Die rekombinante Expression des Vier-Helix-Bündelproteins Sulerythrin, aus Sulfolobus tokodaii-Stamm 7, wurde etabliert und für die Produktion von metallfreiem Apo-Protein optimiert. Durch Metall-Rekonstitution konnten Eisen, Nickel, Kobalt, Mangan und Eisen-57 als Cofaktoren des homo-bimetallischen Zentrums eingebracht werden. Es wurden Zusammenhänge zwischen Eigenschaften, Reaktivitäten und Strukturen verschiedener diMe-SulE-Proteine untersucht. Die Affinität der Bindungen der vier Metalle an das SulE-wt wurde mittels ITC untersucht und deutet auf eine hochaffine und eine niedrigaffine Bindungsstelle im Zentrum hin, was durch Kristallisation von mono-Fe-SulE bestätigt wurde. Die Me-SulE-wt-Proteine zeigten unterschiedliche UV-vis-Spektren im reduzierten und oxidierten Zustand. Es wurden diFe-SulE-Varianten der Koordinationssphäre des aktiven Zentrums erzeugt und deren Oxidationsraten mit Sauerstoff bei 325 nm bestimmt, wobei diFe-SulE-wt langsamer war als alle Varianten (beobachtete Geschwindigkeitskonstanten teilweise 40 bis 50-mal höher). Die reduzierten Zustände der diFe-SulE-Varianten wurden durch Mößbauer-Spektroskopie untersucht (80-100 %ige Fe(II) im high-spin Zustand). Für die diMe-SulE-wt-Varianten wurde die Katalaseaktivität gemessen. Durch die Inkubation von diCo-SulE-wt-Kristallen mit H2O2 konnte ein verbrückender μ2-η2-Peroxid-Ligand in der Proteinstruktur modelliert werden und ein Reaktionsintermediat nachgewiesen werden, dessen O-O-Bindungslänge mit 1,50 Å nahezu identisch mit H2O2 ist. Der Metall-Metall-Abstand im zweikernigen Metallzentrum der Varianten reichte von 3,97 Å für bis 3,15 Å. Anhand der Strukturen der aktiven Stellen der Varianten lassen sich strukturelle Unterschiede erkennen, die als Ursache für die unterschiedlichen Reaktivitäten und Oxidationsraten vermutet werden. Die Daten zeigen eine klare Korrelation zwischen einem abnehmenden Metall-Metall-Abstand und einem Anstieg der Geschwindigkeitskonstante für verschiedene Varianten. / The recombinant expression of the four-helix bundle protein Sulerythrin, from Sulfolobus tokodaii strain 7, was established and optimized for the production of metal-free apo-protein. By metal reconstitution, iron, nickel, cobalt, manganese and iron-57 were incorporated as cofactors of the homo-bimetallic center. Relationships between characteristics, reactivities and structures of different diMe-SulE proteins were investigated. The affinity of the bonds of the four metals to the SulE-wt was investigated by ITC and indicated a high-affinity and a low-affinity binding site in the active site, which was confirmed by crystallization of mono-Fe-SulE. The Me-SulE-wt proteins showed different UV-vis spectra in the reduced and oxidized states. diFe-SulE variants of the active site coordination sphere were generated and their oxidation rates with oxygen at 325 nm were determined, with diFe-SulE-wt being slower than all variants (observed rate constants 40 to 50 times higher in some cases). The reduced states of the diFe-SulE variants were investigated by Mössbauer spectroscopy (80-100 % Fe(II) in high-spin state). The catalase activity was measured for diMe-SulE-wt variants. By incubating diCo-SulE-wt crystals with H2O2, a bridging μ2-η2 peroxide ligand could be modeled in the protein structure and a reaction intermediate was detected whose O-O bond length of 1.50 Å is almost identical to H2O2. The metal-to-metal distance in the binuclear metal center of the variants ranged from 3.97 Å for to 3.15 Å. Based on the structures of the active sites of the variants, structural differences can be identified, which are assumed to be the cause of the different reactivities and oxidation rates. The data further provide a clear correlation between a decreasing distance between the metals and an increase in the rate constant for different variants.

The binding of molecular hydrogen to a metal center leads to the elongation of the H−H bond and subsequently to its cleavage along the reaction coordinate for the oxidative addition of H2. There has been considerable interest in the study of the activation of dihydrogen and map out the reaction coordinate for the homolysis of H2 on a metal center. A large number of H2 complexes reported to date possess H−H distances ranging from 0.8 to 1.0 Å. A relatively fewer examples of elongated dihydrogen complexes wherein the H−H distances fall in the range of 1.0 to 1.5 Å, are known. Study of the elongated dihydrogen complexes is of great significance because of its relevance in important catalytic processes such as hydrogenation, hydrogenolysis, and hydroformylation. Objectives The objectives of this work are as follows: (a) Synthesis and characterization of elongated dihydrogen complexes with chelating phosphine coligands by varying the electron donor ability. (b) Trap the various intermediate states in the process of oxidative addition of H2 to a metal center. (c) Map the reaction coordinate for the oxidative addition for the oxidative addition of H2 to a metal center. Results We have synthesized and characterized two new elongated dihydrogen complexes cis-[Ir(H)(η2-S2CH)(η2-H2)(PR3)2][BF4] (PR3 = PCy3, PPh3) wherein hydrogen atom undergoes site exchange between the H2 and the hydride sites. The dynamics of the exchange was studied using NMR spectroscopy. In addition, a series of ruthenium dihydrogen complexes of the type trans-[Ru(Cl)(η2-H2)(PP)][BF4] (PP = 1,2- Synopsis bis(diarylphosphino)ethane) has been synthesized and characterized wherein the aryl group is a benzyl moiety with a substituent (p-fluoro, H, m-methyl, p-methyl, p-isopropyl); in this series of complexes, a small increment in the electron donor ability (decrease in Hammett substituent constants) of the chelating phosphine ligand resulted in an elongation of the H−H bond by a small, yet significant amount. We also synthesized a series of 16-electron dicationic dihydrogen complexes bearing elongated dihydrogen ligand. In addition, we prepared a series of dihydrogen complexes of the type [RuCp/Cp*(PP)(η2-H2)][OTf] (PP = 1,2-bis(diarylphosphino)ethane, 1,2-bis(diarylphosphino)methane, 1,2-bis(dialkylphosphino)methane) bearing elongated H2 ligand (dHH = 1.0 to 1.17 Å); in this series of complexes as well, we found that the H−H bond distances increased as the donor ability of the chelating phosphines increased in small increments, along the reaction coordinate for the oxidative addition of H2 to a metal center. This investigation therefore, has established a very nice correlation between the H−H bond lengths and the Hammett substitutent constants (donor properties) resulting in the construction of dihydrogen complexes along the reaction coordinate for the oxidative addition of H2 to a metal center.