Metals in biological systems catalyze challenging enzymatic reactions, including bond formation and cleavage, radical chemistry, atom and electron transfer. In this work, two metal-based enzyme systems from archaea, the Ni-containing CO dehydrogenase (CODH) and [NiFe] containing hydrogenase, are structurally characterized.
CODHs catalyze the reversible reduction of CO2 to CO and in archaea this reaction is coupled to the synthesis and cleavage of acetyl-coenzyme A (acetyl-CoA). The overall reaction is catalyzed by the acetyl-CoA decarbonylase/synthase (ACDS) complex and within the complex, the chemistry of reversible carbon-carbon bond formation is accomplished by coordinated activity of five protein subcomponents, at least 33 ironsulfur clusters, 6 cobalt- and 12 nickel-containing active sites. Despite extensive biochemical and spectroscopic characterization, as well as X-ray crystal structures of the individual components from archaeal and bacterial homologs, the structure of the intact protein complex is still unknown.
The first part of this work is dedicated to a structural investigation by transmission electron microscopy of the ACDS complex from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus (AfACDS). Purified ACDS complex could be visualized as an intact globular protein particle by negative stain and vitrification (cryo) techniques. The three-dimensional reconstruction is determined de novo to 29 Å-resolution by singleparticle analysis. Three possible positions for the CODH subunit within the ACDS complex are suggested by rigid-body fitting.
In the second part of this study, the X-ray crystal structure of the CODH subunit of the AfACDS complex is determined. The 220 kDa protein is encoded by the cdhAB operon, and is composed of α- and ε-subunits that form a heterodimer with (α2ε2) stoichiometry (Afα2ε2). While the overall structure of Afα2ε2 resembles the previously reported structure of the α2ε2-subunit from Methanosarcina barkeri (Mbα2ε2), the naturally-occurring exchange of the Cys to Asp and Glu resulted in a depletion of the bridging iron-sulfur cluster. As revealed by sequence analysis, this structural trait is observed in different phyla of bacteria and archaea, including 31 enzymes encoded by cdhAB operons and 14 homologous Ni-CODH enzymes encoded by cooS genes. The phylogenetic analysis suggests that the cluster elimination event could result from gene transfer within the evolutionary lineage, horizontal gene transfer or independent Cys exchange. Additionally, the role of the ε-subunit was investigated by kinetics studies. CO-dependent FAD reduction activity of Afα2ε2 exhibited Michaelis-Menten type kinetics with kcat of 186 s-1 and KM of 76 μM. Same kinetic type, as well as comparable kinetic constants, was demonstrated for the Mbα2ε2-subunit. In contrast, the ε-subunit lacking CODH-II from Carboxydothermus hydrogenoformans (ChCODH-II) showed linear dependency between CO-dependent FAD reduction activity and flavin concentration. The data suggests that the ε-subunit provides a scaffold for the flavin binding.
The third section is dedicated to the F420-reducing hydrogenase from M. barkeri (MbFRH), which belongs to the group 3 [NiFe] hydrogenases and catalyzes the reversible redox reaction between H2 and coenzyme F420. The structure of MbFRH was solved by Xray crystallography and is similar to the structure of FRH from Methanothermobacter marburgensis (MmFRH), revealing a spherical, dodecameric arrangement of approx. 1.2 MDa. Along with the established electron transfer chain observed in MmFRH, consisting of the [NiFe] active site, four [4Fe4S] clusters and FAD, one solvent-exposed [2Fe2S] cluster is detected. This additional metal site is positioned within 7.5 Å between two adjacent electron transfer chains of two protomers and is thought to enable crosstalk between the pathways. The conserved position of the mononuclear metal site (11.5 Å from the [NiFe] active center) is occupied by a single Fe, the function of which is not yet understood. A narrow, 25-Å long hydrophobic channel is observed by xenon derivatization experiments. The position of the channel differs significantly from the canonical hydrophobic channels previously detected in other [NiFe] hydrogenases. Finally, the combined approach of X-ray crystallography and vibrational spectroscopy reveals that MbFRH is isolated in the previously structurally uncharacterized Nia-S state. This state was characterized by the peculiar four Cys-coordinated seesaw-shaped geometry of the Ni ion with two trans S(Cys)-Ni-S(Cys) at 107° and 171°, short Ni–Fe distance of 2.7 Å and an open Ni coordination site.
|Advisor:||Dobbek , Holger , Hildebrandt , Peter , Wendler , Petra|
|School:||Humboldt Universitaet zu Berlin (Germany)|
|Source:||DAI-C 81/7(E), Dissertation Abstracts International|
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