Proton-coupled electron transfer (PCET) is an elementary chemical reaction crucial for biological oxidoreduction. We perform quantum chemical calculations to study the direct and water-mediated PCET ...between two stacked tyrosines, TyrO• + TyrOH → TyrOH + TyrO•, to mimic a key step in the catalytic reaction of class Ia ribonucleotide reductase (RNR). The energy surfaces of electronic ground and excited states are separated by a large gap of ∼20 kcal mol–1, indicative of an electronically adiabatic transfer mechanism. In response to chemical substitutions of the proton donor, the energy of the transition state for direct PCET shifts by exactly half of the change in energetic driving force, resulting in a linear free energy relation with a Brønsted slope of 1/2. In contrast, for water-mediated PCET, we observe integer Brønsted slopes of 1 and 0 for proton acceptor and donor modifications, respectively. Our calculations suggest that the π-stacking of the tyrosine dimer in RNR results in strong electronic coupling and adiabatic PCET. Water participation in the PCET can be identified perturbatively in a Brønsted analysis.
Accurate predictions of photoexcitation properties are a major challenge for modern methods of theoretical chemistry. We show here how approximate coupled-cluster singles and doubles (CC2) ...calculations in combination with the reduced virtual space (RVS) approach can be employed in studies of excited states of large biomolecular systems. The RVS-CC2 approach is used for accurately predicting optical properties of the p-hydroxybenzylidene-dihydroimidazolinone (p-HBDI) chromophore embedded in green fluorescent protein (GFP) models using quantum mechanical calculations in combination with large basis sets. We study the lowest excited states for the isolated and protein-embedded chromophore in two different protonation states, and show how omitting high-lying virtual orbitals in the RVS calculation of excitation energies renders large-scale CC2 studies computationally feasible. We also discuss how the error introduced by the RVS approach can be systematically estimated and controlled. The obtained CC2 excitation energies of 3.13–3.27 and 2.69–2.77 eV for the two protonation states of different protein models are in excellent agreement with the maxima of the experimental absorption spectra of 3.12–3.14 and 2.61–2.64 eV, respectively. Thus, the calculated energy splitting between the excited states of the two protonation states is 0.44–0.52 eV, which agrees very well with the experimental value of 0.48–0.51 eV. The calculations at the RVS-CC2 level on the protein models show the importance of using large QM regions in studies of biochromophores embedded in proteins.
Complex I is a redox-driven proton pump that drives electron transport chains and powers oxidative phosphorylation across all domains of life. Yet, despite recently resolved structures from multiple ...organisms, it still remains unclear how the redox reactions in Complex I trigger proton pumping up to 200 Å away from the active site. Here, we show that the proton-coupled electron transfer reactions during quinone reduction drive long-range conformational changes of conserved loops and trans-membrane (TM) helices in the membrane domain of Complex I from Yarrowia lipolytica. We find that the conformational switching triggers a π → α transition in a TM helix (TM3ND6) and establishes a proton pathway between the quinone chamber and the antiporter-like subunits, responsible for proton pumping. Our large-scale (>20 μs) atomistic molecular dynamics (MD) simulations in combination with quantum/classical (QM/MM) free energy calculations show that the helix transition controls the barrier for proton transfer reactions by wetting transitions and electrostatic effects. The conformational switching is enabled by re-arrangements of ion pairs that propagate from the quinone binding site to the membrane domain via an extended network of conserved residues. We find that these redox-driven changes create a conserved coupling network within the Complex I superfamily, with point mutations leading to drastic activity changes and mitochondrial disorders. On a general level, our findings illustrate how catalysis controls large-scale protein conformational changes and enables ion transport across biological membranes.
In Photosystem II (PSII), the Mn
CaO
-cluster of the active site advances through five sequential oxidation states (S
to S
) before water is oxidized and O
is generated. Here, we have studied the ...transition between the low spin (LS) and high spin (HS) configurations of S
using EPR spectroscopy, quantum chemical calculations using Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy. The EPR experiments show that the equilibrium between S
and S
is pH dependent, with a pK
≈ 8.3 (n ≈ 4) for the native Mn
CaO
and pK
≈ 7.5 (n ≈ 1) for Mn
SrO
. The DFT results suggest that exchanging Ca with Sr modifies the electronic structure of several titratable groups within the active site, including groups that are not direct ligands to Ca/Sr, e.g., W1/W2, Asp61, His332 and His337. This is consistent with the complex modification of the pK
upon the Ca/Sr exchange. EPR also showed that NH
addition reversed the effect of high pH, NH
-S
being present at all pH values studied. Absorption spectroscopy indicates that NH
is no longer bound in the S
Tyr
state, consistent with EPR data showing minor or no NH
-induced modification of S
and S
. In both Ca-PSII and Sr-PSII, S
was capable of advancing to S
at low temperature (198 K). This is an experimental demonstration that the S
is formed first and advances to S
via the S
state without detectable intermediates. We discuss the nature of the changes occurring in the S
to S
transition which allow the S
to S
transition to occur below 200 K. This work also provides a protocol for generating S
in concentrated samples without the need for saturating flashes.
Cytochrome c oxidase (CcO) is a redox‐driven proton pump that powers aerobic respiratory chains. We show here by multi‐scale molecular simulations that a protonated water cluster near the active site ...is likely to serve as the transient proton‐loading site (PLS) that stores a proton during the pumping process. The pKa of this water cluster is sensitive to the redox states of the enzyme, showing distinct similarities to other energy converting proton pumps.
Proton‐coupled electron transfer: Structurally conserved water molecules (see picture) function as a transient proton‐loading site and provide important coupling elements in the proton‐pumping machinery of cytochrome oxidase. Quantum mechanics/molecular mechanics simulations were used to study the biomolecular mechanism.
The membrane-bound hydrogenase (Mbh) from Pyrococcus furiosus is an archaeal member of the Complex I superfamily. It catalyzes the reduction of protons to H2 gas powered by a NiFe active site and ...transduces the free energy into proton pumping and Na+/H+ exchange across the membrane. Despite recent structural advances, the mechanistic principles of H2 catalysis and ion transport in Mbh remain elusive. Here, we probe how the redox chemistry drives the reduction of the proton to H2 and how the catalysis couples to conformational dynamics in the membrane domain of Mbh. By combining large-scale quantum chemical density functional theory (DFT) and correlated ab initio wave function methods with atomistic molecular dynamics simulations, we show that the proton transfer reactions required for the catalysis are gated by electric field effects that direct the protons by water-mediated reactions from Glu21L toward the NiFe site, or alternatively along the nearby His75L pathway that also becomes energetically feasible in certain reaction steps. These local proton-coupled electron transfer (PCET) reactions induce conformational changes around the active site that provide a key coupling element via conserved loop structures to the ion transport activity. We find that H2 forms in a heterolytic proton reduction step, with spin crossovers tuning the energetics along key reaction steps. On a general level, our work showcases the role of electric fields in enzyme catalysis and how these effects are employed by the NiFe active site of Mbh to drive PCET reactions and ion transport.The membrane-bound hydrogenase (Mbh) from Pyrococcus furiosus is an archaeal member of the Complex I superfamily. It catalyzes the reduction of protons to H2 gas powered by a NiFe active site and transduces the free energy into proton pumping and Na+/H+ exchange across the membrane. Despite recent structural advances, the mechanistic principles of H2 catalysis and ion transport in Mbh remain elusive. Here, we probe how the redox chemistry drives the reduction of the proton to H2 and how the catalysis couples to conformational dynamics in the membrane domain of Mbh. By combining large-scale quantum chemical density functional theory (DFT) and correlated ab initio wave function methods with atomistic molecular dynamics simulations, we show that the proton transfer reactions required for the catalysis are gated by electric field effects that direct the protons by water-mediated reactions from Glu21L toward the NiFe site, or alternatively along the nearby His75L pathway that also becomes energetically feasible in certain reaction steps. These local proton-coupled electron transfer (PCET) reactions induce conformational changes around the active site that provide a key coupling element via conserved loop structures to the ion transport activity. We find that H2 forms in a heterolytic proton reduction step, with spin crossovers tuning the energetics along key reaction steps. On a general level, our work showcases the role of electric fields in enzyme catalysis and how these effects are employed by the NiFe active site of Mbh to drive PCET reactions and ion transport.
Zn
is one of the most versatile biologically available metal ions, but accurate modeling of Zn
-containing metalloproteins at the biomolecular force field level can be challenging. Since most Zn
...models are parameterized in bulk solvent, in-depth knowledge about their performance in a protein environment is limited. Thus, we systematically investigate here the behavior of non-polarizable Zn
models for their ability to reproduce experimentally determined metal coordination and ligand binding in metalloproteins. The benchmarking is performed in challenging environments, including mono- (carbonic anhydrase II) and bimetallic (metallo-β-lactamase VIM-2) ligand binding sites. We identify key differences in the performance between the Zn
models with regard to the preferred ligating atoms (charged/non-charged), attraction of water molecules, and the preferred coordination geometry. Based on these results, we suggest suitable simulation conditions for varying Zn
site geometries that could guide the further development of biomolecular Zn
models.
Salmonella infections require the delivery of bacterial effectors into the host cell that alter the regulation of host defense mechanisms. The secreted cysteine protease GtgE from S. Typhimurium ...manipulates vesicular trafficking by modifying the Rab32 subfamily via cleaving the regulatory switch I region. Here we present a comprehensive biochemical, structural, and computational characterization of GtgE in complex with Rab32. Interestingly, GtgE solely processes the inactive GDP-bound GTPase. The crystal structure of the Rab32:GDP substrate in complex with the inactive mutant GtgE
reveals the molecular basis of substrate recognition. In combination with atomistic molecular dynamics simulations, the structural determinants for protein and activity-state specificity are identified. Mutations in a central interaction hub lead to loss of the strict GDP specificity. Our findings shed light on the sequence of host cell manipulation events during Salmonella infection and provide an explanation for the dependence on the co-secreted GTPase activating protein SopD2.
Extensive benchmarking calculations are presented to assess the accuracy of commonly used quantum chemical methods in studying excited state properties of biochromophores. The first few excited ...states of 12 common model chromophores of photoactive yellow protein, green fluorescent protein, and rhodopsin have been studied using approximate second-order coupled-cluster (CC2) and linear-response time-dependent density functional theory (TDDFT) calculations. The study comprises investigations of basis-set dependences on CC2 excitation energies as well as comparisons of the CC2 results with excitation energies obtained at other computational levels and with experimental data. The basis-set study shows that the accuracy of the two lowest excitation energies is generally sufficient when triple-ζ basis sets augmented with polarization functions are employed, whereas the third and higher excited states were found to require diffuse basis functions in the basis set. Augmenting the basis set with diffuse functions contributes less than 0.15 eV to the excitation energies of low-lying excited states, except for some of the studied anionic states and for Rydberg states. Calculations at the TDDFT level using the B3LYP functional show the necessity of stabilizing anions with point charges or counterions when aiming at reliable electronic excitation spectra. The two lowest excitation energies of the green fluorescent protein and rhodopsin chromophores calculated at the CC2 level agree within 0.15 eV with experimental excitation energies, whereas the B3LYP values are somewhat less accurate, with a maximum deviation of 0.27 eV. The computed excitation energies for the photoactive yellow protein chromophore models deviate from available experimental values by 0.3–0.4 eV and 0.1–0.5 eV, at the CC2 and B3LYP levels of theory, respectively.
Directional ion transport across biological membranes plays a central role in many cellular processes. Elucidating the molecular determinants for vectorial ion transport is key to understanding the ...functional mechanism of membrane-bound ion pumps. The extensive investigation of the light-driven proton pump bacteriorhodopsin from Halobacterium salinarum(HsBR) enabled a detailed description of outward proton transport. Although the structure of inward-directed proton pumping rhodopsins is very similar to HsBR, little is known about their protonation pathway, and hence, the molecular reasons for the vectoriality of proton translocation remain unclear. Here, we employ a combined experimental and theoretical approach to tracking protonation steps in the light-driven inward proton pump xenorhodopsin from Nanosalina sp. (NsXeR). Time-resolved infrared spectroscopy reveals the transient deprotonation of D220 concomitantly with deprotonation of the retinal Schiff base. Our molecular dynamics simulations support a proton release pathway from the retinal Schiff base via a hydrogen-bonded water wire leading to D220 that could provide a putative gating point for the proton release and with allosteric interactions to the retinal Schiff base. Our findings support the key role of D220 in mediating proton release to the cytoplasmic side and provide evidence that this residue is not the primary proton acceptor of the proton transiently released by the retinal Schiff base.
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