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The catalytic site of photosynthetic water oxidation, the Mn4CaO5cluster, in photosystem II (PSII) is known to be formed by a light-induced process called photoactivation. However, details of its molecular mechanism remain unresolved. In this study, we monitored the photoactivation process in cyanobacterial PSII using rapid-scan, time-resolved Fourier transform infrared (FTIR) spectroscopy. The Mn3+/Mn2+FTIR difference spectra of PSII, in which D1-D170 was specifically 13C labeled, and PSII from the D1-D170A, D1-E189A, and D1-D342A mutants provide strong evidence that the initial Mn2+is coordinated by D1-D170 and D1-E189. Protein conformational changes and relocation of photo-oxidized Mn3+in the dark rearrangement process were detected as slow-phase signals in the amide I and carboxylate regions, whereas similar signals were not observed in D1-E189A PSII. It is thus proposed that relocation of Mn3+via D1-E189 induces the conformational changes of the proteins to form proper Mn binding sites in the mature protein conformation.

Photosynthetic water oxidation takes place at the Mn4CaO5cluster in photosystem II. Although the atomic structures of its intermediates called S states have recently been reported, the catalytic mechanism of water oxidation has not been well understood. Here, to investigate the involvement of the O4 site of the Mn4CaO5cluster and a water channel from O4 in the water oxidation reaction, we examined the effects of D1-S169A mutation, which perturbs the interaction of a water molecule hydrogen-bonded with O4, by thermoluminescence (TL), Fourier transform infrared (FTIR) difference, and time-resolved infrared (TRIR) measurements. The observed upshifts of TL peaks and some changes in FTIR spectra upon S169A mutation revealed the perturbations of the redox potential of the Mn4CaO5cluster and the interactions of the surrounding hydrogen bond network. In contrast, FTIR oscillation patterns and TRIR traces showed only minor effects of the mutation on the efficiencies and kinetics of individual S-state transitions. It was thus concluded that the O4 site plays a role in retaining the redox potential and the structure of the hydrogen bond network, whereas it is unlikely to be directly involved in the catalytic reaction of substrate water except for proton transfer through the O4 water chain.

The amino acid sequences of 25 archaeal retinal proteins from 13 different strains of extreme halophiles were analyzed to establish their molecular phylogenetic relationship. On the basis of amino acid sequence similarity, these proteins apparently formed a distinct family designated as the archaeal rhodopsin family (ARF), which was not related to other known proteins, including G protein-coupled receptors. The archaeal rhodopsin family was further divided into four clusters with different functions; H+pump (bacteriorhodopsin), Cl−pump (halorhodopsin), and two kinds of sensor (sensory rhodopsin and phoborhodopsin). These four rhodopsin clusters seemed to have occurred by gene duplication(s) before the generic speciation of halophilic archaea, based on phylogenetic analysis. Therefore, the degrees of differences in amino acid sequences within each cluster simply reflected the divergent evolution of halophilic archaea. By comparing the branch lengths after speciation points of the reconstituted tree, we calculated the relative evolution rates of the four archaeal rhodopsins bacteriorhodopsin:halorhodopsin:sensory rhodopsin: phoborhodopsin to be 5:4:3:10. From these values, the degrees of functional and structural restriction of each protein can be inferred. The branching topology of four clusters grouped bacteriorhodopsin and halorhodopsin versussensory rhodopsin and phoborhodopsin by likelihood mapping. Using bacteriorhodopsin (and halorhodopsin) as an outgroup, the gene duplication point of sensory rhodopsin/phoborhodopsin was determined. By calculating the branch lengths between the gene duplication point and each halophilic archaea speciation point, we could speculate upon the relative evolution rate of pre-sensory rhodopsin and pre-phoborhodopsin. The evolution rate of pre-sensory rhodopsin was fivefold faster than that of pre-phoborhodopsin, which suggests that the original function of the ancestral sensor was similar to that of phoborhodopsin, and that sensory rhodopsin evolved from pre-sensory rhodopsin by the accumulation of mutations. The changes in evolution rate by gene duplication and functional differentiation were demonstrated in the archaeal rhodopsin family using the gene duplication date and halobacterial speciation date as common time stamps.

The NDI1gene encodes the internal rotenone‐insensitive NADH‐quinone oxidoreductase localized in the inner mitochondrial membranes of Saccharomyces cerevisiae. The T7 tag‐fused mature NDI1 was overexpressed in Escherichia coli. The overexpressed NDI1 was exclusively found in the membrane fraction. The NDI1‐overexpressed membranes showed significantly increased activities of NADH oxidase and NADH‐ubiquinone‐1 (UQ1) reductase when compared with the control membranes. Flavone, which is a specific inhibitor of the S. cerevisiaeNDI1, inhibited almost completely NADH oxidase and NADH‐UQ1reductase activities of NDI1‐overexpressed membranes but scarcely inhibited these activities of the control membranes. In addition, the NADH oxidase activity of the NDI1‐overexpressed membranes was also inhibited by KCN as well as the control membranes. These results indicate that the overexpressed NDI1 worked as a member of the respiratory chain in the host cells, even though E. colimembranes are different from S. cerevisiaeinner mitochondrial membranes in terms of quinones and lipid composition.

The NDI1gene encodes the internal rotenone-insensitive NADH-quinone oxidoreductase localized in the inner mitochondrial membranes of Saccharomyces cerevisiae. The T7 tag-fused mature NDI1 was overexpressed in Escherichia coli. The overexpressed NDI1 was exclusively found in the membrane fraction. The NDI1-overexpressed membranes showed significantly increased activities of NADH oxidase and NADH-ubiquinone-1 (UQ 1) reductase when compared with the control membranes. Flavone, which is a specific inhibitor of the S. cerevisiaeNDI1, inhibited almost completely NADH oxidase and NADH-UQ 1reductase activities of NDI1-overexpressed membranes but scarcely inhibited these activities of the control membranes. In addition, the NADH oxidase activity of the NDI1-overexpressed membranes was also inhibited by KCN as well as the control membranes. These results indicate that the overexpressed NDI1 worked as a member of the respiratory chain in the host cells, even though E. colimembranes are different from S. cerevisiaeinner mitochondrial membranes in terms of quinones and lipid composition.