Density Functional Study for the Bridged Dinuclear Center Based on a High-Resolution X‑ray Crystal Structure of <i>ba</i>₃ Cytochrome <i>c</i> Oxidase from Thermus thermophilus

Strong electron density for a peroxide type dioxygen species bridging the Fe_a3 and Cu_B dinuclear center (DNC) was observed in the high-resolution (1.8 Å) X-ray crystal structures (PDB entries 3S8G and 3S8F) of <i>ba</i>₃ cytochrome <i>c</i> oxidase (C<i>c</i>O) from Thermus thermophilus. The crystals represent the as-isolated X-ray photoreduced C<i>c</i>O structures. The bridging peroxide was proposed to arise from the recombination of two radiation-produced HO^• radicals formed either very near to or even in the space between the two metals of the DNC. It is unclear whether this peroxide species is in the O₂^2–, O₂^•^–, HO₂^–, or the H₂O₂ form and what is the detailed electronic structure and binding geometry including the DNC. In order to answer what form of this dioxygen species was observed in the DNC of the 1.8 Å X-ray C<i>c</i>O crystal structure (3S8G), we have applied broken-symmetry density functional theory (BS-DFT) geometric and energetic calculations (using OLYP potential) on large DNC cluster models with different Fe_a3–Cu_B oxidation and spin states and with O₂^2–, O₂^•^–, HO₂^–, or H₂O₂ in the bridging position. By comparing the DFT optimized geometries with the X-ray crystal structure (3S8G), we propose that the bridging peroxide is HO₂^–. The X-ray crystal structure is likely to represent the superposition of the Fe_a3²⁺–(HO₂^–)–Cu_B⁺ DNC’s in different states (Fe²⁺ in low spin (LS), intermediate spin (IS), or high spin (HS)) with the majority species having the proton of the HO₂^– residing on the oxygen atom (O1) which is closer to the Fe_a3²⁺ site in the Fe_a3²⁺–(HO–O)⁻–Cu_B⁺ conformation. Our calculations show that the side chain of Tyr237 is likely trapped in the deprotonated Tyr237^– anion form in the 3S8G X-ray crystal structure

Broken Symmetry DFT Calculations/Analysis for Oxidized and Reduced Dinuclear Center in Cytochrome <i>c</i> Oxidase: Relating Structures, Protonation States, Energies, and Mössbauer Properties in <i>ba</i>₃ <i>Thermus thermophilus</i>

The Fe_a3³⁺···Cu_B²⁺ dinuclear center (DNC) structure of the as-isolated oxidized <i>ba</i>₃ cytochrome <i>c</i> oxidase (C<i>c</i>O) from <i>Thermus thermophilus</i> (<i>Tt</i>) is still not fully understood. When the proteins are initially crystallized in the oxidized state, they typically become radiolyticly reduced through X-ray irradiation. Several X-ray crystal structures of reduced <i>ba</i>₃ C<i>c</i>O from <i>Tt</i> are available. However, depending on whether the crystals were prepared in a lipidic cubic phase environment or in detergent micelles, and whether the C<i>c</i>O’s were chemically or radiolyticly reduced, the X-ray diffraction analysis of the crystals showed different Fe_a3²⁺···Cu_B⁺ DNC structures. On the other hand, Mössbauer spectroscopic experiments on reduced and oxidized <i>ba</i>₃ C<i>c</i>Os from <i>Tt</i> (Zimmermann et al., <i>Proc. Natl. Acad. Sci. USA</i> 1988, 85, 5779–5783) revealed multiple ⁵⁷Fe_a3²⁺ and ⁵⁷Fe_a3³⁺ components. Moreover, one of the ⁵⁷Fe_a3³⁺ components observed at 4.2 K transformed from a proposed “low-spin” state to a different high-spin species when the temperature was increased above 190 K, whereas the other high-spin ⁵⁷Fe_a3³⁺ component remained unchanged. In the current Article, in order to understand the heterogeneities of the DNC in both Mössbauer spectra and X-ray crystal structures, the spin crossover of one of the ⁵⁷Fe_a3³⁺ components, and how the coordination and spin states of the Fe_a3^3+/2+ and Cu^2+/1+ sites relate to the heterogeneity of the DNC structures, we have applied density functional OLYP calculations to the DNC clusters established based on the different X-ray crystal structures of <i>ba</i>₃ C<i>c</i>O from <i>Tt</i>. As a result, specific oxidized and reduced DNC structures related to the observed Mössbauer spectra and to spectral changes with temperature have been proposed. Our calculations also show that, in certain intermediate states, the His233 and His283 ligand side chains may dissociate from the Cu_B⁺ site, and they may become potential proton loading sites during the catalytic cycle

A Water Dimer Shift Activates a Proton Pumping Pathway in the <b>P</b>_<b>R</b> → <b>F</b> Transition of <i>ba</i>_<i>3</i> Cytochrome <i>c</i> Oxidase

Broken-symmetry density functional calculations have been performed on the [Fe_a3⁴⁺,Cu_B²⁺] state of the dinuclear center (DNC) for the <b>P</b>_<b>R</b> → <b>F</b> part of the catalytic cycle of <i>ba</i>₃ cytochrome <i>c</i> oxidase (C<i>c</i>O) from Thermus thermophilus (<i>Tt</i>), using the OLYP-D3-BJ functional. The calculations show that the movement of the H₂O molecules in the DNC affects the p<i>K</i>_a values of the residue side chains of Tyr237 and His376⁺, which are crucial for proton transfer/pumping in <i>ba</i>₃ C<i>c</i>O from <i>Tt</i>. The calculated lowest energy structure of the DNC in the [Fe_a3⁴⁺,Cu_B²⁺] state (state <b>F</b>) is of the form Fe_a3⁴⁺O^2–···Cu_B²⁺, in which the H₂O ligand that resulted from protonation of the OH^– ligand in the <b>P</b>_<b>R</b> state is dissociated from the Cu_B²⁺ site. The calculated Fe_a3⁴⁺O^2– distance in <b>F</b> (1.68 Å) is 0.03 Å longer than that in <b>P</b>_<b>R</b> (1.65 Å), which can explain the different Fe_a3⁴⁺O^2– stretching modes in <b>P</b> (804 cm^–1) and <b>F</b> (785 cm^–1) identified by resonance Raman experiments. In this <b>F</b> state, the Cu_B²⁺···O^2– (ferryl–oxygen) distance is only around 2.4 Å. Hence, the subsequent <b>O</b>_<b>H</b> state [Fe_a3³⁺-OH^––Cu_B²⁺] with a μ-hydroxo bridge can be easily formed, as shown by our calculations

The Mössbauer Parameters of the Proximal Cluster of Membrane-Bound Hydrogenase Revisited: A Density Functional Theory Study

An unprecedented [4Fe-3S] cluster proximal to the regular [NiFe] active site has recently been found to be responsible for the ability of membrane-bound hydrogenases (MBHs) to oxidize dihydrogen in the presence of ambient levels of oxygen. Starting from proximal cluster models of a recent DFT study on the redox-dependent structural transformation of the [4Fe-3S] cluster, ⁵⁷Fe Mössbauer parameters (electric field gradients, isomer shifts, and nuclear hyperfine couplings) were calculated using DFT. Our results revise the previously reported correspondence of Mössbauer signals and iron centers in the [4Fe-3S]³⁺ reduced-state proximal cluster. Similar conflicting assignments are also resolved for the [4Fe-3S]⁵⁺ superoxidized state with particular regard to spin-coupling in the broken-symmetry DFT calculations. Calculated ⁵⁷Fe hyperfine coupling (HFC) tensors expose discrepancies in the experimental set of HFC tensors and substantiate the need for additional experimental work on the magnetic properties of the MBH proximal cluster in its reduced and superoxidized redox states

Linking Chemical Electron–Proton Transfer to Proton Pumping in Cytochrome <i>c</i> Oxidase: Broken-Symmetry DFT Exploration of Intermediates along the Catalytic Reaction Pathway of the Iron–Copper Dinuclear Complex

After a summary of the problem of coupling electron and proton transfer to proton pumping in cytochrome <i>c</i> oxidase, we present the results of our earlier and recent density functional theory calculations for the dinuclear Fe-a₃–Cu_B reaction center in this enzyme. A specific catalytic reaction wheel diagram is constructed from the calculations, based on the structures and relative energies of the intermediate states of the reaction cycle. A larger family of tautomers/protonation states is generated compared to our earlier work, and a new lowest-energy pathway is proposed. The entire reaction cycle is calculated for the new smaller model (about 185–190 atoms), and two selected arcs of the wheel are chosen for calculations using a larger model (about 205 atoms). We compare the structural and redox energetics and protonation calculations with available experimental data. The reaction cycle map that we have built is positioned for further improvement and testing against experiment

A Fluorogenic Aryl Fluorosulfate for Intraorganellar Transthyretin Imaging in Living Cells and in <i>Caenorhabditis elegans</i>

Fluorogenic probes, due to their often greater spatial and temporal sensitivity in comparison to permanently fluorescent small molecules, represent powerful tools to study protein localization and function in the context of living systems. Herein, we report fluorogenic probe <b>4</b>, a 1,3,4-oxadiazole designed to bind selectively to transthyretin (TTR). Probe <b>4</b> comprises a fluorosulfate group not previously used in an environment-sensitive fluorophore. The fluorosulfate functional group does not react covalently with TTR on the time scale required for cellular imaging, but does red shift the emission maximum of probe <b>4</b> in comparison to its nonfluorosulfated analogue. We demonstrate that probe <b>4</b> is dark in aqueous buffers, whereas the TTR·<b>4</b> complex exhibits a fluorescence emission maximum at 481 nm. The addition of probe <b>4</b> to living HEK293T cells allows efficient binding to and imaging of exogenous TTR within intracellular organelles, including the mitochondria and the endoplasmic reticulum. Furthermore, live <i>Caenorhabditis elegans</i> expressing human TTR transgenically and treated with probe <b>4</b> display TTR·<b>4</b> fluorescence in macrophage-like coelomocytes. An analogue of fluorosulfate probe <b>4</b> does react selectively with TTR without labeling the remainder of the cellular proteome. Studies on this analogue suggest that certain aryl fluorosulfates, due to their cell and organelle permeability and activatable reactivity, could be considered for the development of protein-selective covalent probes