Autocatalytic Mechanism in the Anaerobic Reduction of Metmyoglobin by Sulfide Species.

The mechanism of the metal centered reduction of metmyoglobin (MbFeIII) by sulfide species (H2S/HS-) under an argon atmosphere has been studied by a combination of spectroscopic, kinetic, and computational methods. Asymmetric S-shaped time-traces for the formation of MbFeII at varying ratios of excess sulfide were observed at pH 5.3 < pH < 8.0 and 25 °C, suggesting an autocatalytic reaction mechanism. An increased rate at more alkaline pHs points to HS- as relevant reactive species for the reduction. The formation of the sulfanyl radical (HS•) in the slow initial phase was assessed using the spin-trap phenyl N-tert-butyl nitrone. This radical initiates the formation of S-S reactive species as disulfanuidyl/ disulfanudi-idyl radical anions and disulfide (HSSH•-/HSS•2- and HSS-, respectively). The autocatalysis has been ascribed to HSS-, formed after HSSH•-/HSS•2- disproportionation, which behaves as a fast reductant toward the intermediate complex MbFeIII(HS-). We propose a reaction mechanism for the sulfide-mediated reduction of metmyoglobin where only ferric heme iron initiates the oxidation of sulfide species. Beside the chemical interest, this insight into the MbFeIII/sulfide reaction under an argon atmosphere is relevant for the interpretation of biochemical aspects of ectopic myoglobins found on hypoxic tissues toward reactive sulfur species.

On the accurate estimation of free energies using the jarzynski equality.

The Jarzynski equality is one of the most widely celebrated and scrutinized nonequilibrium work theorems, relating free energy to the external work performed in nonequilibrium transitions. In practice, the required ensemble average of the Boltzmann weights of infinite nonequilibrium transitions is estimated as a finite sample average, resulting in the so-called Jarzynski estimator, ΔF^J . Alternatively, the second-order approximation of the Jarzynski equality, though seldom invoked, is exact for Gaussian distributions and gives rise to the Fluctuation-Dissipation estimator ΔF^FD . Here we derive the parametric maximum-likelihood estimator (MLE) of the free energy ΔF^ML considering unidirectional work distributions belonging to Gaussian or Gamma families, and compare this estimator to ΔF^J . We further consider bidirectional work distributions belonging to the same families, and compare the corresponding bidirectional ΔF^ML∗ to the Bennett acceptance ratio ( ΔF^BAR ) estimator. We show that, for Gaussian unidirectional work distributions, ΔF^FD is in fact the parametric MLE of the free energy, and as such, the most efficient estimator for this statistical family. We observe that ΔF^ML and ΔF^ML∗ perform better than ΔF^J and ΔF^BAR , for unidirectional and bidirectional distributions, respectively. These results illustrate that the characterization of the underlying work distribution permits an optimal use of the Jarzynski equality. © 2018 Wiley Periodicals, Inc.

Mechanism of Sulfide Binding by Ferric Hemeproteins.

The reaction of hydrogen sulfide (H2S) with hemeproteins is a key physiological reaction; still, its mechanism and implications are not completely understood. In this work, we propose a combination of experimental and theoretical tools to shed light on the reaction in model system microperoxidase 11 (MP11-FeIII) and myoglobin (Mb-FeIII), from the estimation of the intrinsic binding constants of the species H2S and hydrosulfide (HS-), and the computational description of the overall binding process. Our results show that H2S and HS- are the main reactive species in Mb-FeIII and MP11-FeIII, respectively, and that the magnitude of their intrinsic binding constants are similar to most of the binding constants reported so far for hemeproteins systems and model compounds. However, while the binding of HS- to Mb-FeIII was negligible, the binding of H2S to MP11-FeIII was significant, providing a frame for a discriminated analysis of both species and revealing differential mechanistic aspects. A joint inspection of the kinetic data and the free energy profiles of the binding processes suggests that a dissociative mechanism with the release of a coordinated water molecule as rate limiting step is operative in the binding of H2S to Mb-FeIII and that the binding of HS- is prevented in the access to the protein matrix. For the MP11-FeIII case, where no access restrictions for the ligands are present, an associative component in the mechanism seems to be operative. Overall, the results suggest that if accessing the active site then both H2S and HS- are capable of binding a ferric heme moiety.

Reactivity of inorganic sulfide species toward a heme protein model.

The reactivity of inorganic sulfide species toward heme peptides was explored under biorelevant conditions in order to unravel the molecular details of the reactivity of the endogenous hydrogen sulfide toward heme proteins. Unlike ferric porphyrinates, which are reduced by inorganic sulfide, some heme proteins can form stable Fe(III)-sulfide adducts. To isolate the protein factors ruling the redox chemistry, we used as a system model, the undecapeptide microperoxidase (MP11), a heme peptide derived from cytochrome c proteolysis that retains the proximal histidine bound to the Fe(III) atom. Upon addition of gaseous hydrogen sulfide (H2S) at pH 6.8, the UV-vis spectra of MP11 closely resembled those of the low-spin ferric hydroxo complex (only attained at an alkaline pH) and cysteine or alkylthiol derivatives, suggesting that the Fe(III) reduction was prevented. The low-frequency region of the resonance Raman spectrum revealed the presence of an Fe(III)-S band at 366 cm(-1) and the general features of a low-spin hexacoordinated heme. Anhydrous sodium sulfide (Na2S) was the source of sulfide of choice for the kinetic evaluation of the process. Theoretical calculations showed no distal stabilization mechanisms for bound sulfide species in MP11, highlighting a key role of the proximal histidine for the stabilization of the Fe(III)-S adducts of heme compounds devoid of distal counterparts, which is significant with regard to the biochemical reactivity of endogenous hydrogen sulfide.

Sulfide binding properties of truncated hemoglobins.

The truncated hemoglobins from Bacillus subtilis (Bs-trHb) and Thermobifida fusca (Tf-trHb) have been shown to form high-affinity complexes with hydrogen sulfide in their ferric state. The recombinant proteins, as extracted from Escherichia coli cells after overexpression, are indeed partially saturated with sulfide, and even highly purified samples still contain a small but significant amount of iron-bound sulfide. Thus, a complete thermodynamic and kinetic study has been undertaken by means of equilibrium and kinetic displacement experiments to assess the relevant sulfide binding parameters. The body of experimental data indicates that both proteins possess a high affinity for hydrogen sulfide (K = 5.0 x 10(6) and 2.8 x 10(6) M(-1) for Bs-trHb and Tf-trHb, respectively, at pH 7.0), though lower with respect to that reported previously for the sulfide avid Lucina pectinata I hemoglobins (2.9 x 10(8) M(-1)). From the kinetic point of view, the overall high affinity resides in the slow rate of sulfide release, attributed to hydrogen bonding stabilization of the bound ligand by distal residue WG8. A set of point mutants in which these residues have been replaced with Phe indicates that the WG8 residue represents the major kinetic barrier to the escape of the bound sulfide species. Accordingly, classical molecular dynamics simulations of SH(-)-bound ferric Tf-trHb show that WG8 plays a key role in the stabilization of coordinated SH(-) whereas the YCD1 and YB10 contributions are negligible. Interestingly, the triple Tf-trHb mutant bearing only Phe residues in the relevant B10, G8, and CD1 positions is endowed with a higher overall affinity for sulfide characterized by a very fast second-order rate constant and 2 order of magnitude faster kinetics of sulfide release with respect to the wild-type protein. Resonance Raman spectroscopy data indicate that the sulfide adducts are typical of a ferric iron low-spin derivative. In analogy with other low-spin ferric sulfide adducts, the strong band at 375 cm(-1) is tentatively assigned to a Fe-S stretching band. The high affinity for hydrogen sulfide is thought to have a possible physiological significance as H(2)S is produced in bacteria at metabolic steps involved in cysteine biosynthesis and hence in thiol redox homeostasis.

Hemeproteins as Targets for Sulfide Species.

Significance: Sulfides are endogenous and ubiquitous signaling species that share the hemeproteins as biochemical targets with O2, nitric oxide, and carbon monoxide. The description of the binding mechanisms is mandatory to anticipate the biochemical relevance of the interaction. Recent Advances: The binding of sulfide to ferric hemeproteins has been described in more than 40 systems, including native proteins, mutants, and model systems. Mechanisms of sulfide binding to ferric hemeproteins have been examined by a combination of kinetic and computational experiments. The distal control of the association process, dissected into the migration of the ligand to the active site and the binding event, reveals that neutral hydrogen sulfide (H2S) reaches the active site and is the predominant binding ligand, while the HS- is excluded by the protein matrix. Experiments with model compounds, devoid of a protein scaffold, reveal that both H2S and HS- can bind the ferric heme if accessing the site. A critical role of the proximal ligand in the prevention of the metal-centered reduction has been experimentally assessed. For metmyoglobin and methemoglobin, the coordination of sulfide leads to noncanonical functions: sulfide storage and its oxidative detoxification have been evidenced under physiological and excess sulfide concentrations, respectively. Critical Issues: The bound species is suggested to predominate in the monoprotonated form, although spectroscopic evidence is pending. Future Directions: A description of the role of hemeproteins as biochemical targets for inorganic sulfide requires understanding the reactivity of bound sulfide, for example: the metal-centered reduction, the reaction with excess sulfide, oxidants, or other gasotransmitters, among other biomolecules.

Conformational Flexibility Drives Cold Adaptation in Pseudoalteromonas haloplanktis TAC125 Globins.

Significance: Temperature is one of the most important drivers in shaping protein adaptations. Many biochemical and physiological processes are influenced by temperature. Proteins and enzymes from organisms living at low temperature are less stable in comparison to high-temperature adapted proteins. The lower stability is generally due to greater conformational flexibility. Recent Advances: Adaptive changes in the structure of cold-adapted proteins may occur at subunit interfaces, distant from the active site, thus producing energy changes associated with conformational transitions transmitted to the active site by allosteric modulation, valid also for monomeric proteins in which tertiary structural changes may play an essential role. Critical Issues: Despite efforts, the current experimental and computational methods still fail to produce general principles on protein evolution, since many changes are protein and species dependent. Environmental constraints or other biological cellular signals may override the ancestral information included in the structure of the protein, thus introducing inaccuracy in estimates and predictions on the evolutionary adaptations of proteins in response to cold adaptation. Future Directions: In this review, we describe the studies and approaches used to investigate stability and flexibility in the cold-adapted globins of the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. In fact, future research directions will be prescient on more detailed investigation of cold-adapted proteins and the role of fluctuations between different conformational states.

Lessons learned about steered molecular dynamics simulations and free energy calculations.

The calculation of free energy profiles is central in understanding differential enzymatic activity, for instance, involving chemical reactions that require QM-MM tools, ligand migration, and conformational rearrangements that can be modeled using classical potentials. The use of steered molecular dynamics (sMD) together with the Jarzynski equality is a popular approach in calculating free energy profiles. Here, we first briefly review the application of the Jarzynski equality to sMD simulations, then revisit the so-called stiff-spring approximation and the consequent expectation of Gaussian work distributions and, finally, reiterate the practical utility of the second-order cumulant expansion, as it coincides with the parametric maximum-likelihood estimator in this scenario. We illustrate this procedure using simulations of CO, both in aqueous solution and in a carbon nanotube as a model system for biologically relevant nanoheterogeneous environments. We conclude the use of the second-order cumulant expansion permits the use of faster pulling velocities in sMD simulations, without introducing bias due to large dispersion in the non-equilibrium work distribution.

Cold-Adaptation Signatures in the Ligand Rebinding Kinetics to the Truncated Hemoglobin of the Antarctic Bacterium Pseudoalteromonas haloplanktis TAC125.

Cold-adapted organisms have evolved proteins endowed with higher flexibility and lower stability in comparison to their thermophilic homologues, resulting in enhanced reaction rates at low temperatures. In this context, protein-bound water molecules were suggested to play a major role, and their weaker interactions at protein active sites have been associated with cold adaptation. In this work, we tested this hypothesis on truncated hemoglobins (a family of microbial heme-proteins of yet-unclear function) applying molecular dynamics simulations and ligand-rebinding kinetics on a protein from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125 in comparison with its thermophilic Thermobifida fusca homologue. The CO rebinding kinetics of the former highlight several geminate phases, with an unusually long-lived geminate intermediate. An articulated tunnel with at least two distinct docking sites was identified by analysis of molecular dynamics simulations and was suggested to be at the origin of the unusual geminate rebinding phase. Water molecules are present in the distal pocket, but their stabilization by TrpG8, TyrB10, and HisCD1 is much weaker than in thermophilic Thermobifida fusca truncated hemoglobin, resulting in a faster geminate rebinding. Our results support the hypothesis that weaker water-molecule interactions at the reaction site are associated with cold adaptation.

Crystallographic and computational study of a network composed of [ZnCl4]2- anions and triply protonated 4'-functionalized terpyridine cations.

We report herein the synthesis, crystallographic analysis and a study of the noncovalent interactions observed in the new 4'-substituted terpyridine-based derivative bis[4'-(isoquinolin-2-ium-4-yl)-2,2':6',2''-terpyridine-1,1''-diium] tris[tetrachloridozincate(II)] monohydrate, (C24H19N4)2[ZnCl4]3·H2O or (ITPH3)2[ZnCl4]3·H2O, where (ITPH3)3+ is the triply protonated cation derived from 4'-(isoquinolin-4-yl)-2,2':6',2''-terpyridine (ITP) [Granifo et al. (2016). Acta Cryst. C72, 932-938]. The (ITPH3)3+ cation presents a number of interesting similarities and differences compared with its neutral ITP relative, mainly in the role fulfilled in the packing arrangement by the profuse set of D-H...A [D (donor) = C, N or O; A (acceptor) = O or Cl], π-π and anion...π noncovalent interactions present. We discuss these interactions in two different complementary ways, viz. using a point-to-point approach in the light of Bader's theory of Atoms In Molecules (AIM), analyzing the individual significance of each interaction, and in a more `global' analysis, making use of the Hirshfeld surfaces and the associated enrichment ratio (ER) approach, evaluating the surprisingly large co-operative effect of the superabundant weaker contacts.

Access and Binding of H2S to Hemeproteins: The Case of HbI of Lucina pectinata.

Hydrogen sulfide (H2S) was recently discovered as a gasotransmitter, capable of coordinating to the heme iron of hemeproteins. H2S is unique for its ability to render varying concentrations of the nucleophilic conjugate bases (HS(-) or S(2-)), either as free or bound species with expected outcomes on its further reactivity. There is no direct evidence about which species (H2S, HS(-), or S(2-)) coordinates to the iron. We performed computer simulations to address the migration and binding processes of H2S species to the hemoglobin I of Lucina pectinata, which exhibits the highest affinity for the substrate measured to date. We found that H2S is the most favorable species in the migration from the bulk to the active site, through an internal pathway of the protein. After the coordination of H2S, an array of clustered water molecules modifies the active site environment, and assists in the subsequent deprotonation of the ligand, forming Fe(III)-SH(-). The feasibility of the second deprotonation of the coordinated ligand is also discussed.