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The 33 kDa manganese stabilizing protein (MSP) has been proposed to provide ligands to stabilize Mn ions in the water lysis reaction of photosystem II of photosynthesis. In previous research site-directed mutagenesis had been performed on regions of the psbO gene encoding two aspartic acid residues of MSP which were thought to have the potential to form carboxyl bridges with Mn ions. The purpose of this research was to analyze these mutants. Plasmids pUC120-33 (#1,3,5,7,9,11,15) containing mutant psbO genes could not be isolated from E.coli because the expressed MSP was toxic to the cells. However, a psbO mutant gene carried in pPGV5-33 (#7) was isolated from E.coli and transformed into cyanobacterium Svnechococcus PCC 7942. Cyanobacterial cells carrying the MSP mutant showed a susceptibility to intensive light (100 footcandles) with a decrease of 30% in the growth rate within the first 100 hours after inoculation. This result suggested a possible function of the MSP in protecting the oxygen evolving complex from intensive light exposure. However, the mutant appeared to revert after this time probably due to homologous gene recombination with the wild type gene. In order to further analyze the function of mutants without recombination occurring, the construction of an MSP deletion was attempted using insertion of a kanamycin cartridge into the middle of the psbO gene. The inactivated psbO gene was transformed into E.coli and transformants were selected by kanamycin resistance. However, plasmid DNA carrying the interrupted genes could not be isolated, probably due to toxicity of the expression product in E.coli cells. Thus, future studies should be directed to reconstruction of a deletion mutant by direct transformation into cyanobacterial cells. Once a deletion mutant has been constructed analyses of the site-directed mutations could be performed in cyanobacteria. / Department of Biology

Our view of phytoplankton has historically revolved around their inability to control their location in space. The term phytoplankton itself underscores this particular difference between phytoplankton and their sessile terrestrial counterparts. Yet there are other differences between land plants and the phytoplankton that are perhaps equally important, beyond this sessile-planktonic dichotomy, to their growth, survival, and productivity. For example, phytoplankton are microbes and thus are short-lived, with generational scales on the order of days or less. An intriguing question to ask is how today���s pelagic ecology would differ, had this temporal difference between plants and phytoplankton been initially emphasized, perhaps by naming these microbes phytoephemera instead? This dissertation addresses certain aspects of the ecology of phytoplankton that result from their having short generational scales. Because they are so short lived, phytoplankton need to adjust their photosynthetic physiology to cope with more rapid changes in irradiance than may matter to longer-lived plants. Photoacclimation on the hours-plus time scales has been studied extensively in the phytoplankton, because its temporal scales match those of vertical mixing processes in the ocean. Yet most phytoplankton exhibit faster photosynthetic responses as well, down to the time scales of seconds. These photosynthetic responses have received considerably less attention in phytoplankton ecology. This dissertation specifically examines these rapid, seconds-to-hour scale photosynthetic responses in phytoplankton. First, the physiological bases of rapid photosynthetic regulation were examined using a numerical model that shows how specific physiological changes in phytoplankton photosystems either constrain or enhance light harvesting. This model is stochastic, and thus replicates certain nonlinear aspects of light harvesting better than equation-based analytical models. Also in this dissertation, a laboratory study is described that examined rapid photosynthetic regulation in three model phytoplankton. Results suggest that rapid photosynthetic regulation is not only constrained to higher eukaryotic phytoplankton, but also occurs in the two dominant marine photosynthetic prokaryotes, Synechococcus and Prochlorococcus. Finally, rapid photosynthetic responses were examined in field assemblages at Station ALOHA in the North Pacific. This ocean region experiences considerable cloud cover, which may result in a strong degree of rapid photosynthetic responses, even in near-surface assemblages. / Graduation date: 2007

Rhodobacter capsulatus is a Gram negative bacterium that exhibits a variety of growth modes, including chemoheterotrophic growth and photoheterotrophic growth. Upon a shift of cultures from high to low oxygen concentrations the photosynthetic apparatus is synthesized and incorporated into the inner membrane. The puf operon contains genes that encode structural proteins found in the light-harvesting and reaction center complexes. In a preliminary attempt to pinpoint the location of the puf promoter R. capsulatus RNA polymerase was purified by standard techniques and used in in vitro runoff transcription assays. It was found that the polymerase was capable of specific transcription with linearized pUC13 DNA but no specific transcription could be obtained with K capsulatus DNA. It was concluded that some factor or condition necessary for specific transcription with R capsulatus DNA was absent from these assays. The location of the puf promoter was subsequently found through a series of deletions and oligonucleotide-directed mutations in the 5' region of the puf operon. Fragments that contained these mutations were placed translationally in-frame with the lacZ gene of Escherichia coli in plasmids that could be conjugated into K capsulatus. Assays of beta-galactosidase activities under low and high oxygen conditions resulted in localization of the promoter to a position approximately 540 basepairs upstream of what was previously believed to be the first gene of the operon, the pufB gene. RNA 5' end-mapping experiments showed that the quantity of RNA transcripts obtained were comparable to the lacZ activities. The existence of multiple low abundance RNA 5' ends prompted the theory that the primary transcript has a short half-life, and is rapidly processed to yield a more stable transcript with a 5' end that maps just upstream of the pufB gene. It was found that only the 5' end nearest to the promoter could be capped by guanylyl transferase, and this could only be detected when the putative processing sites were deleted. The DNA sequence between the promoter and the pufB gene contains a new gene of the puf operon, the pufO gene. Deletion of this gene showed that it plays an essential role in the formation of mature light-harvesting and reaction center complexes. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Multimodular designs of electron donor-acceptor systems are the ultimate strategy in fabricating antenna-reaction center mimics for artificial photosynthetic applications. The studied photosystems clearly demonstrated efficient energy transfer from the antenna system to the primary electron donor, and charge stabilization of the radical ion pair achieved with the utilization of secondary electron donors that permits either electron migration or hole transfer. Moreover, the molecular arrangement of the photoactive components also influences the route of energy and electron transfer as observed from the aluminum(III) porphyrin-based photosystems. Furthermore, modulation of the photophysical and electronic properties of these photoactive units were illustrated from the thio-aryl substitution of subphthalocyanines yielding red-shifted Q bands of the said chromophore; hence, regulating the rate of charge separation and recombination in the subphthalocyanine-fullerene conjugates. These multicomponent photosystems has the potential to absorb the entire UV-visible-NIR spectrum of the light energy allowing maximum light-harvesting capability. Furthermore, it permits charge stabilization of the radical ion pair enabling the utilization of the transferred electron/s to be used by water oxidizing and proton reducing catalysts in full-scale artificial photosynthetic apparatuses.

Catalysis is an important part of chemistry. This is also reflected in the chemical industry where 85-90 % of all products are made catalytically. Also nature employs catalysts, i.e. enzymes, for its reactions. To improve on the already existing catalysts one can learn a lot from nature which often uses earth-abundant elements in the enzymes which have also been optimized and finely tuned for billions of years. To gain a deeper understanding of both enzymatic and artificial catalysis one needs to investigate the mechanism of the catalytic process. But for very efficient catalysts with turnover frequencies of several thousand per second this is not easy, since an investigation of the mechanism involves resolving intermediates in the catalytic cycle. The intermediates in these instances are short-lived corresponding to their turnover frequencies. A maximum turnover frequency of 1,000 s-1 e.g. means that each catalyst goes through the whole catalytic cycle in 1 ms. Therefore time-resolved techniques are necessary that have a faster detection speed than the turnover frequency of the catalyst. Flash photolysis is a spectroscopic technique with an instrument response function down to 10 ns.  Coupling this technique with mid-infrared probing yields an excellent detection system for probing different redox and protonation states of carbonyl metal complexes. Since many catalysts as well as natural enzymes involved in water splitting are metal carbonyl complexes this is an ideal technique to monitor the intermediates of these catalysts. Chapter 3 covers the investigation of [FeFe] hydrogenases, enzymes that catalyze the reduction of protons to hydrogen in nature. Chapter 4 investigates the intermediates of biomimetic complexes, resembling the active site of natural [FeFe] hydrogenases. Chapter 5 covers the insights gained from investigating other catalysts which are also involved in water splitting and artificial photosynthesis.

The experiment was conducted at the University of Arizona Campus Agricultural Center to evaluate the effect of soil moisture stress on photosynthesis, transpiration, diffusive resistance, temperature differential, leaf temperature, and specific leaf weight of seven sorghum Sorghum bicolor (L.) Moench cytoplasms represented by nine lines. As soil moisture stress increased, diffusive resistance and leaf temperature increased whereas photosynthesis and transpiration decreased. Temperature differential was highest under high soil moisture stress and lowest under medium soil moisture stress. Specific leaf weight was highest under medium soil moisture stress. Three lines, AKS37, AKS38, and A2Tx398, representing two different germplasms under high soil moisture stress exhibited high photosynthesis and transpiration rates, high specific leaf weights, and low diffusive resistance. Differences in photosynthesis rates under non-soil moisture stress between A1 and A2 cytoplasmic sterility systems were significant.

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