Holger Dobbek

Xenobiotic reductase A (XenA) from Pseudomonas putida is a member of the old-yellow-enzyme family of flavin-containing enzymes and catalyzes the NADH/NADPH-dependent reduction of various substrates, including 8-hydroxycoumarin and 2-cyclohexenone. Here we present a kinetic and thermodynamic analysis of XenA. In the reductive half-reaction, complexes of oxidized XenA with NADH or NADPH form charge-transfer (CT) intermediates with increased absorption around 520−560 nm, which occurs with a second-order rate constant of 9.4 × 10⁵ M⁻¹ s⁻¹ with NADH and 6.4 × 10⁵ M⁻¹ s⁻¹ with NADPH, while its disappearance is controlled by a rate constant of 210−250 s⁻¹ with both substrates. Transfer of hydride from NADPH proceeds 24 times more rapidly than from NADH. This modest kinetic preference of XenA for NADPH is unlike the typical discrimination between NADH and NADPH by binding affinity. Docking studies combined with electrostatic energy calculations indicate that the 2′-phosphate group attached to the adenine moiety of NADPH is responsible for this difference. The reductions of 2-cyclohexenone and coumarin in the oxidative half-reaction are both concentration-dependent under the assay conditions and reveal a more than 50-fold larger limiting rate constant for the reduction of 2-cyclohexenone compared to that of coumarin. Our work corroborates the link between XenA and other members of the old-yellow-enzyme family but demonstrates several differences in the reactivity of these enzymes.

Nicotinate dehydrogenase (NDH) from Eubacterium barkeri is a molybdoenzyme catalyzing the hydroxylation of nicotinate to 6-hydroxynicotinate. Reactivity of NDH critically depends on the presence of labile (nonselenocysteine) selenium with an as-yet-unidentified form and function. We have determined the crystal structure of NDH and analyzed its active site by multiple wavelengths anomalous dispersion methods. We show that selenium is bound as a terminal Mo Se ligand to molybdenum and that it occupies the position of the terminal sulfido ligand in other molybdenum hydroxylases. The role of selenium in catalysis has been assessed by model calculations, which indicate an acceleration of the critical hydride transfer from the substrate to the selenido ligand in the course of substrate hydroxylation when compared with an active site containing a sulfido ligand. The MoO(OH)Se active site of NDH shows a novel type of utilization and reactivity of selenium in nature.

Anaerobic CO dehydrogenases catalyze the reversible oxidation of CO to CO ₂ at a complex Ni-, Fe-, and S-containing metal center called cluster C. We report crystal structures of CO dehydrogenase II from Carboxydothermus hydrogenoformans in three different states. In a reduced state, exogenous CO ₂ supplied in solution is bound and reductively activated by cluster C. In the intermediate structure, CO ₂ acts as a bridging ligand between Ni and the asymmetrically coordinated Fe, where it completes the square-planar coordination of the Ni ion. It replaces a water/hydroxo ligand bound to the Fe ion in the other two states. The structures define the mechanism of CO oxidation and CO ₂ reduction at the Ni-Fe site of cluster C.

The cobalt- and iron-containing corrinoid iron–sulfur protein (CoFeSP) is functional in the acetyl-CoA (Ljungdahl–Wood) pathway of autotrophic carbon fixation in various bacteria and archaea, where it is essential for the biosynthesis of acetyl-CoA. CoFeSP acts in two methylation reactions: the transfer of a methyl group from methyltransferase (MeTr)-bound methyltetrahydrofolate to the cob(I)amide of CoFeSP and the transfer of the methyl group of methyl-cob(III)amide to the reduced Ni-Ni-[4Fe-4S] active site cluster A of acetyl-CoA synthase (ACS). We have solved the crystal structure of as-isolated CoFeSP _Ch from the CO-oxidizing hydrogenogenic bacterium Carboxydothermus hydrogenoformans at 1.9-Å resolution. The heterodimeric protein consists of two tightly interacting subunits with pseudo-twofold symmetry. The large CfsA subunit comprises three domains, of which the N-terminal domain binds the [4Fe-4S] cluster, the middle domain is a (βα) ₈ -barrel, and the C-terminal domain shows an open fold and binds Co β-aqua-(5,6-dimethylbenzimidazolylcobamide) in a “base-off” state without a protein ligand at the cobalt ion. The small CfsB subunit also displays a (βα) ₈ -barrel fold and interacts with the upper side of the corrin macrocycle. Structure-based alignments show that both (βα) ₈ -barrel domains are related to the MeTr in the acetyl-CoA pathway and to the folate domain of methionine synthase. We suggest that the C-terminal domain of the large subunit is the mobile element that allows the necessary interaction of CoFeSP _Ch with the active site of ACS _Ch and the methyltetrahydrofolate carrying MeTr. The conformation in the crystal structure shields the two open coordinations of cobalt and likely represents a resting state.