Structural determination and anticholinesterase assay of C-glycosidic ellagitannins from Lawsonia inermis leaves: A study supported by DFT calculations and molecular docking.

An ellagitannin monomer, lythracin M (1), and a dimer, lythracin D (2), along with eight known monomers (3-10) were isolated from Lawsonia inermis (Lythraceae) leaves. Lythracin M (1) is a C-glycosidic ellagitannin with a flavogallonyl dilactone moiety that participates in the creation of a γ-lactone ring with the anomeric carbon of the glucose core. Lythracin D (2) was determined as an atropisomer of the reported lythcarin D. These newly discovered structures (1 and 2) were determined by intensive spectroscopic experiments and by comparing DFT-calculated 1H1H coupling, 1H NMR chemical shifts, and ECD data with experimental values. The anti-acetylcholinesterase assay of the compounds 1-10 revealed that the C-1 ellagitannin epimers [casuarinin (7; IC50 = 34 ± 2 nM) and stachyurin (8; IC50 = 56 ± 3 nM)], and the new dimer (2; IC50 = 61 ± 4 nM) possess enzyme inhibitory effects comparable to the reference drug (donepezil, IC50 = 44 ± 3 nM). Molecular docking of compounds 1-10 with AChE identified the free galloyl moiety as an important pharmacophore in the anticholinesterase activity of tannins.

Modeling Shows that Rotation about the Peroxide O-O Bond Assists Protein and Lipid Functional Groups in Discriminating between H2O2 and H2O.

Long associated with cell death, hydrogen peroxide (H2O2) is now known to perform many physiological roles. Unraveling its biological mechanisms of action requires atomic-level knowledge of its association with proteins and lipids, which we address here. High-level [MP2(full)/6-311++G(3df,3pd)] ab initio calculations reveal skew rotamers as the lowest-energy states of isolated H2O2 (ϕHOOH ∼ 112°) with minimum and maximum electrostatic potentials (kcal/mol) of -24.8 (Vs,min) and 36.5 (Vs,max), respectively. Transition-state, nonpolar trans rotamers (ϕHOOH ∼ 180°) at 1.2 kcal/mol higher in energy are poorer H-bond acceptors (Vs,min = -16.6) than the skew rotamers, while highly polar cis rotamers (ϕHOOH ∼ 0°) at 7.8 kcal/mol are much better H-bond donors (Vs,max = 52.7). Modeling H2O2 association with neutral and charged analogs of protein residues and lipid groups (e.g., ester, phosphate, choline) reveals that skew rotamers (ϕHOOH = 84-122°) are favored in the neutral and cationic complexes, which display gas-phase interaction energies (ECP, kcal/mol) of -1.5 to -18. The neutral and cationic complexes of H2O exhibit a similar range of stabilities (ECP ∼ -1 to -18). However, considerably higher energies (ECP ∼ -14 to -36) are found for the H2O2 complexes of the anionic ligands, which are stabilized by charge-assisted H-bond donation from cis and distorted cis rotamers (ϕHOOH = 0-60°). H2O is a much poorer H-bond donor (Vs,max = 33.4) than cis-H2O2, so its anionic complexes are significantly weaker (ECP ∼ -11 to -20). Thus, by dictating the rotamer preference of H2O2, functional groups in biomolecules can discriminate between H2O2 and H2O. Finally, exploiting the present ab initio data, we calibrated and validated our published molecular mechanics model for H2O2 (Orabi, E. A.; English, A. M. J. Chem. Theory Comput. 2018, 14, 2808-2821) to provide an important tool for simulating H2O2 in biology.

Sublethal Paraquat Confers Multidrug Tolerance in Pseudomonas aeruginosa by Inducing Superoxide Dismutase Activity and Lowering Envelope Permeability.

Stressors and environmental cues shape the physiological state of bacteria, and thus how they subsequently respond to antibiotic toxicity. To understand how superoxide stress can modulate survival to bactericidal antibiotics, we examined the effect of intracellular superoxide generators, paraquat and menadione, on stationary-phase antibiotic tolerance of the opportunistic pathogen, Pseudomonas aeruginosa. We tested how pre-challenge with sublethal paraquat and menadione alters the tolerance to ofloxacin and meropenem in wild-type P. aeruginosa and mutants lacking superoxide dismutase (SOD) activity (sodAB), the paraquat responsive regulator soxR, (p)ppGpp signaling (relA spoT mutant), or the alternative sigma factor rpoS. We confirmed that loss of SOD activity impairs ofloxacin and meropenem tolerance in stationary phase cells, and found that sublethal superoxide generators induce drug tolerance by stimulating SOD activity. This response is rapid, requires de novo protein synthesis, and is RpoS-dependent but does not require (p)ppGpp signaling nor SoxR. We further showed that pre-challenge with sublethal paraquat induces a SOD-dependent reduction in cell-envelope permeability and ofloxacin penetration. Our results highlight a novel mechanism of hormetic protection by superoxide generators, which may have important implications for stress-induced antibiotic tolerance in P. aeruginosa cells.

Quantifying Heme-Protein Maturation from Ratiometric Fluorescence Lifetime Measurements on the Single Fluorophore in Its GFP Fusion.

Protein maturation by heme insertion is a common post-translation modification of key biological importance. Nonetheless, where and when this maturation occurs in eukaryotic cells remain unknown for most heme proteins. Here, we demonstrate for the first time that the maturation of a chromosomally expressed, endogenous heme protein fused to a green fluorescent protein (GFP) can be tracked in live cells. Selecting yeast cytochrome c peroxidase (Ccp1) as our model heme-binding protein, we first characterized the emission in vitro of recombinant Ccp1-GFP with GFP fused C-terminally to Ccp1 by the linker GRRIPGLIN. Time-correlated single-photon counting reveals a single fluorescence lifetime for heme-free apoCcp1-GFP, τ0 = 2.84 ± 0.01 ns. Heme bound to Ccp1 only partially quenches GFP fluorescence since holoCcp1-GFP exhibits two lifetimes, τ1 = 0.95 ± 0.02 and τ2 = 2.46 ± 0.03 ns with fractional amplitudes a1 = 38 ± 1.5% and a2 = 62 ± 1.5%. Also, τ and a are independent of Ccp1-GFP concentration and solution pH between 5.5 and 8.0, and a standard plot of a1 vs % holoCcp1-GFP in mixtures with apoCcp1-GFP is linear, establishing that the fraction of Ccp1-GFP with heme bound can be determined from a1. Fluorescence lifetime imaging microscopy (FLIM) of live yeast cells chromosomally expressing the same Ccp1-GFP fusion revealed 30% holoCcp1-GFP (i.e., mature Ccp1) and 70% apoCcp1-GFP in agreement with biochemical measurements on cell lysates. Thus, ratiometric fluorescence lifetime measurements offer promise for probing heme-protein maturation in live cells, and we can dispense with the reference fluorophore required for ratiometric intensity-based measurements.

Expanding the range of binding energies and oxidizability of biologically relevant S-aromatic interactions: imidazolium and phenolate binding to sulfoxide and sulfone.

Oxidation and protonation/deprotonation strongly impact intermolecular noncovalent interactions. For example, S-aromatic interactions are stabilized up to three-fold in the gas phase on oxidation of the sulfur ligand or protonation/deprotonation of the aromatic. To probe if such stabilizing effects are additive and to model interactions of oxidized methionine (MetOn) with protonated histidine and deprotonated tyrosine residues in proteins, we examined Me2SOn (n = 1, 2) binding to imidazolium, phenolate and their 4-methylated forms. Ab initio MP2(full)/6-311++G(d,p) gas-phase calculations reveal that the Me2SOn-imidazolium complexes adopt edge-on geometry with σ-type (N/C-HarO) H-bonding and interaction energies of -17.2 to -31.1 kcal mol-1. The less stable (-13.8 to -21.0 kcal mol-1) Me2SOn-phenolates possess en-face geometry stabilized by π-type (C-Hπar) H-bonding. Comparing these energies with those reported for the Me2S-neutral aromatics affirms the additive effects of ligand protonation/deprotonation and oxidation on gas-phase stability. However, this is not the case in water although the aqueous complexes retain their preferred gas-phase σ- and π-type H-bonded structures. Binding free energies (kcal mol-1) calculated from molecular dynamics simulations in bulk water (preceded by CHARMM36 force field calibration where necessary) reveal that Me2SO-imidazolium (-4.4) is more stable than Me2SO-phenolate (-2.4) but Me2SO2-imidazolium (-0.6) is less stable than Me2SO2-phenolate (-3.8). Vertical ionization potentials (IPV) calculated for the gas-phase complexes indicate that the Me2SOn-phenolates, but not the Me2SOn-imidazoles, are oxidizable under biological conditions. Charge transfer from the phenolate increases its IPV by ∼20%, decreasing its susceptibility to oxidation. Overall, this work provides fundamental data to predict the behaviour of protein-based MetOn-aromatic-ion interactions.

Ctt1 catalase activity potentiates antifungal azoles in the emerging opportunistic pathogen Saccharomyces cerevisiae.

Fungi respond to antifungal drugs by increasing their antioxidant stress response. How this impacts antifungal efficacy remains controversial and not well understood. Here we examine the role of catalase activity in the resistance of Saccharomyces cerevisiae to the common antifungals, fluconazole and miconazole, for which we report minimum inhibitory concentrations (MICs) of 104 and 19 µM, respectively. At sub-MIC concentrations, fluconazole and miconazole stimulate catalase activity 2-3-fold but, unexpectedly, deletion of cytosolic catalase (ctt1) makes cells more resistant to these azoles and to clotrimazole, itraconazole and posaconazole. On the other hand, upregulating Ctt1 activity by preconditioning with 0.2 mM H2O2 potentiates miconazole 32-fold and fluconazole 4-fold. Since H2O2 preconditioning does not alter the resistance of ctt1Δ cells, which possess negligible catalase activity, we link azole potentiation with Ctt1 upregulation. In contrast, sod2Δ cells deleted for mitochondrial superoxide dismutase are 4-8-fold more azole sensitive than wild-type cells, revealing that Sod2 activity protects cells against azole toxicity. In fact, the ctt1Δ mutant has double the Sod2 activity of wild-type cells so ctt1 deletion increases azole resistance in part by Sod2 upregulation. Notably, deletion of peroxisomal/mitochondrial cta1 or cytosolic sod1 does not alter fluconazole or miconazole potency.

Predicting structural and energetic changes in Met-aromatic motifs on methionine oxidation to the sulfoxide and sulfone.

Noncovalent interactions between Met and aromatic residues define a common Met-aromatic motif in proteins. Met oxidation to MetOn (n = 1 sulfoxide, n = 2 sulfone) alters protein stability and function. To predict the chemical and physical consequences of such oxidations, we modeled the chemistry and redox properties of MetOn-aromatic complexes in depth for comparison with our Met-aromatic models (E. A. Orabi and A. M. English, J. Phys. Chem. B, 2018, 122, 3760). We describe here ab initio quantum mechanical calculations at the MP2(full)/6-311++G(d,p) level of theory on complexes of MetOn (n = 1, 2; modeled by Me2SO and Me2SO2) with models of the side-chains of Phe (benzene, toluene), Trp (indole, 3-methylindole), Tyr (phenol, 4-methylphenol) and His (imidazole, 4-methylimidazole). Binding energies of the global minimum conformers (-3.4 to -11.9 kcal mol-1) indicate that the gas-phase Me2SOn-aromatics are 40-115% more stable than the Me2S-aromatics. Binding of S between the edge and face of the aromatic ring is favored in most complexes as it accommodates both robust σ- and π-type H-bonding. Interactions involving the σ-holes on the S atoms (σ-holeπar and σ-holeNar/Oar), as well as Sπ interactions in the sulfoxides, contribute to complex stability. Complexation modulates the ionization potential (IP) of the interacting fragments with the binding geometry dictating the center oxidized in the Me2SO-aromatics whereas the aromatic is oxidized in the Me2SO2 complexes because of the sulfone's high IP. Potentials of mean force reveal binding free energies of -0.2 to -0.7 kcal mol-1 in bulk water, which indicates that the Me2SOn-aromatics are up to 80% less stable than the corresponding aqueous Me2S-aromatics. Molecular dynamics simulations predict that Me2SOn preferentially interacts with the ring face and expose the dominance of π- vs. σ-type H-bonding in the hydrated complexes as found for the Me2S-aromatics. Our modeling will inform how Met/MetOn-aromatic motifs are determinants of redox-induced changes in proteins.

Superoxide dismutase activity confers (p)ppGpp-mediated antibiotic tolerance to stationary-phase Pseudomonas aeruginosa.

Metabolically quiescent bacteria represent a large proportion of those in natural and host environments, and they are often refractory to antibiotic treatment. Such drug tolerance is also observed in the laboratory during stationary phase, when bacteria face stress and starvation-induced growth arrest. Tolerance requires (p)ppGpp signaling, which mediates the stress and starvation stringent response (SR), but the downstream effectors that confer tolerance are unclear. We previously demonstrated that the SR is linked to increased antioxidant defenses in Pseudomonas aeruginosa We now demonstrate that superoxide dismutase (SOD) activity is a key factor in SR-mediated multidrug tolerance in stationary-phase P. aeruginosa Inactivation of the SR leads to loss of SOD activity and decreased multidrug tolerance during stationary phase. Genetic or chemical complementation of SOD activity of the ΔrelA spoT mutant (ΔSR) is sufficient to restore antibiotic tolerance to WT levels. Remarkably, we observe high membrane permeability and increased drug internalization upon ablation of SOD activity. Combined, our results highlight an unprecedented mode of SR-mediated multidrug tolerance in stationary-phase P. aeruginosa and suggest that inhibition of SOD activity may potentiate current antibiotics.

LC-MS/MS Proteoform Profiling Exposes Cytochrome c Peroxidase Self-Oxidation in Mitochondria and Functionally Important Hole Hopping from Its Heme.

LC-MS/MS profiling reveals that the proteoforms of cytochrome c peroxidase (Ccp1) isolated from respiring yeast mitochondria are oxidized at numerous Met, Trp, and Tyr residues. In vitro oxidation of recombinant Ccp1 by H2O2 in the absence of its reducing substrate, ferrocytochrome c, gives rise to similar proteoforms, indicating uncoupling of Ccp1 oxidation and reduction in mitochondria. The oxidative modifications found in the Ccp1 proteoforms are consistent with radical transfer (hole hopping) from the heme along several chains of redox-active residues (Trp, Met, Tyr). These modifications delineate likely hole-hopping pathways to novel substrate-binding sites. Moreover, a decrease in recombinant Ccp1 oxidation by H2O2 in vitro in the presence of glutathione supports a protective role for hole hopping to this antioxidant. Isolation and characterization of extramitochondrial Ccp1 proteoforms reveals that hole hopping from the heme in these proteoforms results in selective oxidation of the proximal heme ligand (H175) and heme labilization. Previously, we demonstrated that this labilized heme is recruited for catalase maturation (Kathiresan, M.; Martins, D.; English, A. M. Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 17468-17473; DOI: 10.1073/pnas.1409692111 ). Following heme release, apoCcp1 exits mitochondria, yielding the extramitochondrial proteoforms that we characterize here. The targeting of Ccp1 for selective H175 oxidation may be linked to the phosphorylation status of Y153 close to the heme since pY153 is abundant in certain proteoforms. In sum, when insufficient electrons from ferrocytochrome c are available to Ccp1 in mitochondria, hole hopping from its heme expands its physiological functions. Specifically, we observe an unprecedented hole-hopping sequence for heme labilization and identify hole-hopping pathways from the heme to novel substrates and to glutathione at Ccp1's surface. Furthermore, our results underscore the power of proteoform profiling by LC-MS/MS in exploring the cellular roles of oxidoreductases.

A Simple Additive Potential Model for Simulating Hydrogen Peroxide in Chemical and Biological Systems.

Hydrogen peroxide (H2O2) has numerous industrial, environmental, medical, cosmetic, and biological applications. Given its importance, we provide a simple model as an alternative to experiment for studying the properties of pure liquid H2O2 and its concentrated aqueous solutions, which are hazardous, and for understanding the biological roles of H2O2 at the molecular level. A four-site additive model is calibrated for H2O2 based on the ab initio and experimental properties of the gaseous monomer and the density and heat of vaporization of liquid H2O2 at 0 °C. Our model together with the TIP3P water model reproduce the ab initio binding energies of (H2O2) m, H2O2· nH2O, and nH2O2·H2O clusters ( m = 2, 3 and n = 1, 2) calculated at the MP2 level using the 6-311++G(d,p) or the 6-311++G(3df,3pd) basis set. It yields structure, the self-diffusion coefficient, heat capacity, and densities at temperatures up to 200 °C of the pure liquid in good agreement with experiment. The model correctly predicts the hydration free energy of H2O2 and reproduces the experimental density of aqueous H2O2 solutions at 0-96 °C. Investigation of the solvation of H2O2 and H2O in aqueous H2O2 solutions reveals that, as in the gas phase, H2O2 is a better H-bond donor but poorer acceptor than H2O and the bonding stability follows the order Op-Hp···Ow > Ow-Hw···Ow ≥ Op-Hp···Op > Ow-Hw···Op. Stronger H-bonding in H2O/H2O2 mixtures than in the pure liquids is consistent with exothermic heats of mixing and explains why the observed density and vapor pressure of the aqueous solutions are higher and lower, respectively, than expected from ideal mixing. Results also show that H2O2 adopts a skewed equilibrium geometry in gas and liquid phases but more polar cis and nonpolar trans conformations also are accessible and will stabilize H2O2 in environments of different polarity. In sum, our simple model presents a reliable tool for simulating H2O2 in chemistry and biology.

Modeling Protein S-Aromatic Motifs Reveals Their Structural and Redox Flexibility.

S-aromatic motifs are important noncovalent forces for protein stability and function but remain poorly understood. Hence, we performed quantum calculations at the MP2(full)/6-311++G(d,p) level on complexes between Cys (H2S, MeSH) and Met (Me2S) models with models of Phe (benzene, toluene), Trp (indole, 3-methylindole), Tyr (phenol, 4-methylphenol), and His (imidazole, 4-methylimidazole). The most stable gas-phase conformers exhibit binding energies of -2 to -6 kcal/mol, and the S atom lies perpendicular to the ring plane. This reveals preferential interaction with the ring π-system, except in the imidazoles where S binds edge-on to an N atom. Complexation tunes the gas-phase vertical ionization potentials of the ligands over as much as 1 eV, and strong σ- or π-type H-bonding supports charge transfer to the H-bond donor, rendering it more oxidizable. When the S atom acts as an H-bond acceptor (N/O-Har···S), calibration of the CHARMM36 force field (by optimizing pair-specific Lennard-Jones parameters) is required. Implementing the optimized parameters in molecular dynamics simulations in bulk water, we find stable S-aromatic complexes with binding free energies of -0.6 to -1.1 kcal/mol at ligand separations up to 8 Å. The aqueous S-aromatics exhibit flexible binding conformations, but edge-on conformers are less stable in water. Reflecting this, only 0.3 to 10% of the S-indole, S-phenol, and S-imidazole structures are stabilized by N/O-Har···S or S-H···Oar/Nar σ-type H-bonding. The wide range of energies and geometries found for S-aromatic interactions and their tunable redox properties expose the versatility and variability of the S-aromatic motif in proteins and allow us to predict a number of their reported properties.

LC-MS/MS suggests that hole hopping in cytochrome c peroxidase protects its heme from oxidative modification by excess H2O2.

We recently reported that cytochrome c peroxidase (Ccp1) functions as a H2O2 sensor protein when H2O2 levels rise in respiring yeast. The availability of its reducing substrate, ferrocytochrome c (CycII), determines whether Ccp1 acts as a H2O2 sensor or peroxidase. For H2O2 to serve as a signal it must modify its receptor so we employed high-performance LC-MS/MS to investigate in detail the oxidation of Ccp1 by 1, 5 and 10 M eq. of H2O2 in the absence of CycII to prevent peroxidase activity. We observe strictly heme-mediated oxidation, implicating sequential cycles of binding and reduction of H2O2 at Ccp1's heme. This results in the incorporation of ∼20 oxygen atoms predominantly at methionine and tryptophan residues. Extensive intramolecular dityrosine crosslinking involving neighboring residues was uncovered by LC-MS/MS sequencing of the crosslinked peptides. The proximal heme ligand, H175, is converted to oxo-histidine, which labilizes the heme but irreversible heme oxidation is avoided by hole hopping to the polypeptide until oxidation of the catalytic distal H52 in Ccp1 treated with 10 M eq. of H2O2 shuts down heterolytic cleavage of H2O2 at the heme. Mapping of the 24 oxidized residues in Ccp1 reveals that hole hopping from the heme is directed to three polypeptide zones rich in redox-active residues. This unprecedented analysis unveils the remarkable capacity of a polypeptide to direct hole hopping away from its active site, consistent with heme labilization being a key outcome of Ccp1-mediated H2O2 signaling. LC-MS/MS identification of the oxidized residues also exposes the bias of electron paramagnetic resonance (EPR) detection toward transient radicals with low O2 reactivity.

Do Mammalian Cells Really Need to Export and Import Heme?

Heme is a cofactor that is essential to almost all forms of life. The production of heme is a balancing act between the generation of the requisite levels of the end-product and protection of the cell and/or organism against any toxic substrates, intermediates and, in this case, end-product. In this review, we provide an overview of our understanding of the formation and regulation of this metallocofactor and discuss new research on the cell biology of heme homeostasis, with a focus on putative transmembrane transporters now proposed to be important regulators of heme distribution. The main text is complemented by a discussion dedicated to the intricate chemistry and biochemistry of heme, which is often overlooked when new pathways of heme transport are conceived.

Benchmarking Rapid TLES Simulations of Gas Diffusion in Proteins: Mapping O2 Migration and Escape in Myoglobin as a Case Study.

Standard molecular dynamics (MD) simulations of gas diffusion consume considerable computational time and resources even for small proteins. To combat this, temperature-controlled locally enhanced sampling (TLES) examines multiple diffusion trajectories per simulation by accommodating multiple noninteracting copies of a gas molecule that diffuse independently, while the protein and water molecules experience an average interaction from all copies. Furthermore, gas migration within a protein matrix can be accelerated without altering protein dynamics by increasing the effective temperature of the TLES copies. These features of TLES enable rapid simulations of gas diffusion within a protein matrix at significantly reduced (∼98%) computational cost. However, the results of TLES and standard MD simulations have not been systematically compared, which limits the adoption of the TLES approach. We address this drawback here by benchmarking TLES against standard MD in the simulation of O2 diffusion in myoglobin (Mb) as a case study since this model system has been extensively characterized. We find that 2 ns TLES and 108 ns standard simulations map the same network of diffusion tunnels in Mb and uncover the same docking sites, barriers, and escape portals. We further discuss the influence of simulation time as well as the number of independent simulations on the O2 population density within the diffusion tunnels and on the sampling of Mb's conformational space as revealed by principal component analysis. Overall, our comprehensive benchmarking reveals that TLES is an appropriate and robust tool for the rapid mapping of gas diffusion in proteins when the kinetic data provided by standard MD are not required. Furthermore, TLES provides explicit ligand diffusion pathways, unlike most rapid methods.

Quaternary-Linked Changes in Structure and Dynamics That Modulate O2 Migration within Hemoglobin's Gas Diffusion Tunnels.

Atomistic molecular dynamics simulations of diffusion of O2 from the hemes to the external solvent in the α- and ß-subunits of the human hemoglobin (HbA) tetramer reveal transient gas tunnels that are not seen in crystal structures. We find here that the tunnel topology, which encompasses the reported experimental Xe binding cavities, is identical in HbA's T, R, and R2 quaternary states. However, the O2 population in the cavities and the preferred O2 escape portals vary significantly with quaternary structure. For example, most O2 molecules escape from the T ß-subunit via the cavity at the center of the tetramer, but direct exit from the distal heme pocket dominates in the R2 ß-subunit. To understand what triggers the quaternary-linked redistribution of O2 within its tunnels, we examined how the simulated tertiary structure and dynamics of each subunit differs among T, R, and R2 and report that minor adjustments in α-chain dynamics and ß-heme position modulate O2 distribution and escape in HbA. Coupled to the ß-heme position, residue ßF71 undergoes quaternary-linked conformations that strongly regulate O2 migration between the ß-subunit and HbA's central cavity. Remarkably, the distal histidine (HisE7) remains in a closed conformation near the α- and ß-hemes in all states, but this does not prevent an average of 23, 31, and 46% of O2 escapes from the distal heme pockets of T, R, and R2, respectively, via several distal portals, with the balance of escapes occurring via the interior tunnels. Furthermore, preventing or restricting the access of O2 to selected cavities by mutating HisE7 and other heme pocket residues to tryptophan reveals how O2 migration adjusts to the bulky indole ring and sheds light on the experimental ligand binding kinetics of these variants. Overall, our simulations underscore the high gas porosity of HbA in its T, R, and R2 quaternary states and provide new mechanistic insights into why undergoing transitions among these states likely ensures effective O2 delivery by this tetrameric protein.

O2 and Water Migration Pathways between the Solvent and Heme Pockets of Hemoglobin with Open and Closed Conformations of the Distal HisE7.

Hemoglobin transports O2 by binding the gas at its four hemes. Hydrogen bonding between the distal histidine (HisE7) and heme-bound O2 significantly increases the affinity of human hemoglobin (HbA) for this ligand. HisE7 is also proposed to regulate the release of O2 to the solvent via a transient E7 channel. To reveal the O2 escape routes controlled by HisE7 and to evaluate its role in gating heme access, we compare simulations of O2 diffusion from the distal heme pockets of the T and R states of HbA performed with HisE7 in its open (protonated) and closed (neutral) conformations. Irrespective of HisE7's conformation, we observe the same four or five escape routes leading directly from the α- or ß-distal heme pockets to the solvent. Only 21-53% of O2 escapes occur via these routes, with the remainder escaping through routes that encompass multiple internal cavities in HbA. The conformation of the distal HisE7 controls the escape of O2 from the heme by altering the distal pocket architecture in a pH-dependent manner, not by gating the E7 channel. Removal of the HisE7 side chain in the GlyE7 variant exposes the distal pockets to the solvent, and the percentage of O2 escapes to the solvent directly from the α- or ß-distal pockets of the mutant increases to 70-88%. In contrast to O2, the dominant water route from the bulk solvent is gated by HisE7 because protonation and opening of this residue dramatically increase the rate of influx of water into the empty distal heme pockets. The occupancy of the distal heme site by a water molecule, which functions as an additional nonprotein barrier to binding of the ligand to the heme, is also controlled by HisE7. Overall, analysis of gas and water diffusion routes in the subunits of HbA and its GlyE7 variant sheds light on the contribution of distal HisE7 in controlling polar and nonpolar ligand movement between the solvent and the hemes.

Peroxynitrite and hydrogen peroxide elicit similar cellular stress responses mediated by the Ccp1 sensor protein.

Peroxynitrite [ONOO(H)] is an oxidant associated with deleterious effects in cells. Because it is an inorganic peroxide that reacts rapidly with peroxidases, we speculated that cells may respond to ONOO(H) and H2O2 challenge in a similar manner. We exposed yeast cells to SIN-1, a well-characterized ONOO(H) generator, and observed stimulation of catalase and peroxiredoxin (Prx) activities. Previously, we reported that H2O2 challenge increases these activities in wild-type cells and in cells producing the hyperactive mutant H2O2 sensor Ccp1(W191F) but not in Ccp1-knockout cells (ccp1Δ). We find here that the response of ccp1Δ and ccp1(W191F) cells to SIN-1 mirrors that to H2O2, identifying Ccp1 as a sensor of both peroxides. SIN-1 simultaneously releases (•)NO and O2(•-), which react to form ONOO(H), but exposure of the three strains separately to an (•)NO donor (spermine-NONOate) or an O2(•-) generator (paraquat) mainly depresses catalase or Prx activity, whereas co-challenge with the NONOate and paraquat stimulates these activities. Because Ccp1 appears to sense ONOO(H) in cells, we examined its reaction with ONOO(H) in vitro and found that peroxynitrous acid (ONOOH) rapidly (k2>10(6)M(-1)s(-1)) oxidizes purified Ccp1 to an intermediate with spectral and ferrocytochrome-oxidizing properties indistinguishable from those of its well-characterized compound I formed with H2O2. Importantly, the nitrite released from ONOOH is not oxidized to (•)NO2 by Ccp1(׳)s compound I, unlike peroxidases involved in immune defense. Overall, our results reveal that yeast cells mount a common antioxidant response to ONOO(H) and H2O2, with Ccp1 playing a pivotal role as an inorganic peroxide sensor.

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria.

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H2O2 levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H2O2 because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1's heme is labilized by hyperoxidation of the protein during the burst in H2O2 production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H2O2 in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼ 85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H2O2 via a previously unidentified mechanism involving H2O2-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H2O2 detoxification.

SOD1 oxidation and formation of soluble aggregates in yeast: relevance to sporadic ALS development.

Misfolding and aggregation of copper-zinc superoxide dismutase (Sod1) are observed in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Mutations in Sod1 lead to familial ALS (FALS), which is a late-onset disease. Since oxidative damage to proteins increases with age, it had been proposed that oxidation of Sod1 mutants may trigger their misfolding and aggregation in FALS. However, over 90% of ALS cases are sporadic (SALS) with no obvious genetic component. We hypothesized that oxidation could also trigger the misfolding and aggregation of wild-type Sod1 and sought to confirm this in a cellular environment. Using quiescent, stationary-phase yeast cells as a model for non-dividing motor neurons, we probed for post-translational modification (PTM) and aggregation of wild-type Sod1 extracted from these cells. By size-exclusion chromatography (SEC), we isolated two populations of Sod1 from yeast: a low-molecular weight (LMW) fraction that is catalytically active and a catalytically inactive, high-molecular weight (HMW) fraction. High-resolution mass spectrometric analysis revealed that LMW Sod1 displays no PTMs but HMW Sod1 is oxidized at Cys146 and His71, two critical residues for the stability and folding of the enzyme. HMW Sod1 is also oxidized at His120, a copper ligand, which will promote loss of this catalytic metal cofactor essential for SOD activity. Monitoring the fluorescence of a Sod1-green-fluorescent-protein fusion (Sod1-GFP) extracted from yeast chromosomally expressing this fusion, we find that HMW Sod1-GFP levels increase up to 40-fold in old cells. Thus, we speculate that increased misfolding and inclusion into soluble aggregates is a consequence of elevated oxidative modifications of wild-type Sod1 as cells age. Our observations argue that oxidative damage to wild-type Sod1 initiates the protein misfolding mechanisms that give rise to SALS.

Catalase activity is stimulated by H(2)O(2) in rich culture medium and is required for H(2)O(2) resistance and adaptation in yeast.

Catalases are efficient scavengers of H2O2 and protect cells against H2O2 stress. Examination of the H2O2 stimulon in Saccharomyces cerevisiae revealed that the cytosolic catalase T (Ctt1) protein level increases 15-fold on H2O2 challenge in synthetic complete media although previous work revealed that deletion of the CCT1 or CTA1 genes (encoding peroxisomal/mitochondrial catalase A) does not increase the H2O2 sensitivity of yeast challenged in phosphate buffer (pH 7.4). This we attributed to our observation that catalase activity is depressed when yeast are challenged with H2O2 in nutrient-poor media. Hence, we performed a systematic comparison of catalase activity and cell viability of wild-type yeast and of the single catalase knockouts, ctt1∆ and cta1∆, following H2O2 challenge in nutrient-rich medium (YPD) and in phosphate buffer (pH 7.4). Ctt1 but not Cta1 activity is strongly induced by H2O2 when cells are challenged in YPD but suppressed when cells are challenged in buffer. Consistent with the activity results, exponentially growing ctt1∆ cells in YPD are more sensitive to H2O2 than wild-type or cta1∆ cells, whereas in buffer all three strains exhibit comparable H2O2 hypersensitivity. Furthermore, catalase activity is increased during adaptation to sublethal H2O2 concentrations in YPD but not in buffer. We conclude that induction of cytosolic Ctt1 activity is vital in protecting yeast against exogenous H2O2 but this activity is inhibited by H2O2 when cells are challenged in nutrient-free media.