Comparison of the Channel 3 region in cNOR (A) and the periplasmic cavity in <i>cbb</i>₃ oxidase (B).

<p>Crystal structures of cNOR from <i>Ps. aeruginosa</i> and <i>cbb</i>₃ oxidase from <i>Ps. stutzeri</i> (PDB ID: 3MK7) <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002674#pcbi.1002674-Pisliakov1" target="_blank">[56]</a> were aligned on hemes <i>b</i> and <i>b</i>₃. The transparent blue surfaces indicate the positions of hydrophilic cavities. The central residues, which form the hydrophilic cavity (namely Glu135, Glu138, Arg57 in cNOR and Glu122, Glu125, Arg57 in <i>cbb</i>₃ oxidase), are identical and highly conserved in the HCO superfamily. A calcium ion is located in a similar position between hemes.</p

Channel 1, the proposed proton pathway.

<p>(<b>A</b>) A representative MD snapshot (at ∼100 ns) of the Channel 1 region. The pathway starts near Lys53_c, Glu57_c and Asp198 (the entrance from the bulk is indicated by a blue line) and leads to a water cluster near the heme <i>b</i>₃ propionates via a number of conserved charged residues and water molecules (see details in the text). Hemes <i>b</i> (dark-red) and <i>b</i>₃ (pink) and the charged residues lining the channel (colored by atom types, with carbons in yellow) are shown as sticks, Fe_B (orange) and Ca²⁺ (green) ions as spheres, and water molecules present in or near the channel are shown in ball-and-stick representation (red/white). Important helices are labeled (green). The dashed lines indicate the hydrogen bonds within the forming continuous H-bonded networks, which connect the periplasmic surface with the propionates of heme <i>b</i>₃. In the MD simulation the Channel 1 interior region is very well hydrated, as illustrated by the water density averaged over 300 ns (a transparent light-blue isosurface shown at 25% occupancy). Figure reveals two branches of the water channel at both sides of Glu70_c. (<b>B</b>) Water density in Channel 1 shown as a 2D contour map (a projection on the XY-plane was obtained by summing the water density in the Channel 1 region over the vertical Z-axis). Different colors correspond to water residence values, ranging from red (high water residence) to blue (low water residence). Positions of the Channel 1 residues and crystallographic waters in the cNOR X-ray structure are shown for reference as white sticks and purple spheres, respectively. Two branches of the potential proton pathway (indicated by the dashed black lines) go through high-occupancy water sites, which in general superimpose well with the positions of crystallographic waters. (<b>C</b>) Number of water molecules in the Channel 1 region in the MD simulation; black line represents a running average over 30 data points. Water molecules within 4.5 Å of the channel residues were selected.</p

Chemo-Mechanical Coupling in the Transport Cycle of a Heme ABC Transporter

The heme importer from pathogenic bacteria is a member of the ATP-binding cassette (ABC) transporter family, which uses the energy of ATP-binding and hydrolysis for extensive conformational changes. Previous studies have indicated that conformational changes after heme translocation are triggered by ATP-binding to nucleotide binding domains (NBDs) and then, in turn, induce conformational transitions of the transmembrane domains (TMDs). In this study, we applied a template-based iterative all-atom molecular dynamics (MD) simulation to predict the ATP-bound outward-facing conformation of the Burkholderia cenocepacia heme importer BhuUV-T. The resulting model showed a stable conformation of the TMD with the cytoplasmic gate in the closed state and the periplasmic gate in the open state. Furthermore, targeted MD simulation predicted the intermediate structure of an occluded form (Occ) with bound ATP, in which both ends of the heme translocation channel are closed. The MD simulation of the predicted Occ revealed that Ser147 on the ABC signature motifs (LSGG­[Q/E]) of NBDs occasionally flips and loses the active conformation required for ATP-hydrolysis. The flipping motion was found to be coupled to the inter-NBD distance. Our results highlight the functional significance of the signature motif of ABC transporters in regulation of ATPase and chemo-mechanical coupling mechanism

New plausible proton pathway, Channel 3, revealed by the MD simulations.

<p>(<b>A</b>) A representative configuration (MD snapshot at ∼25 ns) of the Channel 3 region when the Asn54-Asn60_c gate is closed and the internal hydrophilic cavity between Asn60_c, Glu138 and Arg57 has no connection to the outside bulk. (The color coding is as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002674#pcbi-1002674-g002" target="_blank">Figure 2a</a>; the water density was averaged over the first 165 ns.) (<b>B</b>) Water density in the Channel 3 region averaged over the first 165 ns, shown as a 2D contour map (a projection on the XY-plane; the color coding is as in Figure 2b.) (<b>C</b>) A representative configuration (MD snapshot at ∼200 ns) when the gating residues, Asn54 and Asn60_c, move away from each other. The water density, which was averaged over the interval 165–225 ns, when the gate is open, shows a newly formed water channel. The dynamic H-bonded water chains connect the bulk to the two important residues, Glu138 and Glu135, and can facilitate PT toward the active site. The new suggested pathway is spatially separated from Channel 1, part of which is shown for reference as a light-gray surface to the left of heme <i>b</i>₃. (<b>D</b>) Same as in B, but the water density was averaged over 165–225 ns. When the Asn-Asn gate is open, the continuous water distribution from the bulk up to Glu135/Ca²⁺ site and Glu138 is observed. The possible water-mediated PT pathway is indicated by the dashed black lines, with the path via Glu138 to the water sites near the BN center being more plausible (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002674#pcbi-1002674-g007" target="_blank">Figure 7</a> and discussion therein).</p

The MD simulation does not support the previously suggested Channel 2 as a possible proton uptake pathway.

<p>(<b>A</b>) A representative MD snapshot (at ∼170 ns) and the calculated water density (the color coding is as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002674#pcbi-1002674-g002" target="_blank">Figure 2a</a>) show that water molecules in the upper hydrophilic region are separated from the water cluster near heme <i>b</i>₃ by two loops (see details in the text). (<b>B</b>) Water density shown as a 2D contour plot (a projection on the YZ-plane; the color coding is as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002674#pcbi-1002674-g002" target="_blank">Figure 2b</a>) and as a density profile along Z-coordinate. (<b>C</b>) Gly340-Gly69_c distance time series from the MD simulation.</p

Gating of Channel 3.

<p>(<b>A–C</b>) The time series of the minimal distances between Asn54-Asn60_c and Glu138-Asn60_c residues and the number of water molecules near sidechains of Glu138 and Asn54 in the MD simulation. The vertical red dashed lines indicate the interval when the Asn-Asn gate is open and the water channel is formed. Glu138 remains well hydrated even after the gate closing. (<b>D–E</b>) Close-up views of the gate region for the closed and open cases. (<b>F</b>) An overlay of the closed (green) and open (orange) structures of the Channel 3 gate region. The conformational changes associated with the gate opening involve rotation of the sidechains of Asn54, Asn60_c and Glu138 and tilting of the helix TM II.</p

No water channels from the cytoplasm are found in cNOR.

<p>The region similar to the K- and D-pathways in oxidases is shown (MD snapshot at ∼100 ns). In cNOR, residues in this region are mostly hydrophobic. The interior region remains minimally hydrated (as shown by the water density averaged over 300 ns), except for a small charged region below the active site with Glu211, Glu280, and Glu215.</p

ONIOM Study on a Missing Piece in Our Understanding of Heme Chemistry: Bacterial Tryptophan 2,3-Dioxygenase with Dual Oxidants

Unique heme-containing tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) catalyze oxidative cleavage of the pyrrole ring of l-tryptophan (Trp). Although these two heme dioxygenases were discovered more than 40 years ago, their reaction mechanisms were still poorly understood. Encouraged by recent X-ray crystal structures, new mechanistic pathways were proposed. We performed ONIOM(B3LYP:Amber) calculations with explicit consideration of the protein environment to study various possible reaction mechanisms for bacterial TDO. The ONIOM calculations do not support the proposed mechanisms (via either formation of the dioxetane intermediate or Criegee-type rearrangement); a mechanism that is exceptional in the hemes emerges. It starts with (1) direct radical addition of a ferric-superoxide intermediate with C2 of the indole of Trp, followed by (2) ring-closure via homolytic O−O cleavage to give epoxide and ferryl-oxo (Cpd II) intermediates, (3) acid-catalyzed regiospecific ring-opening of the epoxide, (4) oxo-attack, and (5) finally C−C bond cleavage concerted with back proton transfer. The involvement of dual oxidants, ferric-superoxide and ferryl-oxo (Cpd II) intermediates, is proposed to be responsible for the dioxygenase reactivity in bacterial TDO. In particular, the not-well-recognized ferric-superoxide porphyrin intermediate is found to be capable of reacting with π-systems via direct radical addition, an uncommon dioxygen activation in the hemes. The comparison between Xanthomonas campestris TDO and some heme as well non-heme oxygenases is also discussed

Interactions of Soluble Guanylate Cyclase with a P‑Site Inhibitor: Effects of Gaseous Heme Ligands, Azide, and Allosteric Activators on the Binding of 2′-Deoxy-3′-GMP

Nitric oxide (NO) elicits a wide variety of physiological responses by binding to the heme in soluble guanylate cyclase (sGC) to stimulate cGMP production. Although nucleotides, such as ATP or GTP analogues, have been reported to regulate the signaling of NO binding from the heme site to the catalytic site, the other regulatory functions of nucleotides remain unexamined. Among the nucleotides tested, we found that 2′-d-3′-GMP acted as a potent noncompetitive inhibitor with respect to Mn-GTP, when the ferrous enzyme combined with NO, CO, or allosteric activator BAY 41-2272. 2′-d-3′-GMP also displayed nearly identical patterns of inhibition for the ferric enzyme, in which the binding of N₃^– or BAY 41-2272 significantly increased the inhibitory effects of the nucleotide. Equilibrium dialysis measurements using the CO-ligated enzyme in the presence of allosteric activators demonstrated that 2′-d-3′-GMP exclusively binds to the catalytic site of sGC. Furthermore, the affinity of 2′-d-3′-GMP for the enzyme was found to increase upon addition of foscarnet, an analogue of PP_i. These findings together with other kinetic results imply that 2′-d-3′-GMP acts as a P-site inhibitor probably by forming a dead-end complex, sGC–2′-d-3′-GMP–PP_i, in the catalytic reaction. The formation of the complex of the enzyme with 2′-d-3′-GMP does not seem to be associated with changes in the Fe–proximal His bond strength, because the CO coordination state or the redox potentials of the enzyme-heme complex are virtually unaffected

ONIOM Study on a Missing Piece in Our Understanding of Heme Chemistry: Bacterial Tryptophan 2,3-Dioxygenase with Dual Oxidants

Unique heme-containing tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) catalyze oxidative cleavage of the pyrrole ring of l-tryptophan (Trp). Although these two heme dioxygenases were discovered more than 40 years ago, their reaction mechanisms were still poorly understood. Encouraged by recent X-ray crystal structures, new mechanistic pathways were proposed. We performed ONIOM(B3LYP:Amber) calculations with explicit consideration of the protein environment to study various possible reaction mechanisms for bacterial TDO. The ONIOM calculations do not support the proposed mechanisms (via either formation of the dioxetane intermediate or Criegee-type rearrangement); a mechanism that is exceptional in the hemes emerges. It starts with (1) direct radical addition of a ferric-superoxide intermediate with C2 of the indole of Trp, followed by (2) ring-closure via homolytic O−O cleavage to give epoxide and ferryl-oxo (Cpd II) intermediates, (3) acid-catalyzed regiospecific ring-opening of the epoxide, (4) oxo-attack, and (5) finally C−C bond cleavage concerted with back proton transfer. The involvement of dual oxidants, ferric-superoxide and ferryl-oxo (Cpd II) intermediates, is proposed to be responsible for the dioxygenase reactivity in bacterial TDO. In particular, the not-well-recognized ferric-superoxide porphyrin intermediate is found to be capable of reacting with π-systems via direct radical addition, an uncommon dioxygen activation in the hemes. The comparison between Xanthomonas campestris TDO and some heme as well non-heme oxygenases is also discussed