Mechanism of the Nitric Oxide Dioxygenase Reaction of

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Mechanism of the Nitric Oxide Dioxygenase Reaction of Mycobacterium tuberculosis Hemoglobin N Lavinia Arielle Carabet, Michel Guertin, Patrick Lagüe, and Guillaume Lamoureux J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b06494 • Publication Date (Web): 24 Aug 2017 Downloaded from http://pubs.acs.org on August 24, 2017

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The Journal of Physical Chemistry

Mechanism of the Nitric Oxide Dioxygenase Reaction of Mycobacterium tuberculosis Hemoglobin N Lavinia A. Carabet,†,§,# Michel Guertin,‡ Patrick Lagüe,‡,§ and Guillaume Lamoureux*,†,§ †

Department of Chemistry and Biochemistry and Centre for Research in Molecular Modeling

(CERMM), Concordia University, Montréal, Québec, Canada ‡

Department of Biochemistry, Microbiology and Bioinformatics, Université Laval, Québec,

Québec, Canada §

Regroupement québécois de recherche sur la fonction, l’ingénierie et l’application des protéines

(PROTEO) * Corresponding author: Email: [email protected] Telephone: (514) 848-2424, ext. 5314

ACS Paragon Plus Environment

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ABSTRACT

Many globins convert •NO to innocuous NO3– through their nitric oxide dioxygenase (NOD) activity. Mycobacterium tuberculosis fights the oxidative and nitrosative stress imposed by its host (the toxic effects of O2•– and •NO species and their OONO– and •NO2 derivatives) through the action of truncated hemoglobin N (trHbN), which catalyzes the NOD reaction with one of the highest rates among globins. The general NOD mechanism comprises the following steps: binding of O2 to the heme, diffusion of •NO into the heme pocket and formation of peroxynitrite (OONO–), isomerization of OONO–, and release of NO3–. Using QM/MM free energy calculations, we show that the NOD reaction in trHbN follows a mechanism in which hemebound OONO– undergoes homolytic cleavage to give FeIV=O2– and the •NO2 radical, but that these potentially harmful intermediates are short-lived and caged by the heme pocket residues. In particular, the simulations show that Tyr33(B10) side-chain is shielded from FeIV=O2– and •NO2 (and protected from irreversible oxidation and nitration) by forming stable hydrogen bonds with Gln58(E11) side-chain and Leu54(E7) backbone. Aromatic residues Phe46(CD1), Phe32(B9), and Tyr33(B10) promote NO3– dissociation via C−H···O bonding and provide stabilizing interactions for the anion along its egress route.


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1. INTRODUCTION Tuberculosis is one of the oldest recorded human afflictions1 and is still declared a major threat worldwide due to the increase in multi-drug resistant Mycobacterium tuberculosis (Mtb) strains and in HIV co-infection, and due to the reluctance of patients to comply with the currently prescribed drug regimens, which are long, expensive, and not always effective.2 The success of Mtb as a lung pathogen has been attributed to its capacity to survive the host immune system by entering, for a prolonged time, a state of latency in which it can resist oxidative and nitrosative species (e.g. O2•– and •NO).3-6 In particular, Mtb’s protection against reactive nitrogen species relies on the oxygenated form of truncated hemoglobin N (trHbN).7 trHbN protects the bacillus’s aerobic respiration from •NO inhibition and prevents its own irreversible oxidation and nitration by rapidly metabolizing •NO to innocuous nitrate (NO3–), through the nitric oxide dioxygenase (NOD) reaction:7,8 trHbN-FeII-O2 + •NO → trHbN-FeIII + NO3– trHbN catalyzes the NOD reaction with a second-order rate constant kNOD = 7.45 × 108 M-1s-1 (at 23 °C),8 corresponding to a rate approaching the diffusion limit.5 The NOD reaction in trHbN is more than twenty times faster than in sperm whale myoglobin (P. catodon Mb) and human hemoglobin (H. sapiens Hb), and only three times slower than the NOD reaction catalyzed by microbial flavoHbs of E. coli and A. eutrophus, the most efficient •NO dioxygenases studied so far.7-10 trHbs are widely distributed in bacteria, plants and unicellular eukaryotes, and form a separate family within the globin superfamily.11-13 They are distantly related to bacterial flavoHbs


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and to the classical eukaryotic Hbs and Mbs, but are 20-40 amino acids shorter.10,11 The tertiary structure of trHbN is based on a 2-over-2 α-helical fold (“BE over GH”) (see Figure 1, left), which is a trimmed version of the common 3-over-3 globin fold (“ABF over EGH”).14,15 A peculiar feature of Mtb trHbN is the presence of the pre-A N-terminal region, which contains a highly polar motif extending out of the rigid 2-over-2 fold14 and which has been recently hypothesized to participate in the association of trHbN to the membrane.16


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Figure 1. Three-dimensional structure of trHbN of M. tuberculosis (PDB ID: 1IDR).12 (Left) The 2-over-2 α-helical trHb fold, surrounding the distal heme pocket (DHP), formed of two antiparallel helix pairs, helices B (blue) and E (green) on one side, and G (yellow) and H (purple) on the other side (shown in ribbon representation). (Right) The trHbN active site: O2-bound heme, proximal His81(F8) residue, distal Tyr33(B10) and Gln58(E11) residues stabilizing the ligand, Phe46(CD1) residue protecting the DHP from the solvent, Phe32(B9) residue highly conserved in all trHbs,13 Leu54(E7) residue participating in the trHbN-specific H-bond network, and an •NO molecule (all represented as sticks).

The active site of Mtb trHbN is shown in Figure 1 (right). Nomenclature for trHbN residues follows the convention established for P. catodon Mb (see Figure S1): each helix is designated by a letter (A through H) and each residue is numbered based on its position in that helix.17 The heme-bound proximal Fe ligand, His81(F8), is conserved across eukaryotic Hbs and


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Mbs, bacterial flavoHbs, and trHbs families. The distal heme pocket (DHP) of Mtb trHbN is characterized by a cluster of residues – Tyr33(B10), Leu54(E7), and Gln58(E11) – that differ in size and polarity compared to eukaryotic Hbs and Mbs, but that show little variation compared to bacterial flavoHbs and bacterial Hbs.10,11,14,18 Residue B10, directly above the heme group, is a tyrosine in most bacterial trHbs and flavoHbs13 (see Figure S1). Residue E7, the distal histidine in most eukaryotic Hbs, is a leucine in Mtb trHbN and a glutamine in flavoHbs.10,11,14,18 Conversely, residue E11 is a glutamine in a majority of trHbNs (including Mtb trHbN) but a leucine in flavoHbs. In the crystal structure of oxygenated Mtb trHbN (PDB ID: 1IDR)14 (Figure 1), the hemebound O2 is stabilized by a hydrogen bond to Tyr33(B10), which is itself hydrogen bonded to Gln58(E11).14 This H-bond network, specific to trHbN, polarizes the dioxygen to a superoxide character (O2•–), which promotes rapid reaction with •NO.14 In most vertebrate Hbs, the hemebound O2 is stabilized by a histidine in position E7 and there is no H-bond network as observed in trHbN or flavoHbs.11 The replacement of TyrB10 for HisE7 in modern Hbs and Mbs makes them less reactive and better adapted for their O2 transport and storage functions.11 A phenylalanine is found at position CD1 (the first position in the sequence linking the C and D helices) in most eukaryotic globins, in bacterial flavoHbs, and in Mtb trHbN, but is substituted by leucine or tyrosine in trHbs that do not function as NODs.10,11,14,18 The main role of PheCD1 is to shield the DHP from the solvent.15 Positions B9 and G8 are occupied by Phe and Val residues in most trHbNs, flavoHbs, and bacterial Hbs, but by two isoleucine residues in P. catodon Mb.10,11,14,18 Along with GlnE11, PheB9 and ValG8 are believed to control ligand diffusion in and out of the DHP.19


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Mtb trHbN displays a network of hydrophobic tunnels that supports diffusion of small, nonpolar ligands (e.g., O2 and •NO) to and from the DHP.19 The crystal structure14 shows two tunnels that connect the buried DHP to the bulk solvent. The long tunnel (LT) has a length of ~20 Å and is situated between the B, E and G helices. The short tunnel (ST) has a length of ~13 Å and runs between the G and H helices.14 Molecular dynamics simulations by Daigle et al20 have revealed two additional substrate diffusion pathways named EH and BE tunnels after the helices flanking them. The solubility of nonpolar O2 and •NO molecules is approximately three times greater in biological membranes than in aqueous solution.21 By contrast, NO3–, the product of the NOD reaction, is essentially insoluble in the membrane.21 A recent spectroscopic and computational study16 has shown that Mtb trHbN is a peripheral membrane protein, and that its association with the membrane occurs via the pre-A, G, and H helices. This mode of association would orient the ST tunnel toward the membrane interior and the other tunnels toward the solvent, which would facilitate the uptake of nonpolar substrate from the membrane and the release of charged product to the solvent. In summary, the high O2 affinity and the hydrophobic tunnel system of trHbN are critical for its efficient NOD activity. The O2 affinity of trHbN is due to the distal residues Tyr33(B10) and Gln58(E11) that stabilize and polarize the O2 ligand. Kinetic analysis of the Tyr33(B10)Phe trHbN mutant has shown a 150-fold increase in O2 dissociation rate and a substantial decrease in O2 affinity.7 The trHbN tunnel system and its possible connection with the membrane allow fast entry of the •NO substrate and provide direct access to the bound O2 in the active site.14,16,19,20 It is commonly agreed5-11,14-20,22 that the diffusion of the ligands to the DHP is the rate-limiting step in the •NO detoxification process, regardless of the NOD reaction mechanism. Indeed, mutations


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designed to obstruct the tunnel entrances (or make them more polar) have a direct influence on the overall NOD reaction rate.19 The proposed overall mechanism of the NOD reaction23-25 comprises five main steps: (1) trHbN-FeII + O2 → trHbN-FeIII-O2•– (2) trHbN-FeIII-O2•– + •NO → trHbN-FeIII-OONO– (3) trHbN-FeIII-OONO– → trHbN-FeIII-[ONO2–] (4) trHbN-FeIII-[ONO2–] → trHbN-FeIII + NO3– (5) trHbN-FeIII + e– → trHbN-FeII It includes the formation of a peroxynitrite (OONO–) intermediate (eq. 2) and its isomerization to a nitrato-complex (eq. 3), followed by the release of the inoffensive NO3– anion (eq. 4). OONO– is highly reactive and toxic as it can oxidize or nitrate all types of biomolecules including DNA, proteins and lipids.5,6 Moreover, the isomerization of OONO– itself involves the formation of transient radical intermediates, which can be harmful if they escape the active site. Therefore, to be effective the isomerization reaction must be rapid and isolated from the solvent. To this day, two mechanisms have been considered for the NOD isomerization reaction (eq. 3):23-25 - a sequential mechanism, trHbN-FeIII-OONO– → trHbN-[FeIV=O2–] + •NO2 → trHbN-FeIII[ONO2–], that involves the homolytic cleavage of the O-O bond to produce oxo-ferryl species (compound II) FeIV=O2– and •NO2 intermediates, followed by the •NO2 radical attack on the oxoferryl species to form the nitrato-complex, and


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- a concerted mechanism, trHbN-FeIII-OONO– → trHbN-FeIII-[ONO2–], that involves an internal rearrangement reaction of the peroxynitrite complex in which the peroxo O-O bond is simultaneously ruptured as the Fe-coordinated O-atom bonds nitrogen to form nitrate. Discrepancies exist in the literature with respect to the isomerization mechanism (eq. 3) and the role of the active site residues in assisting the NOD reaction. Based on DFT calculations on cluster models of the Mb active site, Blomberg et al23 have suggested a sequential isomerization mechanism for mammalian Mbs and Hbs. They could not find a concerted mechanism yielding plausible activation energies, and the sequential mechanism they found displayed a relatively low energy barrier for the O-O bond homolysis and a highly exergonic overall reaction sequence. In Mtb trHbN, both mechanisms have been proposed. QM/MM geometry optimizations by Crespo et al24 support a sequential mechanism in which both elementary steps (O-O bond homolysis and formation of bound nitrate) have very low activation barriers and are highly exergonic. On the other hand, force-field based, adiabatic reactive molecular dynamics (ARMD) simulations by Mishra et al25 support a concerted mechanism in which the OONO– homolytic cleavage and •NO2 dissociation elementary step do not occur. Experimental studies on the •NO dioxygenation in Mb and Hb provide conflicting and inconclusive views on the mechanism and possible intermediates of the NOD reaction. Herold et al,9,26,27 using rapid scan UV-vis spectroscopy, provided evidence for a millisecond-lived peroxynitrite FeIII-OONO– intermediate in both oxy-Mb and oxy-Hb. They were unable to detect any dissociated •NO2 but observed quantitative NO3– formation and suggested that the peroxynitrite coordinated to ferric iron undergoes a rapid rearrangement to nitrate (without nitrating the globin residues). Their analysis is inconclusive, however, on whether the rearrangement is concerted or not since the lack of detection of oxo-ferryl species and •NO2 may


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either indicate that the intermediates are not formed at all or that they react too quickly to accumulate and be detected. Yukl et al,28 using resonance Raman spectroscopy and rapid-freeze quenching techniques, showed that the intermediate found by Herold et al9,26,27 in the reaction of •

NO with oxy-Mb (FeII) is an FeIII-nitrato complex but mention that their results do not invalidate

the sequential mechanism. In addition, Bourassa et al29 proposed O-O homolysis based on stopped-flow kinetic measurements, and Su and Groves30 provided spectroscopic evidence for ferryl-Mb (FeIV) formation from met-Mb (FeIII) and OONO–. As far as possible intermediates of the NOD isomerization reaction are concerned, there have been, to the best of our knowledge, no experimental kinetics studies for trHbN. The role of active site residues in the NOD reaction in trHbN has been investigated mainly by computational approaches and mutagenesis studies. Once again, discrepancies exist between the findings of Crespo et al24 and of Mishra et al25 about the role of the trHbN environment. Mishra et al25 assessed the role of the active site residues through ARMD simulations of Tyr33Ala and Gln58Ala mutants and their combination. As in the wild-type trHbN, the OONO– homolysis reaction did not occur in the mutants. However, the ARMD simulations revealed that the •NO2 rebinding step of the sequential isomerization reaction is influenced by Tyr33(B10) and Gln58(E11) residues, which pre-orient the reactive ligands through an H-bond network. Mishra et al’s concerted rearrangement mechanism25 was found to be strongly influenced by the mutation of Tyr33(B10) residue but not significantly influenced by the mutation of Gln58(E11). Mishra et al’s25 assessment of the essential role of Tyr33(B10) is in agreement with Ouellet et al’s experimental study7 showing that mutant Tyr33Phe mycobacteria cannot metabolize •NO. In contrast, Crespo et al,24 also using a Tyr33Phe mutant, reported that the trHbN environment does not make significant contributions to the heme moiety catalyzed


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reaction. However, Crespo et al24 suggested that the active site residues, especially the Tyr33(B10)/Gln58(E11) pair, might be relevant to the function of trHbN at different levels, including to isolate the reaction intermediates formed in the hydrophobic pocket lined by residues Phe32, Tyr33, Val36, Phe46, and Val94. The presence of Tyr33(B10) in the trHbN active site, which could supply the redox equivalent needed for catalyzing the O-O bond cleavage, led Yeh et al31 to hypothesize a mechanism in which Tyr33(B10) could be directly involved in the OONO– isomerization. This mechanism would involve the heterolytic O-O bond cleavage of the peroxynitrite complex to produce a compound I oxo-ferryl intermediate, a Tyr33(B10) radical cation (Tyr33•+), and a nitrite NO2– anion: trHbN-FeIII-OONO– → trHbN-[Por•+-FeIV=O2–] + NO2– → trHbN-[FeIV=O2–]Tyr33•+ + NO2– → trHbN-[FeIV=O2–] + •NO2 → trHbN-FeIII-[ONO2–] In this alternative reaction path, the compound I complex (with an unpaired electron on the porphyrin ring) would be reduced by the nearby Tyr33(B10). The Tyr33(B10) radical cation would then be reduced by NO2– to form compound II and •NO2 radical, and the reaction would continue as in the sequential mechanism, with an •NO2 attack on compound II to form the bound nitrate. Crespo et al’s study24 indicated that Tyr33(B10) does not play a catalytic role in the NOD reaction, but instead that the reaction occurs mainly by means of the heme group. Herold et al9,26,27 suggested that the relatively slow reaction of •+HbFeIV=O with NO2– (16 M-1s-1) and the rapid reaction of HbFeIV=O with •NO2 (107 M-1s-1) favor the homolytic mechanism producing •

NO2 over the heterolytic mechanism producing NO2–.


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The presence of Tyr33(B10) next to highly reactive intermediates in the active site is also problematic in that it could lead to trHbN degradation. FeIV=O2– is a strong oxidant (~1.0 V)6 that could readily react with Tyr33(B10) to form a tyrosyl radical (Tyr33•) and a hydroxide-bound ferric heme (FeIII-OH–).6,32 This Tyr33• radical could irreversibly interact with •NO2 to form 3nitrotyrosine,6,32 which would lead to permanent trHbN degradation and potentially to the death of the Mtb bacillus. How trHbN prevents these possible secondary reactions (i.e. Tyr33 oxidation and nitration) is an important question and has not been considered to this day in theoretical studies. Release of the nitrate anion, the product of the NOD reaction, involves the breaking of the bond with the heme group and a change in the iron coordination from hexa- to pentacoordinated (presuming no other ligand comes in), followed by the diffusion of the anion through the protein matrix. Martí et al33 simulated the release of nitrate in Mtb trHbN. Their molecular dynamics simulations suggest that the formation of the ferric-nitrato species causes a structural distortion of the DHP walls forming pores for water entry. The structural destabilization affects, among other, Phe46(CD1), that shields the heme from the solvent, which rearranges to open a pathway for water molecules to coordinate the nitrate anion and facilitate its dissociation from the heme iron.33 Martí et al33 find that, once the FeIII-O bond is broken, the NO3– anion exits in ~5 ns via a pathway distinct from O2 and •NO tunnels. Furthermore, Martí et al’s QM/MM energy minimizations33 with Tyr33(B10) and Gln58(E11) residues included in the QM subsystem pointed out that the Tyr33(B10)/Gln58(E11) pair is crucial for breaking the FeIIIO bond. Similarly, Mishra et al’s ARMD simulations25 with wild-type trHbN and its mutants indicated that Tyr33(B10) and Gln58(E11) strongly influence NO3– dissociation.


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Earlier theoretical studies provide conflicting and inconclusive views on the mechanism and on the dynamics of trHbN active site residues during the NOD reaction. Crespo et al24 employed QM/MM restrained geometry optimizations along one-dimensional predefined reaction coordinates for the elementary steps of the isomerization reaction. The method lacks nuclear dynamics and conformational sampling. Mishra et al25 employed the ARMD method that allows for sufficiently long molecular dynamics simulations. However, ARMD is a simple surface-crossing approach to reactive processes that involves the parameterization of potential energy surfaces of the reactants and the products with individual force fields. In the present work, the NOD reaction in Mtb trHbN is investigated using free energy calculations based on constrained QM/MM molecular dynamics simulations, which incorporate the effects of thermal fluctuations and of the rearrangement of trHbN residues at the active site, and which provide extensive sampling along the reaction coordinates. In particular, we elucidate the mechanism of the NOD isomerization reaction and determine the interactions and motions of trHbN active site residues critical in assisting the isomerization reaction and product release, and in preventing the permanent degradation of trHbN itself. 2. COMPUTATIONAL DETAILS QM/MM MD Simulations. Hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) simulations are performed using the open-source CP2K package.34 The energy of the QM subsystem is calculated using the QuickStep module.35 Wavefunctions are described by plane waves, augmented with double-ζ short-range Gaussian basis sets optimized for molecular systems (DZVP-MOLOPT-SR-GTH).36 The plane waves are cut off at 300 Ry and the core electrons are described by Goedecker-Tetter-Hutter (GTH) pseudopotentials.37 BLYP


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density functional38 is used to compute the exchange and correlation energy. The energy of the classical MM subsystem is calculated using the FIST module34 with CHARMM27 force field.39 The linkage between the QM and MM regions (e.g. between the Cβs and Cαs of side-chains) is treated using the Integrated Molecular Orbital Molecular Mechanics (IMOMM) link atom approach.40 QM periodic images are decoupled with a multipole Poisson solver, using atomic point charges derived from the electron density.41 The electrostatic coupling between MM charges and the QM electron density represented as point charges is calculated by a linearscaling real space multi-grid procedure.41 QM/MM MD simulations are performed at constant temperature and volume. Langevin dynamics42 is used to control the temperature at T = 298.15 K, with a friction coefficient of 0.004 fs-1. The QM region includes the iron-porphyrin ring, the O2 ligand, the •NO substrate, and the side-chains of trHbN active site highly conserved residues: the proximal His81(F8) and the distal Tyr33(B10), Gln58(E11) and Phe46(CD1). The peroxynitrite complex QM system has a net charge of +1 and is prepared in a doublet spin state (multiplicity = 2). The QM box is approximately 16 Å × 22 Å × 15 Å, 6–8 Å larger than the extent of the QM fragments in each direction. The surroundings (i.e. rest of the protein, TIP3P water molecules, counter-ions to balance the protein charges and 9 •NO molecules in solution) are treated classically. Link atoms are used in the heme and in the proximal and distal residues to decouple the porphyrin ring from the heme substituents (i.e. propionates, vinyl and methyl groups; C-C truncations) and the sidechains of active site residues from the peptidic backbone (i.e. Cα-Cβ truncations). The final system consists of 96 QM atoms and 36118 MM atoms in a solvent box with dimensions 71 Å × 71 Å × 71 Å. Several replicas of unconstrained QM/MM MD simulations at least 10 ps each were conducted using selected snapshots from classical MD simulations19 as the initial structure.


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Constrained QM/MM MD simulations. Potentials of mean force (PMFs) are calculated using constrained QM/MM MD simulations. For the peroxynitrite isomerization reaction (eq. 3), a two-dimensional (2D) PMF is obtained from multiple simulations carried out with O1-O2 and O1-N distances fixed using Lagrange multipliers, and with the rest of the system free to undergo thermal fluctuations. The O1-O2 distance describes the cleavage of the O-O peroxo bond of the peroxynitrite complex and the O1-N distance describes the formation of the O-N bond of the nitrate complex. Those two reaction coordinates are fixed at values in the 1.25 to 3.75 Å range and scanned in 0.25-Å increments. The full PMF is built from a total of 99 constrained simulations of 10 ps each. The first window is run starting from the coordinates of an unconstrained QM/MM simulation of the peroxynitrite complex, and following windows are run once at least one adjacent window is complete, using the final coordinates of that window as starting point. Average constraint forces are calculated using the last 9 ps of each simulation. The 2D PMF is built using the single-sweep method,43 In the single-sweep method, the free energy landscape is represented as a sum of radial Gaussian functions of uniform width σ centered at each window. The free energy profile is determined by adjusting the value of σ and the height of each Gaussian function. Specifically, the optimal PMF is found by minimizing with respect to all parameters (σ and the heights) an error function that measures at each window the discrepancy between the negative gradient of the PMF and the constraint forces obtained from the simulations. For the 99 windows simulated, the optimum value of σ is found to be 0.475 Å (almost twice the distance between the windows), leading to an average force discrepancy of 0.95 kcal/mol/Å at each window. The PMF for the nitrate release (eq. 4) is generated by carrying out a total of 14 constrained QM/MM simulations of 10 ps each, using the Fe-N distance as reaction coordinate.


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This single reaction coordinate, which describes the dissociation of the nitrate anion from the ferric heme iron, is fixed at values in the 2.75 to 6.00 Å range and scanned in 0.25-Å increments. The optimized structure of heme-bound nitrato-complex – the free energy minimum on the 2D PMF of the peroxynitrite isomerization reaction – is used as a starting point for the nitrate release simulations. The constrained simulations are conducted following the same protocol as for the 2D PMF. While the reconstruction of the 2D PMF for OONO– isomerization requires the more complex single-sweep method, the PMF for NO3– release is constructed by straightforward numerical integration of the average constraint forces, using the trapezoidal rule. 3. RESULTS AND DISCUSSION 3.1 NOD reaction: Peroxynitrite isomerization Mechanism and energetics. To elucidate the mechanism – sequential or concerted – of the OONO– isomerization in trHbN, a two-dimensional potential of mean force (2D PMF) was computed from constrained QM/MM MD simulations (see Figure 2A). Constraints were imposed on two reaction coordinates selected to drive the isomerization reaction in the protein environment: the O1-O2 distance, describing the cleavage of the O-O peroxo bond of the peroxynitrite, and the O1-N distance, describing the formation of the O-N bond of the nitrate. The reaction coordinates were fixed at values between 1.25 and 3.75 Å and scanned in 0.25-Å increments, while the rest of the system was let free to undergo thermal fluctuations. Each black dot on the 2D PMF (Figure 2A) represents an individual 10-ps simulation window with fixed O1-O2 and O1-N distances.


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Figure 2. Panel (A) Two-dimensional potential of mean force (2D PMF) of the NOD isomerization reaction in trHbN environment. Each black dot corresponds to a sampling window where an individual 10-ps constrained QM/MM MD simulation was performed. The O1-O2 distance describes the cleavage of the O-O peroxo bond of the peroxynitrite complex, and the O1-N distance describes the formation of the O-N bond of the nitrate complex. Schematic


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representations of the trHbN active site, including the side-chains of residues Tyr33(B10) and Gln58(E11), are presented for three simulation windows representative of the peroxynitrite reactant, the radical intermediate, and the nitrate product (indicated by black arrows). Free energies are defined relative to that of bound product, the most stable state on the 2D PMF. Panel (B) Free energies at simulation windows closest to each state of the isomerization reaction (i.e. peroxynitrite reactant, radical intermediate, nitrate product, and transition states) relative to that of peroxynitrite. The position of each free energy level on the 2D PMF is indicated above the graph (by red dots) and a schematic of each state as observed in MD simulation is shown below.

The free energy surface shows a fast and highly exergonic sequential mechanism with very short-lived intermediates, starting with rapid formation of OONO– from the attack of free •

NO on the ferric superoxide complex (around dO1-O2 = 1.50 Å and dO1-N = 2.50 Å), followed by

rapid homolytic cleavage of the peroxo bond (O1-O2) and formation of a stable oxo-ferryl (FeIV=O2–(O1)) species and a free •NO2 radical (around dO1-O2 = 3.25 Å and dO1-N = 3.00 Å). The free energy minimum is reached following the formation of heme-bound NO3– (around dO1-O2 = 2.25 Å and dO1-N = 1.50 Å). OONO– isomerization occurs sequentially and involves very short-lived intermediates. As shown in Figure 2B, formation of oxo-ferryl species and •NO2 radical from peroxynitrite is energetically favored by 1.6 kcal/mol, with a low activation barrier of 2.4 kcal/mol. By comparison, the QM/MM energy optimizations from Crespo et al24 yield a potential energy difference of 8.1 kcal/mol in favor of the •NO2 radical, which likely translates into an even higher free energy difference since the dissociated radical state has higher entropy. Rebinding of •NO2 to


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compound II is almost barrierless (1.5 kcal/mol) and the overall isomerization reaction is exergonic by 14.7 kcal/mol. By comparison, Crespo et al24 find a potential energy difference of 18.0 kcal/mol (exothermic). The transition states free energies are estimated from the 2D PMF. Schematic representations of each state (i.e. the 3 minima and 2 transition states) as observed from the constrained simulations are shown at the bottom of Figure 2B. The schematics illustrate that, following OONO– homolysis, •NO2 rotates and positions itself to attack the oxo-ferryl heme and form the NO3– product, in a rebound-type mechanism. The energetics suggest that the isomerization occurs on picosecond timescale (estimated using Eyring’s rate theory44 at room temperature, with a prefactor of 6.21 × 1012 s-1) and, in light of the substrate diffusion studies of Bidon-Chanal et al45 and of Daigle et al,19 is not the rate-limiting step in the overall NOD reaction. Moreover, the concerted mechanism, following the diagonal path from OONO– to bound NO3– on which dO1-N decreases while dO1-O2 increases, can be ruled out due to the large free energy barrier (greater than 30 kcal/mol). Since large barrier is also found in gas-phase calculations (see Figure S2), it suggests that the concerted mechanism observed using the ARMD method25 is the result of an inaccurate representation of the potential energy surface. Unconstrained QM/MM simulations of oxy-trHbN with •NO in the active site yield trajectories consistent with these findings. In these simulations the isomerization reaction occurs spontaneously in a sequential manner: OONO– forms within 1 ps of simulation time and dissociates into oxo-ferryl (FeIV=O2–) species and •NO2 in about 2.5 ps (data not shown). The fast sequential isomerization mechanism suggested by the 2D PMF of Figure 2A is consistent with a 2D potential energy surface (PES) obtained from gas-phase DFT geometry optimizations carried out at the UB3LYP/6-311G(d,p) level of theory using ECP/ LANL2DZ for Fe (see Figure S2). On the gas-phase B3LYP energy profile, the •NO2 rebinding to compound II


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is almost barrierless (1.2 kcal/mol) and the overall isomerization reaction is exothermic by 28.2 kcal/mol (see Figure S2B). On a similar energy profile calculated with the BLYP functional, the functional used for the QM/MM simulations, the barrier for •NO2 rebinding is slightly higher (4.2 kcal/mol instead of 1.2) and the overall reaction is less exothermic (22.2 kcal/mol instead of 28.2) (see Figure S2B). The activation barrier for the homolysis elementary step obtained using the more reliable B3LYP functional is 7 kcal/mol higher than for the BLYP calculations, which suggests that the equivalent barrier on the PMF obtained from QM/MM simulations should be revised upward. However, we expect the correction to be less than 7 kcal/mol due to enthalpyentropy compensation effects. trHbN dynamics. We investigate the role of the trHbN active site residues in the isomerization reaction by analyzing the trajectories obtained from 5 simulation windows along the reaction pathway, corresponding to the red points on the 2D PMFs in Figure 2B. A schematic illustration of the mechanism is provided in Figure 3. In addition to the side-chains of Tyr33(B10) and Gln58(E11) already shown in Figure 2B, the schemes in Figure 3 include the Phe46(CD1) side-chain and the backbone carbonyl of Leu54(E7).


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Figure 3. Schematic illustration of the sequential mechanism of peroxynitrite isomerization in trHbN based on five constrained QM/MM MD simulations along the minimum free energy reaction pathway. The interactions and motions of trHbN active site residues critical in assisting the reaction are indicated with dashed lines and arrows. The aromatic ring of Phe46(CD1) and the backbone carbonyl of Leu54(E7) are included in the schematic in addition to the side-chains of the two polar groups, Tyr33(B10) and Gln58(E11).

The constrained QM/MM simulations show that the trHbN-specific H-bond between Tyr33(B10) and Gln58(E11) is stable throughout the isomerization reaction, with an average Hbonding distance of 2.2 Å. As illustrated schematically in Figure 4, Tyr33(B10) hydroxyl group acts as an H-bond acceptor to the amine group of Gln58(E11) and as an H-bond donor to the carbonyl group of Leu54(E7) backbone. The H-bond formed between Tyr33(B10) and Leu54(E7) backbone is stable throughout the reaction, except for the simulation window corresponding to dissociated •NO2, where it is transiently broken (see Figure 4A-B). In this window, at dO1-O2 = 3.25 Å and dO1-N = 3.00 Å, the •NO2 radical is far from the oxo-ferryl species and competes against the Leu54(E7) backbone carbonyl for H-bonding to Tyr33(B10) (see Figure 4B). The reorientation of •NO2 and its H-bonding to Tyr33(B10) are accompanied by conformational changes in the tyrosine side chain (see Figures 4C-D, regions encircled). The


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subsequent loss of the Tyr33(B10)–•NO2 H-bond and the formation of a Gln58(E11)–•NO2 Hbond further drive the rotation of the •NO2 radical. At this stage, •NO2 is properly oriented to attack compound II.


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Figure 4. Tyr33(B10) dynamics during five constrained QM/MM MD simulations along the reaction pathway. Each 9-ps segment, delimited by vertical lines, corresponds to one of the simulation windows indicated by red grid points on the 2D PMFs of Figure 2B. Panel A represents the H-bond between Tyr33(B10) and Leu54(E7) backbone, and panel B the H-bond between Tyr33(B10) and •NO2 radical. Panels C and D represent the orientation of Tyr33(B10) phenyl and hydroxyl groups, respectively. The persistent H-bond network between Tyr33(B10), Gln58(E11), and the heme-bound O-atom observed from the simulations is indicated with red


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dashed lines in the schematic (top). Continuous horizontal red lines in panels A and B indicate the distances at which Tyr33(B10) hydroxyl hydrogen forms stable H-bonds with Leu54(E7) backbone carbonyl and with •NO2 radical, respectively. In the schematic, the H-bonds formed by Tyr33(B10) are identified as red dashed lines and the rotations of Tyr33(B10) phenyl/hydroxyl groups are indicated as red/green arrows.

Gln58(E11) contributes to maintaining this favorable orientation of •NO2 by forming Hbonds with the O atoms of the •NO2 intermediate and the NO3– product. The H-bonds formed by Gln58(E11) with •NO2 and NO3– are indicated with blue dashed lines on the schematic in Figure 4. The average H-bond length between Gln58(E11) and •NO2 is 2.36 Å, and that between Gln58(E11) and NO3– is 2.45 Å. Apart from assisting •NO2 rebinding via H-bonding, Gln58(E11) also binds atom O1 (bound to Fe) persistently throughout the isomerization reaction, as indicated with red dashed lines on the schematic in Figure 4 (with an average Gln58(E11)–O1 distance of 2.45 Å). Tyr33(B10) changes conformations during OONO– isomerization. It is predominantly oriented outward the active site, forming a stable H-bond with Leu54(E7) backbone carbonyl, but reorients itself when the •NO2 radical intermediate is high enough in the DHP to participate in H-bonding. In the simulation window where •NO2 and compound II are most stable, both the phenyl and hydroxyl groups of Tyr33(B10) rotate inward. The phenyl group rotates by as much as 110°, as shown in the χ2 (CA-CB-CG-CD1) time series of Figure 4C going from a typical gauche+ rotamer (χ2 ~ 100°) to a strained χ2 ~ –10° conformation. Concomitantly, the hydroxyl group rotates by as much as ~150°, as evidenced by the HH-OH-CZ-CE1 dihedral angle going


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from a –130° average to +20° (Figure 4D). These rotations allow the formation of a transient Hbond between the Tyr33(B10) hydroxyl and the •NO2 radical. In the transition state (TS2), Tyr33(B10) reverts to its original conformation once the •NO2 radical has oriented its unpaired electron toward the oxo-ferryl species to form NO3–. Heme-bound NO3– is stabilized by Gln58(E11) as well as by C−H···O bonding with Phe46(CD1). Electronic structure calculations of Bryantsev and Hay46 show that C−H···O bonds between benzene and NO3– can have as much as 57% of the strength of water-NO3– hydrogen bonds. The binding energy for the global minimum of the benzene-NO3– complex found by Bryantsev and Hay,46 in which two C−H groups contact the anion and form an asymmetric complex with one short linear C−H···O bond (H···O distance of 2.20 Å) and one long bent C−H···O bond (H···O distance of 2.40 Å), is –9.26 kcal/mol, compared to –16.03 kcal/mol for the water complex (with corresponding H···O distances of 1.86 and 2.32 Å). Our own calculations at the B3LYP/6-311G(d,p) level of theory show an optimum geometry comparable with the global minimum of Bryantsev and Hay,46 with H···O distances of 2.20 and 2.32 Å. It is therefore expected that the interactions between NO3– and the aromatic side-chains of trHbN active site could stabilize the product and contribute to its release. Indeed, for more than 7 ps of the 9-ps simulation window in which bound NO3– is formed, a sharp drop is observed in the C−H···O distance between Phe46(CD1) and NO3–, going from 5.0 Å to less than 2.4 Å (with a minimum of 1.94 Å) (Figure 3, red dashed line). The simulations show that trHbN residues Tyr33(B10), Gln58(E11), Phe46(CD1), and Phe32(B9) isolate the highly reactive •NO2 and prevent its escape out of the active site and into the protein matrix. By controlling the isomerization reaction, trHbN active site residues prevent


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•

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NO2 escape toward the protein matrix where it could nitrate Tyr residues and lead to trHbN

degradation. (In trHbN there are two Tyr residues besides the distal Tyr33(B10).) Secondary reactions: Tyr33(B10) oxidation and nitration. The OONO– isomerization in trHbN is particularly interesting due to the presence of Tyr33(B10), which could in principle be involved in the reaction by supplying the redox equivalent needed for the heterolytic cleavage of the peroxynitrite O-O bond, forming a compound I oxo-ferryl intermediate, a Tyr33(B10) radical cation (Tyr33•+), and an NO2– anion. However, the present QM/MM MD simulations show homolytic cleavage of the O-O bond, forming a compound II oxo-ferryl intermediate and an •NO2 radical. The distance between Tyr33(B10) and the ferryl iron remains large (between 6 and 8 Å) in all simulations, which would prevent fast electron transfer from Tyr33 to the iron (according to Marcus theory47,48) and preclude the formation of an NO2– anion. Given how shortlived the •NO2 radical is, these results suggest that Tyr33(B10) nitration may not be a competitive pathway in the NOD isomerization reaction, as hypothesized earlier by Yeh et al.31 The presence of Tyr33(B10) and of highly reactive species FeIV=O2– and •NO2 in the trHbN active site is problematic in principle. FeIV=O2– could oxidize Tyr33(B10) to form a tyrosyl radical (Tyr33•) and a hydroxide-bound ferric heme (FeIII-OH–), by electron transfer from the Tyr33 to the ferryl iron (to form a tyrosine radical cation, Tyr33•+) followed by proton transfer from the Tyr33 hydroxyl to the ferric heme (to form the tyrosyl radical, Tyr33•). Tyr33• could then irreversibly interact with •NO2 to form 3-nitrotyrosine, leading to permanent trHbN degradation. A similar mechanism was recently proposed for peroxynitrite-sensitive superoxide dismutase (SOD).49 In the case of trHbN, as mentioned above, the electron transfer step in Tyr33(B10) oxidation leading to the formation of the tyrosine radical cation (Tyr33•+) may be prohibited by the large distance between Tyr33(B10) and the ferryl iron. The distance between


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the H-atom of Tyr33(B10) hydroxyl group and the O-atom of the oxo-ferryl compound (O1) is similarly large (greater than 5 Å, typically) and makes proton abstraction unlikely as well. More importantly, the simulations show that Tyr33(B10) is involved in a persistent H-bond network with Gln58(E11) and Leu54(E7). The hydroxyl group of Tyr33(B10) continuously H-bonds with Leu54(E7) backbone and is oriented outward the active site, except in the short-lived intermediate for which it transiently binds the •NO2 radical. However, Tyr33(B10) predominantly displays conformations in which the ortho position, the site at which nitration damage can occur, is far from the •NO2 radical (5.5 Å on average). The results suggest that the H-bond network between Gln58(E11), Tyr33(B10), and Leu54(E7) prevents the oxidation and nitration of Tyr33(B10) by keeping the hydroxyl and phenyl groups at safe distance and orientation relative to the oxo-ferryl species and the •NO2 radical. An additional constrained QM/MM MD simulation was performed with the distance between the hydroxyl hydrogen of Tyr33(B10) and the oxygen of FeIV=O2– fixed at 0.95 Å to force the hydroxyl group proton abstraction by FeIV=O2– (under the assumption that the electron transfer has occurred), and thus to force the formation of the tyrosyl radical (Tyr33•). A snapshot from the sampling window where •NO2 is nearest the ortho position of Tyr33 was selected as a starting structure. After 30 ps of simulation, •NO2 is closer to the presumed Tyr33• ortho position relative to the starting structure (at 3.5 Å on average), but the Tyr33 phenyl ring retains its outward orientation away from •NO2, and 3-nitrotyrosine does not form. This suggests that the attack of Tyr33 by •NO2 is not a competitive pathway even for a pre-formed Tyr33• radical. 3.2 NOD reaction: Product release


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Additional QM/MM MD simulations were conducted to investigate the dissociation and release of the nitrate anion in trHbN and to understand the role of Tyr33(B10)/Gln58(E11) pair and of Phe46(CD1) and Phe32(B9) residues in promoting NO3– release. A one-dimensional PMF was constructed from constrained simulations using the Fe-N distance as reaction coordinate. To obtain the free energy profile for NO3– dissociation, the Fe-N distance was constrained in 14 sampling windows covering the 2.75–6.00 Å range in increments of 0.25 Å. Energetics. The PMF calculated for NO3– release (see Figure 5) shows a free energy minimum at dFe-N = 3.00 Å, corresponding to the bound NO3–. The maximum at dFe-N = 4.00 Å indicates a free energy barrier of 11.5 kcal/mol, suggesting a microsecond timescale for nitrate dissociation. This estimate is most likely an upper limit given that NO3– dissociation and release may be favored by the inclusion of water molecules in the DHP, which could hydrate the anion (as suggested by Martí el al33), or coordinate the ferric iron.50 No water molecules or •NO molecules from the MM part of the simulation system ever entered the DHP during the QM/MM simulations, which was to be expected given their short timespan. However, water is known to bind ferric trHbN and to be stabilized by Tyr33(B10).50 At Fe-N distances larger than 4.00 Å, the free energy goes downhill to the product state. The free energy profile in trHbN is lowered relative to the potential energy profile for a similar NO3– release process obtained from QM(DFT) calculations in gas phase (see Figure S3). The potential energy surface in gas phase shows the global minimum at dFe-N = 3.00 Å, similar to that obtained by Martí et al33 and to the free energy profile in trHbN (see Figure 5). In gas phase, the energetic difference between bound nitrate and released product is 35 kcal/mol (at dFe-N = 6.00 Å). The free energy profile is considerably lower in trHbN due to favorable contacts of NO3– with the edges of the aromatic side-chains of Phe46(CD1), Phe32(B9) and Tyr33(B10).


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Figure 5. PMF of the NOD product release in trHbN calculated from constrained QM/MM MD simulations with Fe-N distance fixed at values between 2.75 and 6.00 Å. Each point on the free energy profile corresponds to a 10-ps constrained QM/MM MD simulation. The free energy minimum at dFe-N = 3.00 Å corresponds to bound NO3–. The maximum at dFe-N = 4.00 Å indicates a free energy barrier of 11.5 kcal/mol for NO3– dissociation. Schematics of trHbN active site and the interactions that stabilize the NO3– anion at Fe-N distances of 3.00 and 6.00 Å are shown above the PMF.

trHbN dynamics. The simulations of NO3– release substantiate earlier theoretical results25,33 and show that the electrostatic interactions between the nitrate anion and the two polar side-chains of Tyr33(B10) and Gln58(E11) pair promote the breaking of the FeIII-O bond and dissociation of NO3– by lowering the energy barrier of the reaction. Once formed, NO3– is participating in the Gln58(E11)–Tyr33(B10)–Leu54(E7) dynamic H-bond network. At that point, Tyr33(B10) alternatively binds the Leu54(E7) backbone carbonyl and NO3– (see Figure 6B-D). The H-bond between Tyr33(B10) and Gln58(E11) is very stable, with an average distance of 2.2 Å in simulation windows where the Fe-N distance is in the 2.75–4.00 Å, “pre-


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dissociation” range (Figure 6A). At larger Fe-N distances, the Tyr33(B10)–Gln58(E11) H-bond breaks transiently due to H-bonding competition from the NO3– ligand. The H-bond between Tyr33(B10) and the backbone carbonyl of Leu54(E7) is generally stable throughout the dissociation process, with a typical distance of 1.9 Å (Figure 6B). The Gln58(E11)–Tyr33(B10)– Leu54(E7) H-bond network is maintained during the NO3– dissociation, except when the Tyr33(B10) hydroxyl rotates and binds NO3– instead of Leu54(E7) (see Figure 6C, regions encircled). Specifically, the Tyr33(B10)–Leu54(E7) H-bond is lost for several picoseconds in 5 simulations windows identified at the top of Figure 6: dFe-N = 3.00 Å corresponds to NO3– bound to ferric iron; dFe-N = 3.50, 3.75, and 4.25 Å are in the range over which the FeIII-O bond breaks and NO3– dissociates; and dFe-N = 5.00 Å corresponds to fully dissociated NO3–.


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Figure 6. trHbN dynamics during NOD product release. Each 9-ps segment (delimited by vertical dashed lines) corresponds to one of the 14 red points on the PMF of Figure 5, in order of increasing Fe-N distance. Panel A shows H-bonding between Tyr33(B10) and Gln58(E11), panel B shows H-bonding between Tyr33(B10) and Leu54(E7) backbone, panel C shows the conformation of Tyr33(B10) hydroxyl group, and panels D/E show H-bonding between NO3– product and Tyr33(B10)/Gln58(E11) hydroxyl/amide groups. Tyr33(B10) is usually donating a H-bond to Leu54(E7) (panel B) but changes conformations (panel C) and donates a H-bond to NO3– instead (panel D) in 5 simulation windows (identified at the top). (See text for discussion of


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the dFe-N = 5.00 Å window of panel D). Gln58(E11) is a H-bond donor to NO3– and stabilizes NO3– both in its bound and dissociated states. Continuous horizontal red lines indicate the distances at which stable H-bonds form. The rotations of Tyr33(B10) hydroxyl group (correlated with breaking of the Tyr33(B10)–Leu54(E7) H-bond and formation of the Tyr33(B10)–NO3– Hbond) are indicated as green circles.

The Tyr33(B10) hydroxyl group changes its orientation upon losing its H-bond to Leu54(E7) and binding to nitrate (see Figure 6C). During the NO3– dissociation process, though, Tyr33(B10) does not rotate significantly around the CB-CG bond, and remains in its typical gauche+ conformation. The hydroxyl group, on the other hand, rotates inward to assist, in conjunction with Gln58(E11), the breaking of the FeIII-O bond (at dFe-N ≤ 4.25 Å) and to stabilize the NO3– product outside of the active site (at dFe-N ≥ 5.00 Å). The HH-OH-CZ-CE1 dihedral angle of Tyr33(B10) then changes by as much as 120° (see Figure 6C). The distance between the hydroxyl hydrogen atom of Tyr33(B10) and the closest oxygen atom of NO3– is plotted in Figure 6D, showing occasional hydrogen bonding. Interestingly, in the dFe-N = 5.00 Å simulation window, for almost 3 ps, NO3– abstracts the proton from the Tyr33(B10) hydroxyl hydrogen atom to form nitric acid. The tyrosinate then takes the proton back and continues to H-bond the NO3– product (as in the beginning of the simulation) and to assist its release. Gln58(E11) is a H-bond donor to the NO3– oxygen atoms and interacts with the NO3– anion in both bound and dissociated forms (see Figure 6E). These findings show that Gln58(E11) contributes substantially to the breaking of the FeIII-O bond and to the stabilization of the dissociated nitrate on its way out of the heme pocket.


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In addition, aromatic residues Phe46(CD1), Phe32(B9) and Tyr33(B10) contribute to stabilizing the NO3– anion as it gets further away from the heme (see Figure S4). They form C−H···O bonds similar to that reported earlier (between Phe46(CD1) and NO3–), with H···O distances less than 2.4 Å and C···N distances less than 4.0 Å (see Figure S4). While the role of these highly conserved residues in promoting product dissociation and release has never been addressed, such C−H···anion interactions could create additional binding sites for the nitrate product within the protein cavity. A schematic illustrating the stabilization of nitrate by the aromatic rings of Phe46(CD1), Phe32(B9) and Tyr33(B10) at Fe-N distance of 6.00 Å is provided in Figure 5. 4. CONCLUSION In this study, the nitric oxide dioxygenase (NOD) reaction of the truncated hemoglobin N (trHbN) from Mycobacterium tuberculosis (Mtb) was investigated by QM/MM free energy calculations, thus incorporating the effects of thermal fluctuations and of the rearrangement of trHbN residues at the active site. In particular, the mechanism of OONO– isomerization was elucidated by extensive sampling along two reaction coordinates, and the trHbN active site residues critical in assisting the reaction were determined. The free energy surface reveals a fast sequential mechanism with very short-lived FeIV=O2– and •NO2 intermediates, and suggests that the intermediates may be too short-lived to be detected experimentally. Our results rule out the possibility of a concerted mechanism but, instead, provide evidence that the highly conserved residues Tyr33(B10), Gln58(E11), Leu54(E7), Phe46(CD1), Phe32(B9) and Val94(G8) in the DHP are caging the reactive FeIV=O2– and •NO2 intermediates resulting from OONO– homolysis. Our results are in agreement with Ouellet et al’s experimental mutagenesis study7 showing that Tyr33Phe variant mycobacteria cannot eliminate •NO, thus demonstrating the essential role of


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Tyr33(B10) for the high •NO-scavenging efficiency of trHbN. Similarly, Gardner et al22 showed that the substitution of Tyr(B10) for a Phe in E. coli flavoHb decreases the NOD reaction rate by a factor ~30. Furthermore, our results confirm an earlier hypothesis24 that the trHbN active site residues, especially the Tyr33(B10)/Gln58(E11) pair, might isolate the reaction intermediates formed in the hydrophobic pocket lined by residues Phe32, Tyr33, Phe46, and Val94. The simulations show that the H-bond between Tyr33(B10) and Gln58(E11) is very stable throughout the OONO– isomerization. Tyr33(B10) also forms a H-bond with Leu54(E7) backbone, which is stable at all times during the reaction except when the •NO2 radical intermediate is present. In contrast to Mishra et al’s findings,25 indicating that •NO2 being formed continuously participates in H-bonding with both Tyr33(B10) and Gln58(E11), our results show that Tyr33(B10) stabilizes the •NO2 radical transiently, but is predominantly H-bonded to Leu54(E7). Tyr33(B10) changes conformations during the isomerization reaction, to transiently stabilize the •NO2 radical and orient it for rebinding to the oxo-ferryl species. Gln58(E11) assists •

NO2 rebinding via H-bonding and Phe46(CD1) forms favorable C−H···O contacts with the

bound product. Our results also indicate that the H-bond network between Gln58(E11), Tyr33(B10), and Leu54(E7) prevents Tyr33(B10) oxidation and nitration (hence, trHbN degradation) by keeping the hydroxyl and phenyl groups at safe distance and orientation relative to the oxo-ferryl species and the •NO2 radical. A general characteristic of enzymes that use radicals for catalysis is their propensity to self-inactivate during turnover. While we cannot exclude that tyrosine nitration does occur from time to time (leading to a loss of function), it appears that trHbN prevents its own degradation by controlling the isomerization reaction. Similar dynamics of the DHP residues have been observed during NO3– dissociation and release. Our results substantiate earlier theoretical results25,33 that the electrostatic interactions


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between the nitrate anion and the two polar side-chains of Tyr33(B10) and Gln58(E11) pair promote the breaking of the FeIII-O bond, promote NO3– dissociation, and are the main stabilizing residues for the product. In addition, our results show that the C−H···anion interactions between the aromatic residues Phe46(CD1), Phe32(B9) and Tyr33(B10) and the nitrate anion contribute to the stabilization of the dissociated product and to its release. Water entry into the DHP, which could promote NO3– dissociation as suggested by Martí el al,33 was not observed in our short QM/MM MD simulations. Extensive MD simulations of the O2-bound form of the enzyme suggest that the DHP has a very weak but detectable affinity for water (unpublished analysis of the results from Rhéault et al16), but microsecond-long simulations of the nitrate-bound form of the enzyme would have to be performed to estimate how likely water is to stabilize the product during its egress. Based on our current results, we cannot say whether the LT long tunnel or the BE tunnel (or any other alternate pathway) is used for product release. In combination with recent insights into the function of the pre-A helix, which can promote the association of trHbN with the membrane (and facilitate the uptake of O2 and •NO dissolved in the membrane)16 and which can modulate the interaction with a compatible reductase (required for recycling of trHbN to its ferrous state),51 our results provide a detailed description of the highly efficient •NO-scavenging mechanism of Mtb trHbN. The present work suggests that the NOD mechanism in Mtb trHbN would be similar in bacterial flavoHbs, which have distal heme pockets lined with two polar groups (Tyr and Gln) capable of H-bonding and three aromatic side-chains (Tyr, Phe) capable of forming C−H···anion interactions, but would be different in mammalian globins functioning as rudimentary enzymatic NODs, which have a single polar group (His) and three long aliphatic residues (Ile, Leu, Val) in the distal pocket (see Figure S1). The overall NOD mechanism would likely be preserved in


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other globins having distal heme pockets of similar large size and high polarity as trHbN and flavoHbs

ASSOCIATED CONTENT Supporting Information. Additional results. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Present Addresses #

Department of Experimental Medicine, Faculty of Medicine, The University of British

Columbia and The Vancouver Prostate Centre at Vancouver General Hospital, Vancouver, BC, Canada. ACKNOWLEDGMENTS This work was supported by an FRQNT Team research project Grant to M.G., P.L. and G.L., and by an NSERC Discovery Grant to G.L. Computational resources were provided by Calcul Québec and Compute Canada.


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Mechanism of the Nitric Oxide Dioxygenase Reaction of

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