Leonardo Boechi

Modeling heme proteins using atomistic simulations

Heme proteins are found in all living organisms, and perform a wide variety of tasks ranging from electron transport, to the oxidation of organic compounds, to the sensing and transport of small molecules. In this work we review the application of classical and quantum-mechanical atomistic simulation tools to the investigation of several relevant issues in heme proteins chemistry: (i) conformational analysis, ligand migration, and solvation effects studied using classical molecular dynamics simulations; (ii) electronic structure and spin state energetics of the active sites explored using quantum-mechanics (QM) methods; (iii) the interaction of heme proteins with small ligands studied through hybrid quantum mechanics-molecular mechanics (QM-MM) techniques; (iv) and finally chemical reactivity and catalysis tackled by a combination of quantum and classical tools.

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.

Structural determinants of ligand migration in Mycobacterium tuberculosis truncated hemoglobin O

Mycobacterium tuberculosis is the causative agent of human tuberculosis, one of the most prevalent infectious diseases in the world. Its genome hosts the glbN and glbO genes coding for two proteins, truncated hemoglobin N (trHbN) and truncated hemoglobin O (trHbO), that belong to different groups (I and II, respectively) of the recently discovered trHb family of hemeproteins. The different expression pattern and kinetics rates constants for ligand association and NO oxidation rate suggest different functions for these proteins. Previous experimental and theoretical studies showed that, in trHbs, ligand migration along the internal tunnel cavity system is a key issue in determining the ligand‐binding characteristics. The X‐ray structure of trHbO has been solved and shows several internal cavities and secondary‐docking sites. In this work, we present an extensive investigation of the tunnel/cavity system ofM. tuberculosis trHbO by means of computer‐simulation techniques. We have computed the free‐energy profiles for ligand migration along three found tunnels in the oxy and deoxy w.t. and mutant trHbO proteins. Our results show that multiple‐ligand migration paths are possible and that several conserved residues such as TrpG8 play a key role in the ligand‐migration regulation. Proteins 2008.

Unraveling the molecular basis for ligand binding in truncated hemoglobins: The trHbO Bacillus subtilis case

Truncated hemoglobins (trHbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. Their tertiary structure consists in a 2‐over‐2 helical sandwich, which display typically an inner tunnel/cavity system for ligand migration and/or storage. The microorganism Bacillus subtilis contains a peculiar trHb, which does not show an evident tunnel/cavity system connecting the protein active site with the solvent, and exhibits anyway a very high oxygen association rate. Moreover, resonant Raman results of CO bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. To understand these experimental results with atomistic detail, we performed classical molecular dynamics simulations of the oxy, carboxy, and deoxy proteins. The free energy profiles for ligand migration suggest that there is a key residue, GlnE11, that presents an alternate conformation, in which a wide ligand migration tunnel is formed, consistently with the kinetic data. This tunnel is topologically related to the one found in group I trHbs. On the other hand, the results for the CO and O2 bound protein show that GlnE11 is directly involved in the stabilization of the cordinated ligand, playing a similar role as TyrB10 and TrpG8 in other trHbs. Our results not only reconcile the structural data with the kinetic information, but also provide additional insight into the general behaviour of trHbs. Proteins 2010.

Comparing and combining implicit ligand sampling with multiple steered molecular dynamics to study ligand migration processes in heme proteins.

The ubiquitous heme proteins perform a wide variety of tasks that rely on the subtle regulation of their affinity for small ligands like O2, CO, and NO. Ligand affinity is characterized by kinetic association and dissociation rate constants, that partially depend on ligand migration between the solvent and active site, mediated by the presence of internal cavities or tunnels. Different computational methods have been developed to study these processes which can be roughly divided in two strategies: those costly methods in which the ligand is treated explicitly during the simulations, and the free energy landscape of the process is computed; and those faster methods that use prior computed Molecular Dynamics simulation without the ligand, and incorporate it afterwards, called implicit ligand sampling (ILS) methods. To compare both approaches performance and to provide a combined protocol to study ligand migration in heme proteins, we performed ILS and multiple steered molecular dynamics (MSMD) free energy calculations of the ligand migration process in three representative and well theoretically and experimentally studied cases that cover a wide range of complex situations presenting a challenging benchmark for the aim of the present work. Our results show that ILS provides a good description of the tunnel topology and a reasonable approximation to the free energy landscape, while MSMD provides more accurate and detailed free energy profile description of each tunnel. Based on these results, a combined strategy is presented for the study of internal ligand migration in heme proteins.

Role of heme distortion on oxygen affinity in heme proteins: the protoglobin case.

The chemical properties of heme proteins largely reflect the electronic properties of their heme group. Often, the porphyrin ring of the heme exhibits significant distortions from its isolated structure, but the impact of these distortions on the chemical properties of the heme is yet uncertain. A systematic study focused on the effects of the distortion of the macrocycle on the binding affinity for oxygen is presented. The results show that out-of-plane distortions decrease the binding affinity, while in-plane distortions can increase or decrease it. Among in-plane distortions, only the breathing mode, which involves the symmetric compression-expansion of the porphyrin ring, strongly modulates the binding affinity. These findings shed light into the peculiar binding affinity of Methanosarcina acetivorans protoglobin, a protein that contains a highly distorted heme. Overall, the results highlight that in-plane distortions might be exploited by certain classes of heme proteins to modulate the ligand affinity.

Protein dynamics and ligand migration interplay as studied by computer simulation.

Since proteins are dynamic systems in living organisms, the employment of methodologies contemplating this crucial characteristic results fundamental to allow revealing several aspects of their function. In this work, we present results obtained using classical mechanical atomistic simulation tools applied to understand the connection between protein dynamics and ligand migration. Firstly, we will present a review of the different sampling schemes used in the last years to obtain both ligand migration pathways and the thermodynamic information associated with the process. Secondly, we will focus on representative examples in which the schemes previously presented are employed, concerning the following: i) ligand migration, tunnels, and cavities in myoglobin and neuroglobin; ii) ligand migration in truncated hemoglobin members; iii) NO escape and conformational changes in nitrophorins; iv) ligand selectivity in catalase and hydrogenase; and v) larger ligand migration: the P450 and haloalkane dehalogenase cases. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Ligand migration in Methanosarcina acetivorans protoglobin: effects of ligand binding and dimeric assembly.

Protoglobin is the first globin found in Archaea. Its biological role is still unknown, although this protein can bind O(2), CO, and NO reversibly in vitro. The X-ray structure of Methanosarcina acetivorans protoglobin (MaPgb) has shown that access of ligands to the heme, which is completely buried within the protein matrix, can be granted by two apolar tunnels, which are mainly defined by helices G and B (tunnel 1), and helices B and E (tunnel 2). Here we analyze the structural and dynamical behavior of MaPgb through molecular dynamics and computational techniques aimed at shedding light on distinctive features of ligand migration through the tunnels that may be linked to functionality. While tunnel 2 is found to be accessible to diatomic ligands in both deoxygenated and oxygenated forms of the protein, the accessibility of tunnel 1 is controlled through the synergistic effect of both the protein dimeric state and the presence of the heme-bound ligand. Thus, dimerization mainly affects the spatial arrangement of helix G, which influences the shape of tunnel 1. Ligand accessibility through this tunnel is regulated by Phe(145)G8, which can adopt open and closed conformations. Noteworthy, the ratio between open and closed states is modulated by protein dimerization and more strikingly by ligand binding. In particular, sensing of the ligand is mediated by Phe(93)E11, and the steric hindrance between Phe(93)E11 and the heme-bound ligand alters the structural and dynamical behavior of helices B and E, which facilitates opening of tunnel 1. This functional mechanism provides a basis to understand the finding that ligation favors fast rebinding from ligand binding kinetic to MaPgb. Finally, it also suggests that MaPgb might be physiologically involved in a ligand-controlled bimolecular chemical process.

Fluoride as a probe for h-bonding interactions in the active site of heme proteins: The case of thermobifida fusca hemoglobin

The structural and functional properties of the active site of the bacterial hemoglobin from Thermobifida fusca are largely determined by three polar amino acids: TrpG8, TyrCD1, and TyrB10. We have exploited the availability of a combinatorial set of mutants, in each of which these three amino acids have been singly, doubly, or triply replaced by a Phe residue, to perform a detailed study on H-bonding interactions between the protein and heme-bound fluoride. By appropriate choice of the excitation conditions, ν(Fe-F) stretching bands have been detected in the resonance Raman spectra. In the wild-type protein and one of the mutants, two ν(Fe-F) bands have been observed and assigned to the presence of two protein conformers where fluoride is singly or doubly H-bonded. Furthermore, by plotting the CT1 charge-transfer transition energy vs the ν(Fe-F) wavenumbers, an empirical correlation has been found. The data are well fitted by a straight line with a positive slope. The position along the correlation line can be considered as a novel, general spectroscopic indicator of the extent of H-bonding in the active site of heme proteins. In agreement with the spectroscopic results, we have observed that the rate of ligand dissociation in stopped-flow kinetic measurements progressively increases upon substitution of the H-bonding amino acids. Molecular dynamics simulations have been performed on the fluoride complexes of native and mutated forms, indicating the prevalent interactions at the active site. All the techniques yield evidence that TrpG8 and TyrCD1 can form strong H bonds with fluoride, whereas TyrB10 plays only a minor role in the stabilization of the ligand.

Role of PheE15 Gate in Ligand Entry and Nitric Oxide Detoxification Function of Mycobacterium tuberculosis Truncated Hemoglobin N

The truncated hemoglobin N, HbN, of Mycobacterium tuberculosis is endowed with a potent nitric oxide dioxygenase (NOD) activity that allows it to relieve nitrosative stress and enhance in vivo survival of its host. Despite its small size, the protein matrix of HbN hosts a two-branched tunnel, consisting of orthogonal short and long channels, that connects the heme active site to the protein surface. A novel dual-path mechanism has been suggested to drive migration of O2 and NO to the distal heme cavity. While oxygen migrates mainly by the short path, a ligand-induced conformational change regulates opening of the long tunnel branch for NO, via a phenylalanine (PheE15) residue that acts as a gate. Site-directed mutagenesis and molecular simulations have been used to examine the gating role played by PheE15 in modulating the NOD function of HbN. Mutants carrying replacement of PheE15 with alanine, isoleucine, tyrosine and tryptophan have similar O2/CO association kinetics, but display significant reduction in their NOD function. Molecular simulations substantiated that mutation at the PheE15 gate confers significant changes in the long tunnel, and therefore may affect the migration of ligands. These results support the pivotal role of PheE15 gate in modulating the diffusion of NO via the long tunnel branch in the oxygenated protein, and hence the NOD function of HbN.