Of special interest have been further investigations of alterations in this kinetic pattern that occur as the membrane complex is gradually disassembled. The disappearance of the period 4 behavior is a sensitive indicator of the loss of ability to. store oxidizing equivalents; however, under many circumstances rapid rereduction of the primary electron donor continues to occur by components that have yet to be identified. Photo system I reaction centers behave qualitatively differently from those of PS II. Instead . of the large increase in fluorescence yield seen upon closing PS II reaction centers through illumination or chemical reduction of the electron acceptors, it has been difficult to demonstrate any contribution to this variable fluorescence from PS I. Now, using isolated and enriched PS I reaction center preparations we have seen a small decrease in fluorescence yield associated with closing the reaction centers. By contrast with the behavior of PS II, this variable fluorescence’ occurs’ upon oxidation of the primary electron’ donor chlorophyll, P700. These results for both· PS I and PS II are’ consistent with, but· do not prove, a model in which the increased fluorescence yield comes from the reversal of the primary process leading to the repopulation of the chlorophyll excited singlet state following charge recombination. We are carrying out time-resolved fluorescence decay measurements to try to define the mechanism more precisely. !.r Model studies were carried out on electron donor/acceptor complexes synthesized by covalent linkage of a photo-excitable ruthenium complex to a benzoquinone. The linker consisted of oligo peptides containing zero to four prolines.

By monitoring the effect of increasing Si the physical separation of the electron donor from the electron acceptor using fluorescence yield and time-resolved fluorescence relaxation,ebb and flow tray we could follow the competing electron transfer in the range from 10-9 to 10-6 sec. It is clear from these studies, carried out by Miller Research Institute Fellow Dr. Kirk Schanze, that electron transfer can occur rapidly and efficiently across longer non-aromatic bridging groups than had been suspected. In ongoing studies using kinetic EPR spectroscopy we will investigate the lifetime of the charge-separated species that simulates the initial stages of photosynthetic charge separation. Much evidence has now accumulated that the storage of the four oxidizing equivalents needed to produce 02 from water photosynthetically occurs in a complex containing four manganese atoms. The nature of this complex, which is presumed to involve interaction with a protein environment, is still unknown. Even the kinetics is in dispute. Optical spectroscopic changes have been interpreted to result from three successive one-electron oxidations from Mn+3 to Mn+4 prior to O2 release, whereas EPR, X-ray spectroscopy and other approaches favor a mechanism with different oxidation state changes for the three preliminary steps. We have used X-ray absorption and EXAFS, in conjunction with low temperature EPR measurements to investigate these questions. Absorption edge energy studies on Mn are suggestive that, So, the most reduced state of the complex~ has the .same oxidation state for Mn as Sl, where one electron has been removed by the PS II reaction center. Presumably this electron has come from an associated ligand that does not directly involve Mn. The second step, from Sl to S2, definitely involves Mn oxidation, perhaps by two equivalents transferredone to the reaction center and one to rereduce the ligand. The third step, from S2 to S3, again . involves no apparent change in the Mn oxidation state, but the final step S3 to S4 and back to So returns the complex back to the fully reduced state. The state S4, which should be the most oxidized but is unstable, has not yet been trapped for investigation.

EXAFS measurements give information about the local coordination environment of the Mn in the water splitting complex. Two of the three shells previously identified for Sl are attributed to 0 or N atoms, and the third shell to Mn atoms. Further enhancement of signal quality has revealed a fourth shell, apparently also resulting from Mn. A possible model involving a distorted cubic arrangement with Mn and either 0 or N atoms at alternate comers has been proposed by Brudvig and Crabtree [Proc. Natl. Acad. Sci. USA 83, 4586-4588 ] to account for these and other spectroscopic data. We find that the coordination in Sl does not differ significantly from that in S2, by the EXAFS criteria; however, analogous investigations of S3 are still under way. Previous proposals that sulfur or chlorine atoms are involved as Mn ligands are not consistent with the EXAFS data. We have also shown that resolved super hyperfine structure of a low temperature EPR signal associated with S2 does not result from interactions with CI nuclei in the immediate coordination environment. Until the successful isolation of the water-splitting complex has been accomplished, further spectroscopic studies such as these will be our principal source of information about the enzyme that accomplishes this important aspect of photosynthesis. The information gained is expected to be valuable for designing bio-mimetic systems for using light to split water into hydrogen and oxygen as alternative energy sources. It is of interest that the genes for Crt biosyntheis are regulated differently from the other photosynthetic’ genes in response to light intensity and O2 • We have found that under steady-state high O2 the level of mRNA from the Bam Ill-H fragment was relatively high and it increased when the cells were shifted from anaerobic to aerobic conditions. We have evidence suggesting that crt A in the Bam HI-H is activated by O2 • This gene is responsible for the oxidation of spheroidene to spheroidenone. Such regulation in response to O2 may be related to the function of Crt. It has been long known that Crt has two functions: harvesting light and protecting cells including the photosynthetic apparatus from photooxidative damage which only occurs in presence of both high light and O2 • Activation of crt A and other Crt genes by O2 may be part of the protective mechanism by which Crt scavenges O2 radicals in the cell.

Here we have also found another type of protective mechanism in response to high light. That is, in contrast to the decrease in the levels of mRNA for LH, RC and Bchl biosynthesis, an increase in light intensity raised the levels of mRNA from a number of crt genes located in the Bam HI-M, Bam Ill-G and Bam Ill-H fragments. Although the increase in transcription of crt genes in response to high light seems to be a plausible protective mechanism, we do not rule out the possibility that regulation may also occur post-transcriptionally, including the activation of Crt biosynthetic enzymes by light and O2 • The 0.4 kb small transcript from the Bam Ill-I also showed a response to high light in the opposite fasion to the mRNAs for LH and RC. It did not hybridize to either regions genetically mapped as crt F or crt E . The response of this transcript to O2 is very similar to that of mRNAs for LH and RC, but is very different from that of mRNAs for Crt biosynthesis. On the other hand, transcript level increases in response to high light as do the mRNAs for Crt biosynthesis except to a greater extent, but it is very different from that ‘of mRNAs for LH and RC in this respect.Other interesting results in this study are the finding of multiple transcripts for the RC-H as shown by Northern hybridization. The 1.2 and 1.4 kb transcripts of the RC-H gene, 764 bp in size, are probably initiated from the middle of the ORF F1696. We do not know whether they have different initiation sites,4×8 flood tray or if the 1.2 kb transcript is the product of processing of the 1.4 kb transcript. The relatively long 5′ non-coding region may contain important regulatory sequences. Assay for transcripts from all of the ORF putative genes near the LH and RC gene clusters in the Bam Ill-C -EcoRI-B, and Bam Ill-F fragments resulted in the detection of mRNA from only C2397. We suggest that the putative genes in both fragments are either not expressed under the define growth conditions, or their mRNAs, if present, are below the limits of detection by our methods. Our research has been directed towards the understanding of the mechanism controlling the coordinate expression of genes encoding photosynthetic components. While the photosynthetic processes are primarily carried out in the chloroplasts of eucaryotic photosynthetic cells, the genes encoding the components are distributed into both the nuclear and the chloroplast genome. The coordination of expression of these physically separated genomes is the subject of our investigations. We are determining if the chloroplast or its encoded components play a role in the light response of nuclear genes whose produc.ts· are involved in photosynthesis. The photosynthetic apparatus is a highly organized and specialized structure made up of many components. The activity of the system is dependent on the activity and arrangement of the individual components. The cell must balance the production of the individual components to optimize the photosynthetic activity of the system.

This balancing act is not simply a maintainence of equal numbers of all components since some components in the apparatus are needed in great excess over others. Also, control must be exerted on the production of these components as a function of time. For example, the production of Light Harvesting Chlorophyll Binding Protein must not begin before chlorophyll biosynthesis since the protein is rapidly destroyed in the absence of chlorophyll. Even though the chloroplasts of photosynthetic organisms contain their own genome, many of the functions carried out in the organelle are encoded in the nucleus. Some of the proteins which function in chloroplast are encoded partially in the chloroplast and partially in the nucleus. The expression of both these genomes is regulated by light. Light stimulates mRNA synthesis as well as DNA synthesis in Euglena gracilis. Presumably” this light response is mediated by photo pigments in the cells which receive the light and convert it into a signal which ‘control nucleic acid activity in both genome compartments. We are studying a series of bleached mutants of Euglena which lack various photo pigments as well as have different levels of chloroplast DNA. Using these variants, we hope to determine the involvement of the photo pigments and chloroplast DNA in the stimulation of nuclear DNA and RNA synthesis. First, we are characterizing the cell cycle response to light of these various mutants by flow cytometry. Wild type Euglena conditioned in the resting medium in the dark can be stimulated to reenter the cell cycle by light. We are comparing the kinetics of reentry into the cell cycle of the bleached mutants with that of the wild type to discern the role of the photo pigments and chloroplast supplied functions in regulating the DNA replication response in the nucleus. We are also comparing the light stimulated expression of nuclear encoded chloroplast functions in wild type vs bleached mutants to determine if the chloroplast and/or the missing photo pigments play a role in the regulation of the nuclear genes. Can nuclear genes coding for photosynthetic components be regulated by light in the absence of functional chloroplasts? At present we are using heterologous probing to clone the nuclear genes for the light harvesting chlorophyll alb proteins in Euglena. We have been testing conditions that will allow plant protein-DNA complex formation. Tobacco root and leaf protein extracts have been tested for their DNA-binding capacities after • _ separation of the proteins by electrophoresis and transfer to a nitrocellulose membrane. We have seen a number of proteins which show DNA-binding capacities, and have noted large differences in the DNA-binding pattern of extracts from the two tissues. We are presently characterizing the nature of these proteins and assessing the contribution of chloroplast DNA-binding proteins to the binding pattern observed in the case of the leaf extracts. Even if a major contribution in this difference comes from the leaf chloroplastic proteins, the two smaller MW proteins present only in the root extract represent a true difference in the DNA binding patterns of the proteins from root and leaf tissue.