineages of LHC proteins, and (3) Type I and III LHCI proteins are conserved between green algae and higher plants, while Type II and IV are not. These findings are discussed in the context of evolution of multiple diverse antenna complexes.

Thiocyanate hydrolase (SCNase) purified from Thiobacillus thioparus THI115 hydrolyzes thiocyanate to carbonyl sulfide and ammonia. DNA sequences of the cloned genes revealed the close relation of SCNase to nitrile hydratase (NHase). The consensus sequences for coordination of the metal ion found in NHases were also conserved in the gamma subunit of SCNase. Here, we showed that the SCNase contained one cobalt atom per alphabetagamma heterotrimer. UV-vis absorption spectrum suggested that the cobalt exists as a non-corrin ion. Reduced SCNase showed an ESR signal characteristic of low-spin Co2+, which closely resembled that of the Co-type NHases. Mass spectrometry for the peptide fragment containing the metal-binding motif of the SCNase gamma subunit indicated that the cysteine residue at position 131 was post-translationally oxidized to a cysteine-sulfinic acid. From these results, we concluded that SCNases and NHases form a novel non-corrin and/or non-heme protein family having post-translationally modified cysteine ligands.