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.

Kaskadinė fermentinė reakcija yra tokia cheminių reakcijų grandinė, kai vienos fermentinės reakcijos produktas yra panaudojamas kitose fermentinėse reakcijose tol, kol gaunamas galutinis rezultatas. Tokių reakcijų pavyzdžiai gamtoje yra kraujo krešėjimo reakcija, celiulozės skaidymas bei signalų perdavimas neuronuose. Norint, kad kaskadinė fermentinė reakcija vyktų efektyviai, fermentai, reikalingi reakcijai vykti, turi būti išsidėstę taip, kad po kiekvieno reakcijos etapo tarpinis produktas efektyviai pasiektų kitą reakcijai reikalingą fermentą. Tokių reakcijų efektyvumą galima bandyti pagerinti sutelkiant visus reikalingus reakcijai fermentus šalia vienas kito. Vienas iš variantų kaip būtų galima sukurti daugiafermentį kompleksą yra panaudojant segmentuotus metalinius nanostrypelius kaip koduojančią matricą bei genetiškai modifikuotas fermentų molekules. Prie fermentų molekulių būtų prijungiamos dideli giminiškumą reikalingam nanostrypelio metaliniam segmentui turinčios oligopeptidinės uodegėlės, kurios sukurtų sąlygas fermentams savaime organizuotis ant segmentuoto nanostrypelio paviršiaus. Magistrinio darbo užduotys buvo charakterizuoti susintetintus nanostrypelius, patikrinti ar modifikuotas giminiškomis sidabrui peptidinėmis uodegėlėmis streptavidinas sugeba prisijungti biotiną bei palyginti modifikuoto ir ir nemodifikuoto streptavidino giminiškumą sidabro paviršiui. Atlikus eksperimentus buvo nustatyta, kad naudojantis atominės jėgos mikroskopija nepavyko patikimai... [toliau žr. visą tekstą] / Biochemical enzyme cascade is a series of chemical reactions in which the products of one reaction are consumed in the next reaction. If one could organize all the necessary enzymes for the reaction in close quarters this could possibly lead to more effective cascade reactions. One way of organizing enzymes is by fusing them on barcoded nanowire matrices. This could be achieved by tayloring enzyme molecules with genetically engineered proteins for inorganics (GEPIs). My assignment was to characterise possible nanowire candidates for these biohybrid complexes using AFM and examine silver binding characteristics of GEPI taylored streptavidin using SERS. I could not realiably characterise nanowires because the interaction between AFM probe and nanowires was to interfering. Also the nanowires used to aggregate and it was difficult to separate them using ultrasound. 15nm diameter nanowires aggregated more thant 30nm diameter nanowires. Streptavidin taylored with Ag binding GEPIs showed stronger interaction with Ag electrode surface than ordinary streptavidin. Also this modified streptavidin was capable of binding with biotin. This proves that added oligopeptide chains did not negatively affect the chemical structure of streptavidin.

Polyhydroxyalkanoates (PHAs) are polyesters composed of (R)-3-hydroxy-fatty acids. A variety of gram-positive as well as gram-negative bacteria and some archaea are able to produce these biopolymers as energy and carbon storage materials. In times of unbalanced growth, when carbon is available in excess but other nutrients are limited, PHA inclusions are formed. These granules are water-insoluble, stored intracellularly and can be maintained outside the cell as beads. The key enzyme for the formation of PHA inclusions is the PHA synthase PhaC, which catalyses the polymerization of (R)- 3-hydroxyacyl-CoA to PHA with the concomitant release of CoA. The PHA synthase from Ralstonia eutropha (currently Cupriavidus necator), which is covalently bound to the PHA granule surface, tolerates fusions to its N terminus without loss of activity. In this study it was investigated if it would also tolerate translational fusions to its C terminus. A specially designed linker was employed, aiming at maintaining the hydrophobic surroundings of the R. eutropha synthase C terminus to allow proper folding and activity. Two reporter proteins were tested as fusion partners, the maltose binding protein MalE and the green fluorescent protein GFP. As GFP is a hydrophobic protein itself, no additional linker between the PHA synthase and the reporter protein was necessary to produce PHA granules displaying the functional fusion protein on the surface. Principally, the PHA synthase PhaC tolerates translational fusions to its C terminus but the nature of the fusion partner influences the functionality. Recently, PHA granules have often been acknowledged as bio-beads. A one-step production allows the formation of functionalised beads without the need for further cross-linking to impart desired surface properties. PHA beads displaying a gold- or silica-binding peptide at the N terminus of PhaC were constructed and tested for their applicability. Additionally, these beads were able to bind IgG due to the ZZ domain of the IgG binding protein A, which was employed as a linker sequence. These functionalised beads can be used as molecular tools in bioimaging and biomedicine, combining organic core with inorganic-binding shell structures. In a different biomimetic approach, the display of ten lysine residues at the granule surface was achieved using the phasin protein PhaP as the anchoring matrix. Extensive work was performed in an attempt to also employ the synthase protein, but was unsuccessful. These positively charged bio-beads can be used for dispersion or crosslinking experiments as well as silica binding.