Imaging the Migration Pathways for O2, CO, NO, and Xe Inside Myoglobin Jordi Cohen, Anton Arkhipov, Rosemary Braun, Klaus Schulten  Biophysical Journal  Volume 91, Issue 5, Pages 1844-1857 (September 2006) DOI: 10.1529/biophysj.106.085746 Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 1 Schematic diagram showing the probability of occurrence of all the protein states, during a simulation of the protein reference system (solid line), or those desired in order to get a proper PMF for the protein-ligand system (dashed line). The introduction of a ligand inside the protein at a given position perturbs the energies of all the protein states from the reference ensemble, and consequently alters their probability of occurrence. Biophysical Journal 2006 91, 1844-1857DOI: (10.1529/biophysj.106.085746) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 2 Predicted and actual Xe binding sites for the sperm whale Mb D122N mutant (shown in ribbon representation with the heme drawn as licorice). The predictions, shown as red isosurfaces representing the areas where the Xe PMF is <−4.9kcal/mol (points on this surface have an error of ±0.8kcal/mol), are based on a 4-ns equilibrium simulation of a Xe-less Mb structure (PDB accession No. 2MBW). The four experimental Xe locations, represented by labeled circles, are taken from a structure of the same protein under 7atm Xe pressure (1J52) (37). Biophysical Journal 2006 91, 1844-1857DOI: (10.1529/biophysj.106.085746) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 3 Implicit ligand PMF for CO migration inside sperm whale Mb, based on a 5-ns equilibrium simulation of the 1DUK PDB structure, shown from four views looking toward the heme (a–d). The three energy isosurfaces represent PMF values of −1.5kcal/mol (red), 1kcal/mol (blue cavities), and 5kcal/mol (green). The empty white space corresponds to regions of measured PMF above 5kcal/mol; the zero energy value corresponds to the ligand in vacuum. Practically speaking, the red surfaces show gas docking sites, the inner blue surfaces show the areas inside the protein that are more favorable to CO than the external aqueous solution, and the green surfaces highlight the regions of lowest energy barriers between the various cavities. The low energy barrier exits according to the displayed PMF map are indicated by red lines and circles, and dashed indicators mean that the exits are in the back. The error on points lying on the three PMF isosurfaces are ±0.3, +0.3/−0.4, and +0.3/−3.6kcal/mol for red, blue, and green, respectively. The Mb’s static surface is represented in white-inside-blue-outside color and the heme is displayed with its bound proximal histidine. Biophysical Journal 2006 91, 1844-1857DOI: (10.1529/biophysj.106.085746) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 4 Amino acids whose substitutions significantly affect O2 or CO migration properties during geminate rebinding in Mb, as determined by Huang and Boxer (40). The heme is drawn with the attached proximal His-93. Residues forming the commonly recognized distal pathway are shown in yellow. The amino acids that are found at the exits from the Mb interior, according to the PMF maps, are shown in red. Small amino acids that line a constriction between cavities, and large amino acids that directly block passages between neighboring ligand-accessible areas, are colored in blue. Residues that were shown to affect ligand migration properties, but do not have any visible influence on the gas pathway according to the PMF maps are colored in green (some of these residues do cap α-helices and may play a structural role). The location of the gas migration pathways is drawn schematically, with light gray and dark gray, respectively, representing likely and highly likely regions for the ligand. Thick dashed lines indicate the exits that go out of the plane of the figure toward the viewer; thin dashed lines correspond to the exits behind the plane. Red arrows have been added to indicate the exits from Mb, dashed arrows represent exits behind the plane. All residues except those lining the distal pocket (yellow) are labeled. Biophysical Journal 2006 91, 1844-1857DOI: (10.1529/biophysj.106.085746) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 5 PMF profiles experienced by ligands exiting Mb along (a) the distal pathway and (b,c) the two other most favorable exit paths between the heme binding site and the external solution. The path profiles were determined by finding the path, between two predefined endpoints (one at the heme binding site, and one near an Mb exit), that exhibits the smallest energy barriers. The values of the PMF at each point along these paths are then plotted as a function of the ligands’ distance from the heme binding site. The procedure is repeated for O2, NO, CO, and Xe ligands, using the same endpoints for a given exit. The solvation energies of each ligand in water, as given in Table 2, are represented as horizontal dashed lines, and the location of the distal pocket (DP) and Xe binding site Xe4 are indicated. (d) The actual points along the three paths in relation to the Mb PMF maps are plotted in green (for the distal path a), red (path b), and yellow (path c). Biophysical Journal 2006 91, 1844-1857DOI: (10.1529/biophysj.106.085746) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 6 Comparison of the implicit ligand PMF maps in Mbs of different species. The implicit ligand PMF map of O2 for (a) pig, (b) yellowfin tuna, and (c) sea hare Mbs (red) are compared with that for the sperm whale Mb (blue). The isosurfaces are drawn using a PMF value of 1.8 kcal/mol (points on these contours have an error of +0.2/−0.4kcal/mol). The sperm whale Mb’s heme with the connected proximal histidine is shown along with the protein’s external surface (gray). Biophysical Journal 2006 91, 1844-1857DOI: (10.1529/biophysj.106.085746) Copyright © 2006 The Biophysical Society Terms and Conditions