**3. Structure of urate oxidase under inert gas pressure**

#### **3.1 Structure of urate oxidase under pressure of xenon and nitrous oxide and comparison with** *in-vivo* **pharmacology effects**

*Aspergillus flavus* urate oxidase (EC 1.7.3.3) is a homotetrameric enzyme of 301 residues per subunit which is involved in the oxidation of uric acid in presence of molecular oxygen. It crystallizes in the orthorhombic space group I222 with one monomer per asymmetric unit (cell: a = 79.8 Å, b = 96.2 Å, c = 105.4 Å, = = = 90°). X-ray structures of urate oxidase under various pressures of xenon and nitrous oxide have been determined. Both gases were bound mainly in an internal cavity close to the active site of the enzyme, this cavity being empty in the native gas-less structure (Figure 2). This cavity, completely buried within the monomer, is highly hydrophobic, with 86 % of the atoms lining the cavity being carbons. Both gases were bound also very weakly to a second location, a small extension of a solvent-accessible pocket quite hydrophobic (lined by 75 % carbons). The gas occupancy in this second binding site remained very low (less than 30 % at 30 bar of pressure). Gas occupancies in the main binding site were high, reaching saturation at 100 % for xenon and 60 % for nitrous oxide (Table 2). The main effect of the gas was to expand the volume of the cavity where it binds. This expansion increased with gas occupancy and hence with gas pressure.

Protein-Noble Gas Interactions Investigated by Crystallography

Koblin et al., 1998).

noxious stimuli (Colloc'h et al., 2007).

**comparison with** *in-vitro* **activity assays** 

indirect mechanism (Marassio et al., 2011).

to allow a structural fit for the ligand in the active site.

**solubility in lipids** 

on Three Enzymes - Implication on Anesthesia and Neuroprotection Mechanisms 291

nitrous oxide was close to 1, a value that corresponded to the ratio of anesthetic potency of xenon compared to nitrous oxide, as assessed by their MAC-immobility (Russell et al., 1992;

These relationships between gas-induced structural effect and gas-induced narcotic effect allowed proposing a step-by-step mechanism of anesthesia. Gas would first bind to globular cytosolic or extracellular proteins which possess suitable gas binding site easily accessible. Gas-induced disruption of their function would lead to the early stages of anesthesia, i.e. amnesia and hypnosis. When all easily accessible gas binding sites are occupied, gas would then bind to neuronal channel which possess smaller gas binding sites, the disruption of their function would lead to surgical anesthesia, i.e. deep sedation and lack of responses to

**3.2 Structure of urate oxidase under pressure of xenon and nitrous oxide and** 

As mentioned above, the main gas binding site in urate oxidase is very close to the active site (Figure 2). To investigate if gas occupancy and gas-induced volume expansion may have some functional relevance, we performed activity assays on urate oxidase in presence either of air, and either of a mixture of 75 vol % xenon or nitrous oxide and 25 vol % oxygen. To evaluate the alternative therapeutic strategy of using a mixture of xenon and nitrous oxide to combine the efficiency of xenon and the low cost and availability of nitrous oxide, we have also determined the structure of urate oxidase with various pressure of an equimolar mixture of xenon and nitrous oxide, and we have performed an activity assay in presence of a mixture of 37.5 vol% xenon, 37.5 vol% N2O and 25 vol% oxygen. We found that Xe:N2O induced a higher expansion of the cavity volume than pure xenon, which in turn induced a higher expansion than N2O as seen above. *In-vitro* activity assays revealed that Xe:N2Oinduced inhibition was higher than Xe-induced inhibition, itself higher than N2O-induced inhibition. The relationship between structural effect of the gas, i.e. gas-induced volume expansion, and the functional effect of the gas, i.e. gas-induced inhibition of the enzymatic reaction, highlighted the way by which gases might disrupt protein function through an

The role of the void hydrophobic cavity in the catalytic mechanism was thus demonstrated by the activity assays in presence of gas. This functional role was also suggested by the highpressure structural and functional study of urate oxidase. Under high hydrostatic pressure (150 Ma; 1500 bar) the volume of the cavity was reduced as expected, while the volume of the active site was expanded. High pressure also inhibited the catalytic mechanism of urate

In both cases (gas or pressure), there was a loss of flexibility of the cavity, either by the gas presence which induced an expansion and inhibited its contraction, either by high pressure which induced a cavity contraction but inhibited its expansion. The role of the cavity in the functional mechanism of urate oxidase seems then to give some flexibility to the active site

**3.3 Structure of urate oxidase under pressure of krypton and comparison with gas** 

Structures of urate oxidase under krypton pressure of 2 to 30 bar were determined in the present study. Krypton was bound to the exact same location than xenon, in the

oxidase, this loss of activity being a loss of substrate affinity (Girard et al., 2010).

Fig. 2. Hydrophobic cavity in urate oxidase where xenon is bound (shown as an orange sphere). This cavity is close to the active site, where the competitive inhibitor 8-azaxanthine is located (colored in stick by atomic type). The solvent-accessible surface is shown in mesh representation colored by atomic type.

The ratio of gas-induced expansion of the hydrophobic cavity volume for xenon and nitrous oxide in urate oxidase, which could be considered as a model of globular proteins where inert gases bind and whose activity is disrupted by their presence, ranged between 1.1 and 1.5, depending on the applied pressure (Table 2). For the pressures estimated to correspond to physiological conditions (i.e. 5-10 bar), this ratio ranged between 1.3 and 1.5.


Table 2. Gas pressure, xenon and nitrous oxide occupancies in the main binding site, gasinduced expansion of the main gas binding site, ratio of expansion, and xenon and nitrous oxide occupancies in the secondary binding site.

If we compared these data with *in-vivo* pharmacology studies, we noticed that this ratio corresponded to the ratio of the narcotic potency of xenon compared to nitrous oxide (about 1.38) as estimated by the concentration of gas necessary to induce loss of righting reflex in rodents (Koblin et al., 1998; David et al., 2003), considered to be a behavioural endpoint closely related to MAC-awake (Campagna et al., 2003).

In comparison, the ratio of gas-induced volume expansion for xenon and nitrous oxide in annexin V, a protein which could be considered as a prototype of NMDA receptor for its properties of ion selectivity and voltage gating (Demange et al., 1994), did not correspond to the ratio of anesthetic potency of xenon and nitrous oxide. However, when considering urate oxidase and annexin V together as a model of simultaneous occupancy of globular proteins and ion-channel receptors, the ratio of gas-induced expansion for xenon and

Fig. 2. Hydrophobic cavity in urate oxidase where xenon is bound (shown as an orange sphere). This cavity is close to the active site, where the competitive inhibitor 8-azaxanthine is located (colored in stick by atomic type). The solvent-accessible surface is shown in mesh

**Xenon**

to physiological conditions (i.e. 5-10 bar), this ratio ranged between 1.3 and 1.5.

N2O occ. (%) main

The ratio of gas-induced expansion of the hydrophobic cavity volume for xenon and nitrous oxide in urate oxidase, which could be considered as a model of globular proteins where inert gases bind and whose activity is disrupted by their presence, ranged between 1.1 and 1.5, depending on the applied pressure (Table 2). For the pressures estimated to correspond

5 18 10.8 0 8.5 1.3 0 0 10 60 18.8 40 12.4 1.5 10 0 15 100 19.5 50 17.8 1.1 20 0 20 100 23.1 60 18.4 1.3 22 0 30 100 23.2 60 20.1 1.2 27 25

Table 2. Gas pressure, xenon and nitrous oxide occupancies in the main binding site, gasinduced expansion of the main gas binding site, ratio of expansion, and xenon and nitrous

If we compared these data with *in-vivo* pharmacology studies, we noticed that this ratio corresponded to the ratio of the narcotic potency of xenon compared to nitrous oxide (about 1.38) as estimated by the concentration of gas necessary to induce loss of righting reflex in rodents (Koblin et al., 1998; David et al., 2003), considered to be a behavioural endpoint

In comparison, the ratio of gas-induced volume expansion for xenon and nitrous oxide in annexin V, a protein which could be considered as a prototype of NMDA receptor for its properties of ion selectivity and voltage gating (Demange et al., 1994), did not correspond to the ratio of anesthetic potency of xenon and nitrous oxide. However, when considering urate oxidase and annexin V together as a model of simultaneous occupancy of globular proteins and ion-channel receptors, the ratio of gas-induced expansion for xenon and

N2Oinduced expansion (%)

Ratio Xe/N2Oinduced expansion

**8-azaxanthine** 

Xe occ. (%) 2nd N2O occ. (%) 2nd

representation colored by atomic type.

Xe occ. (%) Main

Xeinduced expansion

oxide occupancies in the secondary binding site.

closely related to MAC-awake (Campagna et al., 2003).

Xenon pressure (bar)

nitrous oxide was close to 1, a value that corresponded to the ratio of anesthetic potency of xenon compared to nitrous oxide, as assessed by their MAC-immobility (Russell et al., 1992; Koblin et al., 1998).

These relationships between gas-induced structural effect and gas-induced narcotic effect allowed proposing a step-by-step mechanism of anesthesia. Gas would first bind to globular cytosolic or extracellular proteins which possess suitable gas binding site easily accessible. Gas-induced disruption of their function would lead to the early stages of anesthesia, i.e. amnesia and hypnosis. When all easily accessible gas binding sites are occupied, gas would then bind to neuronal channel which possess smaller gas binding sites, the disruption of their function would lead to surgical anesthesia, i.e. deep sedation and lack of responses to noxious stimuli (Colloc'h et al., 2007).

#### **3.2 Structure of urate oxidase under pressure of xenon and nitrous oxide and comparison with** *in-vitro* **activity assays**

As mentioned above, the main gas binding site in urate oxidase is very close to the active site (Figure 2). To investigate if gas occupancy and gas-induced volume expansion may have some functional relevance, we performed activity assays on urate oxidase in presence either of air, and either of a mixture of 75 vol % xenon or nitrous oxide and 25 vol % oxygen. To evaluate the alternative therapeutic strategy of using a mixture of xenon and nitrous oxide to combine the efficiency of xenon and the low cost and availability of nitrous oxide, we have also determined the structure of urate oxidase with various pressure of an equimolar mixture of xenon and nitrous oxide, and we have performed an activity assay in presence of a mixture of 37.5 vol% xenon, 37.5 vol% N2O and 25 vol% oxygen. We found that Xe:N2O induced a higher expansion of the cavity volume than pure xenon, which in turn induced a higher expansion than N2O as seen above. *In-vitro* activity assays revealed that Xe:N2Oinduced inhibition was higher than Xe-induced inhibition, itself higher than N2O-induced inhibition. The relationship between structural effect of the gas, i.e. gas-induced volume expansion, and the functional effect of the gas, i.e. gas-induced inhibition of the enzymatic reaction, highlighted the way by which gases might disrupt protein function through an indirect mechanism (Marassio et al., 2011).

The role of the void hydrophobic cavity in the catalytic mechanism was thus demonstrated by the activity assays in presence of gas. This functional role was also suggested by the highpressure structural and functional study of urate oxidase. Under high hydrostatic pressure (150 Ma; 1500 bar) the volume of the cavity was reduced as expected, while the volume of the active site was expanded. High pressure also inhibited the catalytic mechanism of urate oxidase, this loss of activity being a loss of substrate affinity (Girard et al., 2010).

In both cases (gas or pressure), there was a loss of flexibility of the cavity, either by the gas presence which induced an expansion and inhibited its contraction, either by high pressure which induced a cavity contraction but inhibited its expansion. The role of the cavity in the functional mechanism of urate oxidase seems then to give some flexibility to the active site to allow a structural fit for the ligand in the active site.

#### **3.3 Structure of urate oxidase under pressure of krypton and comparison with gas solubility in lipids**

Structures of urate oxidase under krypton pressure of 2 to 30 bar were determined in the present study. Krypton was bound to the exact same location than xenon, in the

Protein-Noble Gas Interactions Investigated by Crystallography

25 1.60 20 13.4

35 1.60 20 14.1 40 1.75 20 10.3 45 1.60 25 11.1 55 1.60 30 10.7 65 1.60 40 16.4

Resolution (Å)

Argon pressure (bar)

induced volume expansion.

binding site as a function of pressure.

0 10 20 30 40 50 60 70 **Pressure (bar)**

**Xe** 

**Kr Ar** 

Figure 3B).

**Occupancy (%)**

on Three Enzymes - Implication on Anesthesia and Neuroprotection Mechanisms 293

10 1.65 0 7.1 20.7 2.9 20 1.90 0 11.0 24.6 2.2

30 1.60 20 12.3 24.6 2

Table 4. Argon pressure, resolution of the crystallographic structure, argon occupancy, argon-induced and xenon-induced cavity volume expansion, and ratio of the Xe and Ar-

Fig. 3. A. Gas occupancy as a function of pressure. B. Gas-induced expansion of the main

As for the two other nobles gas, the main effect of argon was to expand the volume of the cavity where it was bound. However, due to its quite low occupancy factor and its small size, the argon-induced expansion remained low, around 10 % of expansion except for the pressure of 65 bar where expansion reached 16 % (Table 4). For pressure of 10 and 20 bar where argon was not detectable in the electron density map, there was already a volume expansion indicating with little doubt the presence of argon within the cavity (Table 4,

**Expansion (%)**

The ratio of Xe- and Ar-induced expansions of the cavity volume was 2.9 for a pressure of 10 bar, which did not correspond to their inverse ratio of MAC-immobility (27/1.61 = 16.8) nor their ratio of solubility in lipids (1.17 / 0.14 = 8.4) (Table 1). However, argon is not narcotic

In order to allow comparison of the effects of argon and xenon in urate oxidase, we further calculated the theoretical expansion of the gas binding cavity produced by argon at 100 % occupancy according to a linear regression model. For occupancy of 100% of argon, the corresponding volume expansion would be of 23.3 % (Figure 4A). According to a linear

at ambient pressure and needs to be pressurised to have some narcotic action.

Ar-induced expansion (%)

Xeinduced expansion (%)

0 10 20 30 40 50 60 70 **Pressure (bar)**

**Xe** 

**Kr**

Ratio Xe / Ar Indiced expansion

**Ar** 

Occ. in main binding site (%)

hydrophobic cavity close to the active site. Krypton occupancy increased with the applied pressure up to 45 % at 30 bar (Table 3). Like xenon, krypton was also weakly bound to a secondary binding site at the bottom of a solvent-accessible pocket, but only at pressure above 20 bar.

For an identical pressure, krypton occupancy was always lower than xenon occupancy (Figure 3A). Xenon which has a higher number of electrons than krypton has a higher polarizability (Table 1) and binds thus with a higher occupancy, as already observed in the case of phage T4 lysozyme (Quillin et al., 2000).


Table 3. Krypton pressure, resolution of the crystallographic structure, krypton occupancy in the main binding site, krypton-induced and xenon-induced volume expansion of the main binding site, ratio of the Xe and Kr-induced volume expansion and krypton occupancy in the secondary binding site.

If one refers to the Meyer-Overton rule, the narcotic potency of a gas would be related to its solubility in lipids. The ratio of solubility in lipids of xenon compared to krypton is 1.17 / 0.14 = 2.7 (Table 1), a value which correspond to the ratio of Xe and Kr-induced volume expansion at the pressure of 5 bar, well within the range of pressure estimated to correspond to physiological condition. This result confirmed what was shown previously when comparing the structural-induced effect of xenon and nitrous oxide on urate oxidase to their in-vivo effect as evaluated by their MAC-awake (Colloc'h et al., 2007). However, the MAC-immobility which prevents response to noxious stimuli for xenon in man is about 4.5 higher than the MAC-immobility of krypton (Table 1), which does not correspond to the structural Xe- and Kr-induced structural effect in urate oxidase, considered as a model for globular protein whose function is disrupted by the presence of gas.

#### **3.4 Structure of urate oxidase under pressure of argon and comparison with** *in-vivo* **pharmacology study**

Structures of urate oxidase under argon pressure of 10 to 65 bar were determined in the present study. Argon was bound to the exact same location than xenon and krypton, in the large hydrophobic cavity close to the active site. Argon became visible in the electron density map at a pressure of 30 bar and above, with an occupancy factor of 40 % at a pressure of 65 bar (Table 4). 65 bar is the maximum pressure which could be reach in the quartz capillary; above that pressure, the risk of breakage of the capillary became very high. At the same pressure, argon occupancy was always lower than krypton and xenon occupancies (Figure 3A). In the secondary binding site where xenon and krypton bind very weakly, no argon is detectable in the electron density map, even at a pressure of 65 bar.

hydrophobic cavity close to the active site. Krypton occupancy increased with the applied pressure up to 45 % at 30 bar (Table 3). Like xenon, krypton was also weakly bound to a secondary binding site at the bottom of a solvent-accessible pocket, but only at pressure

For an identical pressure, krypton occupancy was always lower than xenon occupancy (Figure 3A). Xenon which has a higher number of electrons than krypton has a higher polarizability (Table 1) and binds thus with a higher occupancy, as already observed in the

> Krinduced expansion (%)

2 1.60 10 3.2 3.3 1.0 0 5 1.60 15 4.1 10.8 2.6 0 10 1.55 20 11.4 18.8 1.6 0 20 1.65 40 12.8 23.1 1.8 10 30 1.65 45 15.2 23.2 1.5 15 Table 3. Krypton pressure, resolution of the crystallographic structure, krypton occupancy in the main binding site, krypton-induced and xenon-induced volume expansion of the main binding site, ratio of the Xe and Kr-induced volume expansion and krypton occupancy

If one refers to the Meyer-Overton rule, the narcotic potency of a gas would be related to its solubility in lipids. The ratio of solubility in lipids of xenon compared to krypton is 1.17 / 0.14 = 2.7 (Table 1), a value which correspond to the ratio of Xe and Kr-induced volume expansion at the pressure of 5 bar, well within the range of pressure estimated to correspond to physiological condition. This result confirmed what was shown previously when comparing the structural-induced effect of xenon and nitrous oxide on urate oxidase to their in-vivo effect as evaluated by their MAC-awake (Colloc'h et al., 2007). However, the MAC-immobility which prevents response to noxious stimuli for xenon in man is about 4.5 higher than the MAC-immobility of krypton (Table 1), which does not correspond to the structural Xe- and Kr-induced structural effect in urate oxidase, considered as a model for

**3.4 Structure of urate oxidase under pressure of argon and comparison with** *in-vivo*

Structures of urate oxidase under argon pressure of 10 to 65 bar were determined in the present study. Argon was bound to the exact same location than xenon and krypton, in the large hydrophobic cavity close to the active site. Argon became visible in the electron density map at a pressure of 30 bar and above, with an occupancy factor of 40 % at a pressure of 65 bar (Table 4). 65 bar is the maximum pressure which could be reach in the quartz capillary; above that pressure, the risk of breakage of the capillary became very high. At the same pressure, argon occupancy was always lower than krypton and xenon occupancies (Figure 3A). In the secondary binding site where xenon and krypton bind very weakly, no argon is detectable in the electron density map, even at a pressure of 65 bar.

Xeinduced expansion (%)

Ratio Xe / Kr induced expansion

Occ. in 2nd binding site (%)

above 20 bar.

Krypton pressure (bar)

case of phage T4 lysozyme (Quillin et al., 2000).

Occ. in main binding site (%)

globular protein whose function is disrupted by the presence of gas.

Resolution (Å)

in the secondary binding site.

**pharmacology study** 


Table 4. Argon pressure, resolution of the crystallographic structure, argon occupancy, argon-induced and xenon-induced cavity volume expansion, and ratio of the Xe and Arinduced volume expansion.

Fig. 3. A. Gas occupancy as a function of pressure. B. Gas-induced expansion of the main binding site as a function of pressure.

As for the two other nobles gas, the main effect of argon was to expand the volume of the cavity where it was bound. However, due to its quite low occupancy factor and its small size, the argon-induced expansion remained low, around 10 % of expansion except for the pressure of 65 bar where expansion reached 16 % (Table 4). For pressure of 10 and 20 bar where argon was not detectable in the electron density map, there was already a volume expansion indicating with little doubt the presence of argon within the cavity (Table 4, Figure 3B).

The ratio of Xe- and Ar-induced expansions of the cavity volume was 2.9 for a pressure of 10 bar, which did not correspond to their inverse ratio of MAC-immobility (27/1.61 = 16.8) nor their ratio of solubility in lipids (1.17 / 0.14 = 8.4) (Table 1). However, argon is not narcotic at ambient pressure and needs to be pressurised to have some narcotic action.

In order to allow comparison of the effects of argon and xenon in urate oxidase, we further calculated the theoretical expansion of the gas binding cavity produced by argon at 100 % occupancy according to a linear regression model. For occupancy of 100% of argon, the corresponding volume expansion would be of 23.3 % (Figure 4A). According to a linear

Protein-Noble Gas Interactions Investigated by Crystallography

**4. Structure of elastase under inert gas pressure** 

which is specific for recognition of the peptidic substrate.

hydrophobic, lined with 60% carbons.

**A B**

cyan in stick representation).

**4.1 Introduction on elastase structures under inert gas pressure** 

pressure of 10 bar (Table 4).

on Three Enzymes - Implication on Anesthesia and Neuroprotection Mechanisms 295

expansion in the range 5-10 bar (Table 3). Argon-induced inhibition should be very low or inexistent, according to the very low gas occupancy and argon-induced expansion at a

Pancreatic porcine elastase (EC 3.4.21.36) is a serine protease of 266 residues which hydrolyzes peptide bonds in proteins, its main substrate being elastine. The catalytic triad of elastase is composed, as for all serine proteases, of an activated serine (Ser 195) assisted by a proton relay (His 57), which acts as a general base, and stabilized through an hydrogen bond by an aspartic acid (Asp 102). Pancreatic porcine elastase crystallizes in the orthorhombic space group P212121 with one monomeric enzyme per asymmetric unit (cell : a = 51.4 Å, b = 58.0 Å, c = 75.3 Å, = = = 90°). In the crystallographic structure, a sulphate or an acetate ion is bound in the oxyanion hole, depending on the concentration of the precipitating agents. The primary specificity pocket S1 is a hydrophobic pocket located below the oxyanion hole and the Ser 195

In the crystallographic structures of apo elastase, the S1 pocket was either empty, either filled by a water molecule hydrogen-bonded to a water molecule outside of the S1 pocket and to an oxygen atom of the sulphate ion (Figure 5A). When this water molecule (termed W-S1) was present, its B-factor was quite elevated. In the different crystallographic structures of elestase in complex with xenon deposited in the Protein Data Bank, 1C1M (Schiltz et al., 1995), 1L1G and 1L0Z (Panjikar et al., 2002), 1UO6 and 2A7C (Mueller-Dieckmann et al., 2004) and 2OQU (Kim et al., 2007), xenon was bound within the specificity pocket S1 in the active site of elastase (Figure 5B). This xenon binding site is moderately

Fig. 5. Elastase shown with its solvent-accessible surface colored by atomic type. A. Native gas-less elastase with the water molecule W-S1 in the S1 pocket. B. Elastase in complex with xenon (shown as an orange sphere) or in complex with a peptidic inhibitor TFLA (shown in

**W-S1 Xe**

**TFLA**

In the present study, structures of native elastase (gas-less) were determined in the same conditions than structures under inert gas pressure, i.e. at room temperature in a quartz

regression model, this expansion would be reach for a pressure of 164 bar (Figure 4B). This pressure corresponds to about ten fold the pressure of 14 to 17 bar at which argon is known to produces narcosis in rodents (Abraini et al., 1998; Koblin et al., 1998).

Fig. 4. Linear regression model for cavity volume expansion as a function of occupancy (A) and as a function of pressure (B).

In addition, it should be mentioned that these estimated values for argon were also consistent with crystallographic data that have demonstrated that xenon at full occupancy (pressure of about 20 bar) produces a similar maximal expansion around 23-25 % of the gas binding site (Table 2). This is consistent with the fact that the ratio between the efficient estimated pressure of argon and the efficient experimental pressure of xenon at producing full occupancy and maximal expansion of the gas binding site (164 / 20 = 8.2) is similar to the ratio of their solubility in lipids (1.17 / 0.14 = 8.4) as predicted by the Meyer-Overton rule (Abraini et al., 2003; Campagna et al., 2003).

Argon is narcotic only in hyperbaric condition. At ambient pressure, argon may thus have a very limited influence on its target function. Since one of the major effect of hydrostatic pressure is to contract the volume of internal cavities (Girard et al., 2010), it may explain why argon needs hyperbaric condition to exert its influence.

#### **3.5 Conclusion on urate oxidase structures under inert gas pressure**

The three noble gases were bound to an identical location in urate oxidase, within an internal hydrophobic cavity. The gas occupancies increased in the sequence argon < krypton < xenon, as it was the case for T4 lysozyme (Quillin et al., 2000), who noticed that smaller gases do not bind as well as larger ones as a result of their attenuated polarizability. Xenon and krypton were bound also weakly in a secondary binding site, while argon was not observed even at high pressure.

The main effect of the gas was to expand the cavity volume where it binds. The ratio of expansion was related to the narcotic potency of the gas, as evaluated by their MAC-awake or their solubility in lipids. The presence of xenon within the cavity induced an inhibition of the catalytic mechanism, with a relationship between gas-induced expansion and gasinduced inhibition, as shown by the comparison between xenon and nitrous oxide structural and functional effects. No activity assays were performed in presence of krypton or argon, but we can predict, based on the present structural results, that krypton should induce an inhibition of the catalytic mechanism of urate oxidase. However, krypton-induced inhibition should be lower than xenon-induced inhibition, according to their relative induced expansion in the range 5-10 bar (Table 3). Argon-induced inhibition should be very low or inexistent, according to the very low gas occupancy and argon-induced expansion at a pressure of 10 bar (Table 4).
