Photodamage to Oxygen Evolving Complex - An Initial Event in Photoinhibition of Photosystem II.

Photosystem II (PSII) of oxygenic photosynthesis is susceptible to photoinhibition. Photoinhibition is defined as light induced damage resulting in turnover of the D1 protein subunit of the reaction center of PSII. Both visible and ultraviolet (UV) light cause photoinhibition. Photoinhibition induced by UV light damages the oxygen evolving complex (OEC) via absorption of UV photons by the Mn ion(s) of OEC. Under visible light, most of the earlier hypotheses assume that photoinhibition occurs when the rate of photon absorption by PSII antenna exceeds the use of the absorbed energy in photosynthesis. However, photoinhibition occurs at all light intensities with the same efficiency per photon. The aim of my thesis work was to build a model of photoinhibition that fits the experimental features of photoinhibition. I studied the role of electron transfer reactions of PSII in photoinhibition and found that changing the electron transfer rate had only minor influence on photoinhibition if light intensity was kept constant. Furthermore, quenching of antenna excitations protected less efficiently than it would protect if antenna chlorophylls were the only photoreceptors of photoinhibition. To identify photoreceptors of photoinhibition, I measured the action spectrum of photoinhibition. The action spectrum showed resemblance to the absorption spectra of Mn model compounds suggesting that the Mn cluster of OEC acts as a photoreceptor of photoinhibition under visible light, too. The role of Mn in photoinhibition was further supported by experiments showing that during photoinhibition OEC is damaged before electron transfer activity at the acceptor side of PSII is lost. Mn enzymes were found to be photosensitive under visible and UV light indicating that Mn-containing compounds, including OEC, are capable of functioning as photosensitizers both in visible and UV light. The experimental results above led to the Mn hypothesis of the mechanism of continuous-light-induced photoinhibition. According to the Mn hypothesis, excitation of Mn of OEC results in inhibition of electron donation from OEC to the oxidized primary donor P680+ both under UV and visible light. P680 is oxidized by photons absorbed by chlorophyll, and if not reduced by OEC, P680+ may cause harmful oxidation of other PSII components. Photoinhibition was also induced with intense laser pulses and it was found that the photoinhibitory efficiency increased in proportion to the square of pulse intensity suggesting that laser-pulse-induced photoinhibition is a two-photon reaction. I further developed the Mn hypothesis suggesting that the initial event in photoinhibition under both continuous and pulsed light is the same: Mn excitation that leads to the inhibition of electron donation from OEC to P680+. Under laser-pulse-illumination, another Mn-mediated inhibitory photoreaction occurs within the duration of the same pulse, whereas under continuous light, secondary damage is chlorophyll mediated. A mathematical model based on the Mn hypothesis was found to explain photoinhibition under continuous light, under flash illumination and under the combination of these two.

Type II aromatic polyketide biosynthetic tailoring enzymes: diversity and adaptation in Streptomyces secondary metabolism.

Members of the bacterial genus Streptomyces are well known for their ability to produce an exceptionally wide selection of diverse secondary metabolites. These include natural bioactive chemical compounds which have potential applications in medicine, agriculture and other fields of commerce. The outstanding biosynthetic capacity derives from the characteristic genetic flexibility of Streptomyces secondary metabolism pathways: i) Clustering of the biosynthetic genes in chromosome regions redundant for vital primary functions, and ii) the presence of numerous genetic elements within these regions which facilitate DNA rearrangement and transfer between non-progeny species. Decades of intensive genetic research on the organization and function of the biosynthetic routes has led to a variety of molecular biology applications, which can be used to expand the diversity of compounds synthesized. These include techniques which, for example, allow modification and artificial construction of novel pathways, and enable gene-level detection of silent secondary metabolite clusters. Over the years the research has expanded to cover molecular-level analysis of the enzymes responsible for the individual catalytic reactions. In vitro studies of the enzymes provide a detailed insight into their catalytic functions, mechanisms, substrate specificities, interactions and stereochemical determinants. These are factors that are essential for the thorough understanding and rational design of novel biosynthetic routes. The current study is a part of a more extensive research project (Antibiotic Biosynthetic Enzymes; www.sci.utu.fi/projects/biokemia/abe), which focuses on the post-PKS tailoring enzymes involved in various type II aromatic polyketide biosynthetic pathways in Streptomyces bacteria. The initiative here was to investigate specific catalytic steps in anthracycline and angucycline biosynthesis through in vitro biochemical enzyme characterization and structural enzymology. The objectives were to elucidate detailed mechanisms and enzyme-level interactions which cannot be resolved by in vivo genetic studies alone. The first part of the experimental work concerns the homologous polyketide cyclases SnoaL and AknH. These catalyze the closure of the last carbon ring of the tetracyclic carbon frame common to all anthracycline-type compounds. The second part of the study primarily deals with tailoring enzymes PgaE (and its homolog CabE) and PgaM, which are responsible for a cascade of sequential modification reactions in angucycline biosynthesis. The results complemented earlier in vivo findings and confirmed the enzyme functions in vitro. Importantly, we were able to identify the amino acid -level determinants that influence AknH and SnoaL stereoselectivity and to determine the complex biosynthetic steps of the angucycline oxygenation cascade of PgaE and PgaM. In addition, the findings revealed interesting cases of enzyme-level adaptation, as some of the catalytic mechanisms did not coincide with those described for characterised homologs or enzymes of known function. Specifically, SnoaL and AknH were shown to employ a novel acid-base mechanism for aldol condenzation, whereas the hydroxylation reaction catalysed by PgaM involved unexpected oxygen chemistry. Owing to a gene-level fusion of two ancestral reading frames, PgaM was also shown to adopt an unusual quaternary sturucture, a non-covalent fusion complex of two alternative forms of the protein. Furthermore, the work highlighted some common themes encountered in polyketide biosynthetic pathways such as enzyme substrate specificity and intermediate reactivity. These are discussed in the final chapters of the work.

Photoinhibition of Photosystem II. Kinetics, Photoprotection and Mechanism

Photosystem II (PSII) is susceptible to light-induced damage defined as photoinhibition. In natural conditions, plants are capable of repairing the photoinhibited PSII by on-going degradation and re-synthesis of the D1 reaction centre protein of PSII. Photoinhibition is induced by both visible and ultraviolet light and photoinhibition occurs under all light intensities with the same efficiency per photon. In my thesis work, I studied the reaction kinetics and mechanism of photoinhibition of PSII, as well as photoprotection in leaves of higher plants. Action spectroscopy was used to identify photoreceptors of photoinhibition. I found that the action spectrum of photoinhibition in vivo shows resemblance to the absorption spectra of manganese model compounds of the oxygen evolving complex (OEC) suggesting a role for manganese as a photoreceptor of photoinhibition under UV and visible light. In order to study the protective effect of non-photochemical quenching, the action spectrum was measured from leaves of wild type Arabidopsis thaliana and two mutants impaired in nonphotochemical quenching of chlorophyll a excitations. The findings of action spectroscopy and simulations of chlorophyll-based photoinhibition mechanisms suggested that quenching of antenna excitations protects less efficiently than would be expected if antenna chlorophylls were the only photoreceptors of photoinhibition. The reaction kinetics of prolonged photoinhibition was studied in leaves of Cucurbita maxima and Capsicum annuum. The results indicated that photoinhibitory decrease in both the oxygen evolution activity and ratio of variable to maximum fluorescence follows firstorder kinetics in vivo. The persistence of first-order kinetics suggests that already photoinhibited reaction centres do not protect against photoinhibition and that the mechanism of photoinhibition does not have a reversible intermediate. When Cucurbita maxima leaves were photoinhibited with saturating single-turnover flashes and continuous light, the light response curve of photoinhibition was found to be essentially a straight line with both types of illumination, suggesting that similar photoinhibition mechanisms might function during illumination with continuous light and during illumination with short flashes.

Functional studies on bacterial nucleotide-regulated inorganic pyrophosphatases

CBS domains are ~60 amino acid tandemly repeated regulatory modules forming a widely distributed domain superfamily. Found in thousands of proteins from all kingdoms of life, CBS domains have adopted a variety of functions during evolution, one of which is regulation of enzyme activity through binding of adenylate-containing compounds in a hydrophobic cavity. Mutations in human CBS domain-containing proteins cause hereditary diseases. Inorganic pyrophosphatases (PPases) are ubiquitous enzymes, which pull pyrophosphate (PPi) producing reactions forward by hydrolyzing PPi into phosphate. Of the two nonhomologous soluble PPases, dimeric family II PPases, belonging to the DHH family of phosphoesterases, require a transition metal and magnesium for maximal activity. A quarter of the almost 500 family II PPases, found in bacteria and archaea, contain a 120-250 amino acid N-terminal insertion, comprised of two CBS domains separated in sequence by a DRTGG domain. These enzymes are thus named CBS-PPases. The function of the DRTGG domain in proteins is unknown. The aim of this PhD thesis was to elucidate the structural and functional differences of CBS-PPases in comparison to family II PPases lacking the regulatory insert. To this end, we expressed, purified and characterized the CBS-PPases from Clostridium perfringens (cpCBS-PPase) and Moorella thermoacetica (mtCBS-PPase), the latter lacking a DRTGG domain. Both enzymes are homodimers in solution and display maximal activity against PPi in the presence of Co2+ and Mg2+. Uniquely, the DRTGG domain was found to enable tripolyphosphate hydrolysis at rates similar to that of PPi. Additionally, we found that AMP and ADP inhibit, while ATP and AP4A activate CBSPPases, thus enabling regulation in response to changes in cellular energy status. We then observed substrate- and nucleotide-induced conformational transitions in mtCBS-PPase and found that the enzyme exists in two differentially active conformations, interconverted through substrate binding and resulting in a 2.5-fold enzyme activation. AMP binding was shown to produce an alternate conformation, which is reached through a different pathway than the substrate-induced conformation. We solved the structure of the regulatory insert from cpCBS-PPase in complex with AMP and AP4A and proposed that conformational changes in the loops connecting the catalytic and regulatory domains enable activity regulation. We examined the effects of mutations in the CBS domains of mtCBS-PPase on catalytic activity, as well as, nucleotide binding and inhibition.

Development of an environmentally friendly method of starch oxidation by hydrogen peroxide and a complex water-soluble Iron catalyst

Oxidized starch is a key component in the paper industry, where it is used as both surfacing sizer and filler. Large quantities are annually used for this purpose; however, the methods for the oxidation are not environmentally friendly. In our research, we have studied the possibility to replace the harmful oxidation agents, such as hypochlorite or iodates and transition metal catalysts, with a more environmentally friendly oxidant, hydrogen peroxide (H2O2), and a special metal complex catalyst (FePcS), of which only a small amount is needed. The work comprised batch and semi-batch studies by H2O2, ultrasound studies of starch particles, determination of low-molecular by-products and determination of the decomposition kinetics of H2O2 in the presence of starch and the catalyst. This resulted in a waste-free oxidation method, which only produces water and oxygen as side products. The starch oxidation was studied in both semi-batch and batch modes in respective to the oxidant (H2O2) addition. The semi-batch mode proved to yield a sufficient degree of substitution (COOH groups) for industrial purposes. Treatment of starch granules by ultrasound was found to improve the reactivity of starch. The kinetic results were found out to have a rather complex pattern – several oxidation phases were observed, apparently due to the fact that the oxidation reaction in the beginning only took place on the surface, whereas after a prolonged reaction time, partial degradation of the solid starch granules allowed further reaction in the interior parts. Batch-mode experiments enabled a more detailed study of the mechanisms of starch in the presence of H2O2 and the catalyst, but yielded less oxidized starch due to rapid decomposition of H2O2 due to its high concentrations. The effect of the solid-liquid (S/L) ratio in the reaction system was studied in batch experiments. These studies revealed that the presence of the catalyst and the starch enhance the H2O2 decomposition.

Welding of additively manufactured stainless steel parts: Comparative study between sheet metal and selective laser melted parts

Additive manufacturing is a fast growing manufacturing technology capable of producing complex objects without the need for conventional manufacturing process planning. During the process the work piece is built by adding material one layer at a time according to a digital 3D CAD model. At first additive manufacturing was mainly used to make prototypes but the development of the technology has made it possible to also make final products. Welding is the most common joining method for metallic materials. As the maximum part size of additive manufacturing is often limited, it may sometimes be required to join two or more additively manufactured parts together. However there has been almost no research on the welding of additively manufactured parts so far, which means that there has been very little information available on the possible differences compared to the welding of sheet metal parts. The aim of this study was to compare the weld joint properties of additively manufactured parts to those of sheet metal parts. The welding process that was used was TIG welding and the test material was 316L austenitic stainless steel. Weld joint properties were studied by making tensile, bend and hardness tests and by studying the weld microstructures with a microscope. Results show that there are certain characteristics in the welds of additively manufactured parts. The building direction of the test pieces has some impact on the mechanical properties of the weld. Nevertheless all the welds exhibited higher yield strength than the sheet metal welds but at the same time elongation at break was lower. It was concluded that TIG welding is a feasible process for welding additively manufactured parts.