Table of Contents:
The methane formation from H2 and CO2 is the only energy providing reaction in many methanogenic archaea. Per mol produced methane four mol H2 are consumed. Therefore hydrogen is activated by hydrogenase activity. Most hydrogenotrophic methanogens harbor three different [NiFe]-hydrogenases and one [Fe]-hydrogenase. From these four hydrogenases the F420-reducing [NiFe]-hydrogenase (FrhABG) is the quantitative most important one, because it provides via reduced F420H2 four of the eight reducing equivalents that are necessary for CO2 reduction. F420 is a 5´-deazaflavinderivate that resembles structurally a flavin but behaves more like a pyridinnucleotide by transferring a hydride rather then single electrons. The redox potential Eo´ of F420 is -360 mV and with this -40 mV lower than that from NAD and NADP, respectively. The reduction of F420 with H2 (Eo ́= -414 mV) takes place near the thermodynamical equilibrium, particular under physiological conditions. Here the H2 partial pressure from cultures of methanogenic archaea is much lower then one bar. The goal of the present work was solving the structure of FrhABG, wherefore different motives were present. On the one hand the functional reversibility distinguish this enzyme from the structural known hydrogenases. On the other hand FrhABG belongs to the physiological group 3 of [NiFe]-hydrogenases, from this group no structure is available so far. All structural analyzed [NiFe]-hydrogenases belong to group 1. And finally, because FrhABG contains the subunit B which is phylogenetically related with subunits of other F420-dependent enzymes like F420-dependent formate dehydrogenase, F420-dependent glutamate synthase, F420- dependent sulfite reductase, F420H2:quinone oxidoreductase and F420H2:phenazine oxidoreductase complex, respectively. From all of these enzymes no structure has been solved. The F420 reducing FrhABG complex was purified from Methanothermobacer marburgensis. Homogeneous preparation with a specific activity of 280 U/mg and an apparent KM for F420 of 40 μM was reached after a purification fold of 100. The preparation with an apparent molecular mass of >1000 kDa contains the three subunits A, B, and G in a ratio 1 to 1 to 1. The macromolecular structure of FrhABG gave the ability to characterize this multimeric complex firstly with Cryo-electron microscopy (Cryo-EM). The structure was solved with a unique resolution of ~4 Å. Because of this high resolution, it was possible to model the carbon backbone into the electron density. It turned out that the heterotrimer builds up a complex of (FrhABG)12 with a molecular mass of 1215 kDa. The trimeric and multmerization is stabilized mainly by salt bridges. Parallel to analysis by Cryo-EM crystallization experiments from FrhABG were performed. The X-ray crystal structure was solved with a resolution of 1.8 Å. With this received structure detailed analysis of the containing metal centers were possible. The [NiFe]-center of the FrhA subunit is in detail more visible in the crystal structure than in the Cryo-EM structure. The nickel atom is coordinated by four cysteine thiolates, thereby two of this cysteines forming an additional bridge to the iron atom. Furthermore, the iron is coordinated additionally by three diatomic ligands; one CO and two CN-. Interestingly, despite the fact that the [NiFe]-center is highly conserved, the surrounding revealed some interesting amino acid exchanges which enlarge our view on hydrogenases. FrhG contains three [4Fe4S]-clusters. The proximal [4Fe4S]-cluster has a unique coordination with one aspartate instead of a cystein; this aspartate is not described in any [NiFe]-hydrogenase structure so far. Moreover, the medial cluster is a [4Fe4S]- and not a [3Fe4S]-cluster and together with the distal [4Fe4S]-cluster bound in a ferredoxin-like domain. In the FrhB subunit the [4Fe4S]-cluster and the FAD, that shows a unique compressed confirmation, are clearly visible. The distance between them is 7.5 Å. FAD is arranged in van-der-Waals contact to F420. So FAD could accept a hydride from F420H2 from the pro-S side; this information arises from post modeling of F420. To show that FrhB is indeed the F420 reducing subunit, the gen frhB from Methanocaldococcus jannaschii was over produced in Escherichia coli, purified and characterized. The heterologous produced subunit has a molecular mass of 31 kDa and contains FAD and an iron-sulfur cluster, respectively. The recombinant enzyme catalyzes the oxidation of F420H2 and simultaneously the reduction of benzyl viologen with a specific activity of 15 U/mg. The protein loses the activity fast, especially after freezing. This and preliminary electron paramagnetic resonance (EPR) analysis of the redox potential of the single [4Fe4S]-cluster from FrhABG are presented in this work.