On the ATP-Dependent Activation of the Radical Enzyme (<i>R</i>)‑2-Hydroxyisocaproyl-CoA Dehydratase

Members of the 2-hydroxyacyl-CoA dehydratase enzyme family catalyze the β,α-dehydration of various CoA-esters in the fermentation of amino acids by clostridia. Abstraction of the nonacidic β-proton of the 2-hydroxyacyl-CoA compounds is achieved by the reductive generation of ketyl radicals on the substrate, which is initiated by the transfer of an electron at low redox potentials. The highly energetic electron needed on the dehydratase is donated by a [4Fe-4S] cluster containing ATPase, termed activator. We investigated the activator of the 2-hydroxyisocaproyl-CoA dehydratase from <i>Clostridium difficile</i>. The activator is a homodimeric protein structurally related to acetate and sugar kinases, Hsc70 and actin, and has a [4Fe-4S] cluster bound in the dimer interface. The crystal structures of the Mg-ADP, Mg-ADPNP, and nucleotide-free states of the reduced activator have been solved at 1.6–3.0 Å resolution, allowing us to define the position of Mg²⁺ and water molecules in the vicinity of the nucleotides and the [4Fe-4S] cluster. The structures reveal redox- and nucleotide dependent changes agreeing with the modulation of the reduction potential of the [4Fe-4S] cluster by conformational changes. We also investigated the propensity of the activator to form a complex with its cognate dehydratase in the presence of Mg-ADP and Mg-ADPNP and together with the structural data present a refined mechanistic scheme for the ATP-dependent electron transfer between activator and dehydratase

Substrate Activation at the Ni,Fe Cluster of CO Dehydrogenases: The Influence of the Protein Matrix

Carbon monoxide dehydrogenases catalyze the reversible conversion of CO2 with two electrons to CO and water at a unique Ni- and Fe-containing cluster (cluster C). Structural studies indicate that several highly conserved amino acids in the second coordination sphere of cluster C support the activation of the substrates, CO/CO2 and water, and may be mandatory for catalytic turnover. However, their contribution to substrate activation has been poorly explored. We replaced the three residues with potential direct interaction with the substrates (I567, H93, and K563) and one residue essential for proton/water transfer (H96) and analyzed the associated changes in the structure and reactivity of the enzyme. In addition to the expected exchange of side chains, we observed rearrangements of water molecules as well as the appearance of additional water molecules at the active site. These changes also affect the coordination of cluster C and the hydroxo ligand at Fe, with additional hydroxo/water ligands at Ni. Subsequently, we were able to convert cluster C from a [NiFe4(OH)(μ3-S)4] cluster to a [Fe4(μ3-S)4] cluster by exchanging K563 and a primary coordinating C295. Therefore, the second coordination sphere is important not only for the affinity of the substrates but also for the stability of cluster C. Thus, beyond substrate activation, the residues in the second coordination sphere of cluster C also determine its coordination and stability

HOMO and LUMO energies and natural population analysis charges from DFT^a.

<p>HOMO and LUMO energies and natural population analysis charges from DFT<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158681#t005fn001" target="_blank">^a</a>.</p

EXAFS simulation parameters^a.

<p>EXAFS simulation parameters<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158681#t002fn001" target="_blank">^a</a>.</p

EXAFS spectra of cobalamin systems.

<p>Panel (A) shows Fourier-transforms (FTs) of the EXAFS oscillations in panel (B) for indicated solution Cbl or CoFeSP-Cbl samples. Black lines, experimental data; coloured lines, simulations with parameters in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158681#pone.0158681.t002" target="_blank">Table 2</a> (fits 2, 5, 7, 10, 12, 14, 16, 19, 21); spectra in (A) and (B) were vertically shifted for comparison.</p

Comparison of DFT calculated and experimental ctv features.

<p>Lines, spectra from DFT; dots, experimental data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158681#pone.0158681.g003" target="_blank">Fig 3</a>); spectra were vertically shifted for comparison; note the doubled y-scale in (B). Calculated spectra represent the indicated model structures; solid lines and coloured annotations denote calculated spectra for the indicated structures, which show superior agreement with the experimental data (broken lines show calculation results less in agreement with the experimental data).</p

Cobalt-ligand bond lengths from crystallography, EXAFS, and DFT.

<p>Cobalt-ligand bond lengths from crystallography, EXAFS, and DFT.</p

Core-to-valence electronic transition characters.

<p>Core-to-valence electronic transition characters.</p

Molecular orbitals in Cbl model structures from DFT.

<p>LUMO, lowest unoccupied MO corresponding to the lowest energy core-to-valence electronic transition in the pre-edge absorption X-ray spectral region; ctv_max, MO corresponding to the highest-intensity ctv transition of the pre-edge absorption. Cobalt oxidation state and axial ligation are indicated.</p

Metal content and cobalt oxidation state in the CoFeSP samples^a.

<p>Metal content and cobalt oxidation state in the CoFeSP samples<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158681#t001fn001" target="_blank">^a</a>.</p