O₂ Migration Pathways Are Not Conserved across Proteins of a Similar Fold

Abstract

Recent advances in computational biology have made it possible to map the complete network and energy profile of gas migration pathways inside proteins. Although networks of O₂ pathways have already been characterized for a small number of proteins, the general properties and locations of these pathways have not been previously compared between proteins. In this study, maps of the O₂ pathways inside 12 monomeric globins were computed. It is found that, despite the conserved tertiary structure fold of the studied globins, the shape and topology of O₂ pathway networks exhibit a large variability between different globins, except when two globins are nearly identical. The locations of the O₂ pathways are, however, found to be correlated with the location of large hydrophobic residues, and a similar correlation is observed in two unrelated protein families: monomeric globins and copper-containing amine oxidases. The results have implications for the evolution of gas pathways in proteins and for protein engineering applications involving modifications of these pathways.

INTRODUCTION

For many classes of proteins, enzymatic reaction with, or binding to, gas molecules is an essential component of their function. Such proteins often bind gas molecules by means of buried active sites consisting of metal ions or metal-containing compounds. Gas molecules such as O₂ must make their way across the protein's interior to reach these active sites. In the majority of cases, permanent gas channels can neither be detected nor found present in the protein's static structure; instead, the migrating gas molecules take advantage of a network of transient pathways, which encompass a number of highly favorable gas-holding sites connected by less favorable regions which are used by gas molecules to hop between the holding sites. These pathways are due to thermal fluctuations of the protein (1). By monitoring the occurrence of these transient pathways over time and making use of recent methodological advances such as volumetric gas accessibility maps (2) and implicit ligand sampling (3), one can comprehensively map and describe networks of gas migration pathways inside proteins.

For a large number of proteins that interact with O₂, finding the location of O₂ migration pathways has important implications. For example, locating the O₂ pathways in oxygenases and O₂-consuming oxidases provides important clues regarding their enzymatic activity and operating mechanism. Also, the elucidation of O₂ pathways in hydrogenases is assisting efforts aiming to block O₂ access to the hydrogenase active sites, thus making this protein useful for commercial hydrogen gas production (4–6). To date, complete maps of O₂ pathway networks have only been computed for a small number of proteins, including CpI hydrogenase (2), sperm whale myoglobin (Mb) (3), AQP1 aquaporin (7), and a set of copper amine oxidases (8). As more proteins are scrutinized in terms of their O₂ migration pathways, one will gain a better grasp of how gases are transported inside proteins, discern patterns of how such pathways are conserved across protein families, and develop rules of thumb for quickly identifying these pathways. In this article, we address the question of O₂ pathway conservancy within a given protein fold by computing and comparing maps of the O₂ pathway networks across a range of proteins from the globin superfamily. The comprehensive comparison provides a basic understanding as to how O₂ pathways are formed inside proteins and, in turn, provides clues as to how to engineer gas accessibility in proteins.

Globins are a large and ancient family of proteins for which all members, with few exceptions, share an exceptionally well-conserved tertiary structure: the globin fold. At the heart of this fold lies a prosthetic group, the heme, universally used by globins to reversibly bind to, and to temporarily hold, O₂ and other gas ligands. This investigation focuses on three types of pentacoordinated globins: monomeric hemoglobins (Hbs), which transport O₂ throughout entire organisms, Mbs, which store and transport O₂ within muscle cells, and leghemoglobins (Lbs), which store, transport, and/or scavenge O₂ to maintain a population of symbiotic bacteroids in the root nodules of symbiotic plants (9). We stay clear of hexacoordinated globins such as neuroglobin and cytoglobin, which exhibit poorly understood behavior regarding how O₂ binds to their heme.

In addition to binding O₂, many globins have other physiological functions. For example, although the role of Mb has been long considered to be well established (10–12), recent studies have shown Mb to also be involved in secondary roles (12,13) like scavenging and inactivating nitric oxide (NO) (14) and weak peroxidase activity (15). Many invertebrate Hbs also possess a number of interesting characteristics, such as the ability to react with sulfide (16) and the ability to tune their affinities to O₂ depending on their environment. By forming multimeric assemblies (16–18), many Hb monomers can bind gases such as O₂ and CO₂ cooperatively by making their affinity for O₂ depend on the O₂ occupancy of the neighboring monomers, either through intermonomer conformational changes or through multimeric association/dissociation. It is clear that despite the well-studied nature of the globin family, there remains a large number of globin properties relating to their structure and gas-conduction properties that is still poorly understood and possibly not even known.

In this article, we focus on the O₂-transporting role of the globin protein matrix. A globin's main function is performed by its heme, which binds gas ligands for extended periods of time. Consequently, one can regard the conserved protein fold surrounding the heme as merely a shell. Nevertheless, this “shell” provides important functionality. First, the protein shell protects the heme from oxidizing into an inactive ferric state, which would happen if the heme were to float freely in solution. Second, the protein component strongly modulates the environment of the heme-bound ligand and, thus, influences its binding affinity, its binding rates, and the globin's relative selectivity for various ligands. Finally, the protein matrix provides cavities and pathways for gas ligands to travel from the exterior solution to the heme and vice versa. Since all globins have an identical heme, the protein shell is what differentiates globins in regard to globin behavior and properties.

A large body of work, both experimental and theoretical, has focused on finding gas ligand pathways inside globins, particularly sperm whale Mb and certain Hbs. In particular, x-ray crystallography in the presence of xenon (19), x-ray crystallography of intermediate states (20), time-resolved x-ray crystallography (21–25), spectroscopy of the geminate recombination process (26–32), and molecular dynamics simulation (33–37) all provide important clues as to how gas molecules make their way into proteins. In terms of the pathways' locations, only the areas of the pathways that are most favorable to O₂, often termed “cavities”, “xenon-binding sites”, or “holding regions”, can be observed experimentally since only in these regions is O₂ concentrated enough to be observed. The effects of point mutations on overall O₂ association and dissociation rates in globins can also be observed experimentally. We complement this work by computing the complete map of O₂ pathways inside a broad range of monomeric globins.

METHOD

Simulations were run for a selected set of monomeric globins of known structure, taken from the Protein Data Bank (PDB). In every case, a deoxy form of the globin was created from the PDB coordinates, and any water or gas ligand present in the distal pocket (DP) was removed. For the cases in which only the ferric state of the globin is available (whether unbound or bound to small compounds), the coordinates of the ferric heme were used as starting points, but the hemes were modeled using parameters for the ferrous state.

For every globin, the simulation system was built from the PDB coordinates by adding hydrogen atoms, binding the globin's proximal histidine to the hemes, and picking an appropriate titration state for every histidine based on its immediate environment. The Dowser water placement program (38) was then used to internally solvate the globin atomic structures, though rarely resulting in the placement of additional water molecules. The heme-protein complex was solvated using a water box whose sides exceeded those of the protein by at least 20 Å in all dimensions. A total of 50 mM of NaCl was added, adjusting the relative concentrations of Na⁺ and Cl⁻ to make the whole system chargeless.

The equilibration protocol used two preequilibration stages: an initial 30 ps simulation stage had the protein and heme fixed and allowed the solvent to relax at constant temperature (300 K) and pressure (1 atm); a 50 ps stage then allowed both protein side chains and solvent to equilibrate while constraining the protein backbone. The entire protein-solvent systems were equilibrated for another 950 ps. Finally, an additional 10 ns of simulation at the same NPT conditions was performed for analysis. The 10 ns simulations were processed using the implicit ligand sampling method, reviewed in Cohen et al. (3) and included with the VMD visualization program (39). The implicit ligand sampling method takes advantage of the fact that weakly interacting particles such as gas molecules embedded in a protein do not distort the protein much. In this approximation, it is possible to estimate the free energy of placing a gas molecule at any point inside the protein by treating the gas ligand as a perturbation and by using a one-step free energy perturbation scheme. This allows us to infer the free energy of placing a gas molecule at any given point in space from the energies of placing the molecule directly into protein conformations taken from the frames of an equilibrium simulation trajectory of the dynamics of the protein without gas. The end result is a three-dimensional potential of mean force (PMF) map of the complete network of O₂ migration pathways for each globin.

All simulations were performed using the molecular dynamics program NAMD (40) in combination with the NAMD-G simulation automation engine (41). Simulation parameters were taken from the CHARMM22 force field (42). Particle mesh Ewald, with a resolution of at least 1 Å, was used everywhere for long-range electrostatics. Langevin dynamics and a Langevin piston were used to maintain constant temperature and pressure, respectively. Finally, integration time steps of 1, 2, and 4 ps, respectively, were used for bonded, nonbonded, and long-range electrostatics interactions.

To determine which residue types promote the formation of O₂ cavities and pathways, we performed a scan of the O₂ PMF profile maps to assess residue proximity to O₂ cavities. This was done by computing, for each location on the O₂ PMF grid, the number of residues of each type whose side chains lie within 2 Å of that grid point. This quantity is then averaged over all PMF grid points, using the O₂ occupancy at each grid point as the weight. Finally, this average is divided by the proportional population of each given residue type to remove the bias caused by having certain residue types being more abundant than others. The propensity for each residue type to lie near an O₂ pathways can thus be evaluated as

(1)

where the sum over i is over every PMF grid point within a specified region (corresponding to either the interior or the surface of the protein); G(r_i) is the PMF at i; is the number of residues of type “res” whose side chains lie within 2 Å of i; and T_res is the total population of residues of type “res” which lie within 2 Å of any PMF grid point in the region of interest.

Using residue locations from the crystal structure, as opposed to trajectory snapshots, produced no discernible change to The relative propensities are also not significantly changed when compensated to account for residue size (except for the case of Gly) or when using a different value for the 2 Å distance threshold (tested up to 4 Å).

RESULTS AND DISCUSSION

O₂ pathways in monomeric globins

Although the O₂ pathways and cavities in Mbs (particularly sperm whale Mb) have been studied extensively, those in most other globins remain unknown. We investigated the networks of O₂ pathways in a broad set of pentacoordinated monomeric globins, listed in Table 1, for which atomic coordinates are available. For each protein, its O₂ PMF map was computed using the implicit ligand sampling method (3) (see Methods). These maps provided the locations and energy barriers of the complete network of O₂ pathways inside every simulated globin.

TABLE 1.

List of pentacoordinated monomeric globins investigated in this study

Globin	Species	PDB code
Myoglobins
Sperm whale Mb	Physeter catodon	1A6M, 1A6N (60)
Sperm whale Mb (YQR mutant)	Physeter catodon	1MYZ (24)
Horse heart Mb	Equus caballus	1WLA (61)
Sea hare Mb	Aplysia limacina	1MBA (62)
Invertebrate hemoglobins
Pig roundworm Hb domain I	Ascaris suum	1ASH (63)
Trematode Hb	Paramphistomum epiclitum	1H97 (64)
Marine bloodworm Hb component III	Glycera dibranchiata	1JF3 (65)
Midge HbIII	Chironomus thummi thummi	1ECO (66)
Clam HbI	Lucina pectinata	1FLP (67)
Leghemoglobins
Yellow lupin Lb II	Lupinus luteus	1GDJ (68)
Soybean Lb A	Glycine max	1BIN (69)

For the case of Mbs, we computed the O₂ PMFs for sperm whale, horse heart, and sea hare Mb. In the case of sperm whale Mb, we also looked at two additional variants: deoxy-Mb, in which the distal pocket (DP) contains a water molecule, and the sperm whale (L29Y, H64Q, T67R) “YQR-Mb” mutant, designed to mimic the slower association/dissociation rates observed in the Ascaris nematode Hb (43).

The PMF maps for sperm whale oxy-Mb, deoxy-Mb, and YQR-Mb were considered to test the reproducibility of the implicit ligand sampling approach, as well as to observe the magnitude of the changes caused by the presence of water in the DP and by point mutations. The O₂ PMF maps for sperm whale oxy-Mb and deoxy-Mbs matched particularly well: every favorable O₂ holding region (red, in Fig. 1, a and b) and the O₂ pathway interconnections (blue, in Fig. 1, a and b) exhibit an excellent correspondence between the two maps, both in shape and in size, as they should. The YQR-Mb mutant, also, exhibits strong similarities with the other sperm whale Mb, except for the shape of the DP, as expected, which is where the three “YQR” point mutations are located. The variation in shape of the O₂ pathways near the Xe1 binding site in YQR-Mb is due to the presence of a crystal water molecule at that location which is not present in the other Mbs. Also, a reduction in the size of the pathways far away from the heme (bottom of Mb in Fig. 1 c) appears to be due to statistical variations in the presence of water molecules inside all Mbs near those locations over the course of the simulations. All in all, despite the effect of trapped water molecules inside the proteins, the maps for all three sperm whale Mbs were the most similar to each other of all globin maps, and the details of these maps were remarkably well reproduced between each other (as well as with the independently computed CO PMF map for sperm whale presented in Cohen et al. (3)). These results lend further credibility to the reproducibility of the implicit ligand sampling approach for mapping gas migration pathways.

FIGURE 1.

O₂ PMF maps for various monomeric globins. Shown are the 0 kcal/mol (red) and 1.6 kcal/mol (blue) O₂ free energy contours, along with the four sperm whale Mb xenon binding sites as green spheres. The globins are sperm whale (a) oxyMb, (b) deoxyMb, and (c) YQR mutant Mb, (d) horse, and (e) sea hare Mbs, (f) soy, and (i) lupin Lbs, (g) roundworm, (h) trematode, (j) bloodworm, (k) clam, and (l) midge Hbs. The Xe binding sites of sperm whale Mb are shown as green spheres, and the proteins' α-helices are displayed as black lines.

When comparing Mb O₂ PMF maps across species, we again see a good agreement between sperm whale Mb and horse Mb as we did between the various sperm whale Mbs, reflecting the fact that these globins are all, to a practical extent, almost the same protein. The comparison becomes interesting, however, when one looks at the O₂ PMF for sea hare Mb (Fig. 1 d). Despite the very strong similarities in both function and structure between sperm whale and sea hare Mb, the location of O₂ pathways is, surprisingly, very different for these two Mbs.

The mapped O₂ pathways provide new insights into the behavior of Mbs. A closer examination of the YQR-Mb O₂ PMF map reveals that its DP is much more unfavorable to O₂ than the DP of sperm whale oxy-Mb, namely, by ∼3 kcal/mol. Paradoxically, the shape and energy features of the DP do not resemble those of the Ascaris roundworm Hb, which served as a template for YQR-Mb. According to the O₂ maps, YQR-Mb's low association/dissociation constants are due to a DP which is unfavorable to O₂, resulting in a lower probability of O₂ occupation in the DP and lower chance of binding to the heme, rather than having higher energy barriers to reach the DP through the protein matrix, given that the latter is not observed here.

In particular, we find it interesting that the minima of the three-dimensional energy maps occur at the sperm whale Mb's DP, both in the presence (deoxy-Mb) and absence (oxy-Mb) of a water molecule inside it, implying that water does not prevent O₂ from reaching the DP. The deoxy-Mb DP is, however, measured to be less favorable to O₂ by 3 kcal/mol. Surprisingly, we did not observe any opening of the distal channel (defined as the pathways going through the swinging His-64 “gate”) during any of the 10 ns simulations of sperm whale Mb (which we extended to 25 ns for the case of oxy-Mb) or of horse Mb, resulting in the absence of this pathway in the maps presented in this article. The distal pathway was observed in a previous PMF map of Mb (3); however the initial structure of Mb used in that study contained large crystal deformations (with respect to the other known structures of whale Mb) that may have contributed to this discrepancy in the distal pathway behavior. Other computational studies have, however, reported observing the spontaneous swinging of the distal histidine “gate” in Mb on 10–100 ns timescales (44).

When we extend the comparison to include the O₂ pathways for various monomeric invertebrate Hbs, we note a surprising fact. Fig. 1 illustrates the O₂ pathways for the 12 simulated globins. Aside from a prominent DP, the various monomeric globins exhibit O₂ pathway and cavity locations which are completely different from one Hb to another. These variations are significant and reproducible and cannot at all be attributed to errors in the evaluation of the PMF. Since the globin fold is well conserved among the studied globins and their tertiary structure is near identical (see Fig. 2), our results suggest that the locations of O₂ pathways (which in general are the same as those for CO and NO (3)) are not determined by the protein's secondary and tertiary structures, which are conserved.

FIGURE 2.

Structure of 10 monomeric globins are aligned and superimposed, demonstrating the very strong conservation of their secondary structure globin fold. The structures are sperm whale (blue), horse (green), and sea hare (cyan) Mbs, soy (black) and lupin (white) Lbs, roundworm (yellow), trematode (red), and bloodworm (orange) Hbs, clam (pink), and midge (purple) Hbs. The Xe binding sites of sperm whale Mb are shown as green spheres, and the proteins' α-helices are displayed as black lines.

A second notable observation arising from the O₂ PMF maps for the invertebrate monomeric Hbs is the large number of exits and pathways present in each globin. Our results suggest that multiple exits and an overall porousness to O₂ might be the norm in globins. This is surprising and contrasts with the common assumption of a single O₂ entryway in many kinetic models of O₂ migration in Mb (22,30), as well as with the O₂ PMF maps of the other proteins for which O₂ pathways have been mapped: CpI hydrogenase, which was found to be largely impermeable to O₂ except along two well-defined and very localized pathways (2), and AQP1 aquaporin (7), for which O₂ pathways are only found at the interface of the protein's constituent monomers.

Finally, we studied two Lbs, from lupin and soy, both of which exhibit very similar O₂ PMF maps (see Fig. 3 b). The O₂ PMF maps for both Lbs show them to be mostly inaccessible to O₂, except for a very short and direct exit between the DP and the external solution. The main exit seen in both lupin and soy Lbs (in lupin Lb: Ala-37, Leu-43, His-106, Val-109; in soy Lb: Pro-38, Leu-43, Gln-101, Val-104) is the same as the one reported in lupin Lb by Czerminski and Elber (45) using locally enhanced sampling simulation. Despite the presence of a distal histidine in Lb at the same location as the gating histidine in Mbs, the distal pathway is not present at all in the Lb O₂ PMF maps (the distal pathway was in fact observed here for every Mb, even though it was in some cases seen to be blocked by a closed histidine “gate”).

FIGURE 3.

Comparisons of the 1.6 kcal/mol O₂ PMF surfaces for similar monomeric globins. The globins are (a) sperm whale Mb (blue) versus horse heart Mb (red), and (b) lupin (blue) versus soy Lb (red).

In comparison with the other monomeric globins from Fig. 1, both Lbs are exceptional in having a single, and conserved, dominant exit next to the heme. The only possible other exit, according to the O₂ PMF maps, is an energetically less accessible secondary exit that is still located right next to the primary exit. This is in stark contrast to all the other globins in this study, which are very porous to O₂, offering it multiple exits. Both soybean and lupin Lbs must transport O₂ to symbiotic Rhizobium bacteroids inside root nodules in the plant, while simultaneously ensuring that as little as possible of this O₂ reaches the Rhizobia's nitrogenase enzymes, which are essential to the plant host and which are intolerant to O₂. By having a unique exit, the possibility is raised that this exit could be blocked during transport and/or that Lb could deliver O₂ to a precise target while ensuring minimal O₂ leakage. Our results do not prove such a conclusion; nevertheless, they do show that Lbs have a peculiar O₂ pathway arrangement which is compatible with the possibility of their role in sequestering O₂ in a way that is not realized by any other globin examined in this study.

Specific residues promoting O₂ pathways

From the comparison of the various globin O₂ pathway maps performed in this study, it is evident that O₂ pathways are not defined by a protein's overall fold, which is conserved across all globins; instead, O₂ pathways are found to correlate with the location of certain residue types. Although there is no guarantee that specific residue types have any individual effect on the location of O₂ pathways in the protein, we find that certain residues, on average, are more likely than others to be found near O₂ pathways. By collecting statistical information regarding the propensity of different residue types to form or not form O₂ pathways, one can guide future efforts to manipulate O₂ pathways inside proteins (46–48).

Fig. 4 shows the propensity of residues to form O₂ pathways and cavities normalized by residue abundance (using Eq. 1), sorted by residue type. The analysis was repeated for two different sets of proteins: a set of nine monomeric globins and a set of four copper-containing amine oxidases from yeast (Hansenula polymorpha, Pichia pastoris) (8,49), bacteria (Arthrobacter globiformis), and plant (Pisum sativum) (50). As can be seen, the propensity of given residue types to be near an O₂ pocket is loosely correlated with its hydrophobicity. Large flexible hydrophobic residues and those possessing aromatic rings are most often seen near O₂ pathways, indicating that the large size and mobility of these residues most likely promote the formation of cavities, rather than fill them up as is often assumed.

FIGURE 4.

Relative propensities of residue types to form pathways, computed using Eq. 1, for O₂ pathways (a) inside and (b) on the surface of proteins. The data were collected for a set of nine monomeric globins (excluding whale deoxy-Mb, YQR-Mb, and soy Lb, which are redundant), and four copper amine oxidases. The relative (%) (a) internal and (b) surface residue composition of the combined proteins is displayed as a dotted line (correspondingly, residues which are less abundant are also less well sampled).

It is illuminating that the propensities plotted in Fig. 4 are similar between protein families, which suggests that we are observing a universal behavior. Furthermore, the propensities are not correlated with residue populations in the protein. These results have meaningful relevance to protein engineering applications since they state that to alter the O₂ pathway and cavity networks in proteins, one should focus on the residue side chains near that location, rather than altering the protein's fold.

In the proteins' interiors, the residues which form O₂ pathways are bulky and hydrophobic, whereas residues that are unlikely to form pathways are polar and/or charged (Fig. 4 a). These results are independent of the fact that hydrophobic residues form the core of globular proteins. A priori, it is not obvious whether large mobile residues with nonspecific interactions predominantly create pathways through suboptimal protein packing or whether they fill pathways by occupying volume inside the protein. Based on our results, it appears that the former behavior is the dominant one. However, the correlation between propensities and residue type observed inside the proteins is not readily visible on the proteins' surface, where the solvation energy of O₂ in water dominates the O₂ PMF (Fig. 4 a).

Significance of the O₂ pathway networks

There has been, over the course of decades, a large body of work dedicated to understanding and characterizing O₂ migration kinetics inside a small number of representative globins. Our results clearly show that the shapes of the networks of O₂ pathways inside globins vary greatly from one globin to the next and that whatever conclusion can be experimentally drawn for one specific globin is likely to be only applicable to that specific globin. One might even wonder if the actual location of pathways and cavities inside a globin bear much relevance to its function. As long as the global properties of the O₂ pathways match the desired function of the protein, there may well be no incentive for the protein to conserve or even tune O₂ pathway locations, especially given that most globins appear to possess a large number of O₂ access routes. It is not clear to us that O₂ pathways are critically important. A discussion of the role of O₂ pathways should therefore start with what they are not.

O₂ pathway networks do not affect O₂ binding affinities. A differentiating property of individual globins is their affinity to gas ligands such as O₂. Maintaining fine-tuned affinities to O₂ is crucial to organism function. For example, most Hbs undergo conformational changes to vary their O₂ affinity through cooperative binding: they require high affinities near an O₂ source such as the lungs along with a decreased affinity in low O₂ environments. Similarly, vertebrate Mb as well as secondary Hbs in invertebrates require a relatively high O₂ affinity to uptake O₂ from the primary Hb carrier. Globin O₂ affinity, however, is not a property of the O₂ pathway network; it only depends on the free energy of O₂ binding at the heme, which is almost exclusively influenced by interaction with the residues located in the DP, as evidenced by the high sensitivity of globin-gas affinities to mutations of DP residues (51–53). The pathway taken by O₂ to reach the DP are themselves not expected to have an influence on binding affinities but only on binding rates.

The properties of pathways in the protein matrix, in principle, affect the O₂ on/off rates between heme and exterior. Given a fixed O₂ affinity (which is related to the ratio of on and off rates), altering the energy barriers along, and shapes of, the O₂ pathways could slow or hasten O₂'s migration speeds. Although most globins bind to O₂ for short times (with a 1–100 ms half-life), the Hbs of the Paramphistomum trematode and Ascaris roundworm both display exceptionally long O₂ binding half-lives (21 s and 175 s, respectively) (16). Despite the fact that both the roundworm and trematode Hbs exhibit the two highest free energy barriers for O₂ exit of all the globins studied here, the barriers that we measure are too low to explain by themselves the 4–5 orders of magnitude difference in O₂ binding times for these proteins compared to that of the average globin. Assuming an Arrhenius process, such a difference in binding times would require an energy barrier of at least 9 k_BT, which would have been readily measured by the implicit ligand sampling calculation. Both roundworm and trematode O₂ dissociation rates are well explained by the O₂ affinities of their hemes (and DP), and the details of their O₂ pathway networks have very little effect on these rates. In practice, the effect of the O₂ pathway shapes and locations thus appears to be of minor importance relative to the effect of the bound ligand's environment at the heme, even for extreme cases.

The different pathways in globins may, however, have important roles not yet appreciated or understood. For one, these pathways may have enzymatic functions such as kinetic proofreading as a means to increase the selectivity of Mb binding to various gases, as suggested by Radding and Phillips (54), or as a means to promote the NO to reaction catalyzed by some oxyHbs and oxyMbs (55). Most obviously, such pathways would provide many ways for O₂ to enter and escape globins, and many entrances in the globin surface would also increase the capture (and release) rates of gas molecules by globins. Ever since the hypothesis by Perutz and Mathews (56) that O₂ enters Mb and some Hbs through a conserved swinging histidine gate, this pathway has often been considered the dominant pathway for O₂ to enter these globins. This hypothesis has remained popular because the His gate is and has been the only visible O₂ pathway in static crystal structures of Mbs/Hbs. However, when thermal fluctuations and protein dynamics are accounted for, numerous other possible O₂ pathways are revealed, and it is likely that the His gate is just one pathway among many (3,34,57). In all likelihood, the swinging of the His gate (as opposed to the direct interaction of the gating His with the bound ligand in the DP) is not a critical factor in the regulation of O₂ entry or exit from the protein. We, furthermore, postulate that the conserved His gate appears to gate a water channel, which could permit, for example, the escape of the charged product from the DP. At this point, the roles of such pathways in Mb and other globins, including the histidine gate, are still partially speculative, but in light of new developments in the localization of the O₂ pathways in globins, it is now appropriate to carefully reconsider the assumptions made in the past.

CONCLUSION

Although there has been a large amount of progress in recent years in first identifying major gas-holding packing defects (19,55) and later complete maps of O₂ pathways inside many proteins (2,3,36,44,58,59), a more fundamental understanding of how these pathways occur in general has been lacking. In this study, we characterized the network of O₂ pathways inside a large range of proteins from the globin superfamily. Despite the fact that our results are reproducible and similar for very closely related proteins, we find a complete lack of conservancy of the location, topology, or sizes of the O₂ pathway networks from one individual monomeric globin to the next, despite the similar folds of the proteins. On the one hand, this suggests that the specific details and locations of the O₂ pathways in proteins do not matter much for the protein's function, as long as the pathways are present and provide adequate transport to gas molecules across the protein matrix. On the other hand, this implies that although these pathways are rather independent of the protein's secondary and tertiary structural features, they may actually be dependent on the specific composition of residues inside the protein. This hypothesis was tested, and it was found that the propensity of certain residues to be adjacent or not to O₂ pathways is well reproduced across two protein families: monomeric globins and copper-containing amine oxidases. Such results can be used to plan gas migration pathway-altering mutations inside proteins by substituting residues which have a predisposition to create O₂ favorable regions with those that do not and vice versa.