Electron Transport in a Dioxygenase-Ferredoxin Complex: Long Range Charge Coupling between the Rieske and Non-Heme Iron Center

<div><p>Dioxygenase (dOx) utilizes stereospecific oxidation on aromatic molecules; consequently, dOx has potential applications in bioremediation and stereospecific oxidation synthesis. The reactive components of dOx comprise a Rieske structure Cys₂[2Fe-2S]His₂ and a non-heme reactive oxygen center (ROC). Between the Rieske structure and the ROC, a universally conserved Asp residue appears to bridge the two structures forming a Rieske-Asp-ROC triad, where the Asp is known to be essential for electron transfer processes. The Rieske and ROC share hydrogen bonds with Asp through their His ligands; suggesting an ideal network for electron transfer via the carboxyl side chain of Asp. Associated with the dOx is an itinerant charge carrying protein Ferredoxin (Fdx). Depending on the specific cognate, Fdx may also possess either the Rieske structure or a related structure known as 4-Cys-[2Fe-2S] (4-Cys). In this study, we extensively explore, at different levels of theory, the behavior of the individual components (Rieske and ROC) and their interaction together via the Asp using a variety of density function methods, basis sets, and a method known as Generalized Ionic Fragment Approach (GIFA) that permits setting up spin configurations manually. We also report results on the 4-Cys structure for comparison. The individual optimized structures are compared with observed spectroscopic data from the Rieske, 4-Cys and ROC structures (where information is available). The separate pieces are then combined together into a large Rieske-Asp-ROC (donor/bridge/acceptor) complex to estimate the overall coupling between individual components, based on changes to the partial charges. The results suggest that the partial charges are significantly altered when Asp bridges the Rieske and the ROC; hence, long range coupling through hydrogen bonding effects via the intercalated Asp bridge can drastically affect the partial charge distributions compared to the individual isolated structures. The results are consistent with a proton coupled electron transfer mechanism.</p></div

Break down of normal modes for the ROC.

<p><b>(Left) Modes for the ROC with Asp in the monodentate configuration (including optimized unbound water molecules found in the chamber).</b> (Right) Modes for the ROC with Asp in the bidentate configuration (typically these chambers contain no water).</p

The core reactive domains of the 1,9a-dioxygenase protein/Ferredoxin complex (PDB id: 2DE5).

<p>(a) The key components from the trimer complex (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162031#pone.0162031.g001" target="_blank">Fig 1C</a>). On the left is the non-Heme Iron redox center (ROC: Fe is the green atom) and near the center is the iron-sulfur (Cys)₂[2Fe-2S](His)₂ structure (the Rieske domain). Between the Rieske and the ROC is the universally conserved Asp. (b) The core structure of the ROC including the two His residues (methyl imidazole), one Asp residue (acetate), a bound water molecule and four additional optimized water molecules (found in the chamber) configured around the bound water and the Asp residue. (c) The Rieske domain including the two Cys residues (thio methylate) and two His residues (methyl imidazole).</p

High symmetry structures of the Rieske complex, specifically (Imidazole)₂[2Fe-2S](SMe)₂.

<p>(a and b) oxidized structure with maximum <b>C</b>_2v symmetry and (c and d) the reduced structure with <b>C</b>₁ symmetry.</p

Partial charge and spin for the main components in the Rieske-Asp-ROC complex.

<p>Partial charge and spin for the main components in the Rieske-Asp-ROC complex.</p

Observed and calculated frequencies of normal modes for the Rieske structure in the oxidized and reduced states.

<p>Observed and calculated frequencies of normal modes for the Rieske structure in the oxidized and reduced states.</p

Snapshots taken after a short simulation time showing the variation of the structure from the original PDB file (id: 1WW9).

<p>The simulations were carried out using using the force field parmeters developed in this work. (a) The subsequence of dOx comparing the minimized initial structure with the PDB structure (transparent residues). (b) The same substructure of 1WW9 after a short 4 ns MD simulation. Whereas the <i>truncated structures</i> tend to distort slightly during the simulation (as expected), this shows that the structure around the Fe center remains stable and relatively unchanged from the original PDB structure (transparent residues). (c) The same study for the Rieske region using a subsequence fragment of a related 1,9a-dioxygenase (PDB id: 3GKQ). The transparent residues are associated with the initial pdb data. (d) The total RMS deviation from the x-ray structure for a full MD simulation of the 3GKQ fragment using the developed force field parameters.</p

Results of optimization of the Rieske–Asp–non-heme Fe reactive oxygen center (ROC) complex computed with a minimum of two distance constraints: one between the Rieske:Fe (on the His side) and the ROC:Fe, and the other between H183:Cβ and H93:Cδ2.

<p>The orientation of the Rieske and ROC and their separation distances reflect what was found in the crystal structures of these compounds.</p

A comparison of the estimated stretching and bending constants for the reduced state of the 4-Cys-[2Fe-2S] and Rieske structures, based on calculated normal modes.

<p>A comparison of the estimated stretching and bending constants for the reduced state of the 4-Cys-[2Fe-2S] and Rieske structures, based on calculated normal modes.</p

Normal modes found in the ROC structure for the hydrated monodentate structure.

<p>Normal modes found in the ROC structure for the hydrated monodentate structure.</p