Migration Rates in [NiFe] - American Chemical Society

Article pubs.acs.org/JPCB

O2 Migration Rates in [NiFe] Hydrogenases. A Joint Approach Combining Free-Energy Calculations and Kinetic Modeling Jérémie Topin, Julien Diharce, Sébastien Fiorucci, Serge Antonczak,* and Jérôme Golebiowski* Institut de Chimie de Nice, UMR 7272, Université de Nice-Sophia Antipolis, CNRS, Parc Valrose, 06108 Nice Cedex 2, France S Supporting Information *

ABSTRACT: Hydrogenases are promising candidates for the catalytic production of green energy by means of biological ways. The major impediment to such a production is rooted in their inhibition under aerobic conditions. In this work, we model dioxygen migration rates in mutants of a hydrogenase of Desulfovibrio f ructusovorans. The approach relies on the calculation of the whole potential of mean force for O2 migration within the wild-type as well as in V74M, V74F, and V74Q mutant channels. The three free-energy barriers along the entire migration pathway are converted into chemical rates through modeling based on Transition State Theory. The use of such a model recovers the trend of O2 migration rates among the series.

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INTRODUCTION Hydrogenase enzymes are extensively studied for their promising catalytic efficiency to both oxidize and reduce molecular hydrogen.1 Their turnover rates are comparable to those of platinum, which is the most efficient chemical catalyst used in fuel cells.2,3 As such, they raise hopes to establish a hydrogen-based economy.4,5 However, a major complication for using hydrogenases as catalysts originates in the diffusion of molecular oxygen into the protein matrix, which inhibits H2 conversion at the active site.6 Several studies, both theoretical and experimental, focused on unravelling the mechanism of inhibition by molecular oxygen in order to design aero-tolerant mutants.7−15 Among the diversity of hydrogenases, those whose active site contains both a nickel and an iron atom appear to be more tolerant for molecular dioxygen. Their active site is deeply buried in the large subunit of the heterodimer. The two metallic atoms (nickel and iron) are connected by two cysteine residues. The nickel atom is coordinated by two more cysteine residues, while the iron atom has one CO and two CN ligands. The small subunit contains three iron−sulfur clusters that play the role of relays for electron transfer through the protein matrix.6,16,17 The hydrogenase of Desulfovibrio fructosovorans is particularly studied due to its relatively high aero tolerance. The inhibition of this enzyme by molecular oxygen is notably governed by its access to the active site.18 Crystallographic structures of the protein (wild-type (WT) and V74M mutant) indeed revealed a hydrophobic channel forming the pathway for gas migration.11,19 Several mutants of [NiFe] hydrogenase from D. fructosovorans have been tested to understand the mechanism of gas diffusion into the protein matrix.9−11,18,20 These mutations have been carried out in order to design a molecular sieve between positions 74 (valine) and 122 (leucine) of the large © 2013 American Chemical Society

subunit. These two residues are considered as a gate regulating the access to the active site. Mutations at position 74 of the large subunit indeed led to a modulation of the rate of inhibition.18 Despite extensive experimental efforts, the exact origin of the observed modulation remains uncovered. The resistance would stem from two factors, the rate of O2 diffusion through the protein matrix and its reaction at the active site.9 Recently, theoretical investigations have suggested that a mutant’s resistance originates from the shrinkage of the channel between positions 74 and 476 of the large subunit.7,8,14,21 Figure 1 shows the trend of the inhibition rate with respect to the molecular volume of residue 74. Counterintuitively, there is no direct correlation between the volume of the residue and the inhibition rate. An accurate prediction of the inhibition by O2 cannot be achieved on the sole basis of a volume analysis. More subtle effects have to be taken into account, such as the impact of dynamics and physicochemical properties of the residues constituting the channel.22 Molecular dynamics emphasized that the size of the channel together with the energetic and dynamic features has to be considered for describing enzymes inhibition.7,8,14,15 A model of gas hoping from one cavity to another allowed reproduction of experimental gas migration rates on the wt (WT enzyme).7 An additional study showed that the gas hoping occurred between networks of accessible pathways to the active site. These pathways all converged to a critical point where residue 74 was strongly involved, emphasizing the crucial role of this position on the phenotype.14 Equivalent studies were also conducted to propose a thorough model for CO diffusion.8 Received: September 20, 2013 Revised: November 29, 2013 Published: December 30, 2013 676

dx.doi.org/10.1021/jp4093964 | J. Phys. Chem. B 2014, 118, 676−681

The Journal of Physical Chemistry B

Article

To simulate the unconstrained migration through the protein matrix, the protocol described by Wang et al.7 was applied to our system as follows: on an equilibrium structure of the enzyme, we started by randomly replacing 600 water molecules from the solvent with 300 O2 molecules. The system was then equilibrated, and a 50 ns long simulation was carried out. Finally, the behavior of O2 was visualized. Kinetic simulations were carried out using KinTek Explorer Software.25 Simulations were run assuming a three-step model associated with cavities hoping within the channel. All parameters were set to default values. The initial concentration of O2 was set to 1 mol·L−1 in the cavity connected to the bulk. The global inhibition rate was obtained by fitting the time evolution of O2 at the active site by a first-order kinetic law.

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RESULTS AND DISCUSSION In a previous study, we observed that the PMF calculated for the wt and its most aero-tolerant mutant (V74Q) were not only affected locally by mutation at position 74. By replacing valine 74 by an arginine, this position becomes less favorable for dioxygen.15 The result of the mutation appears to be a translation of the most stable position of O2 from the vicinity of the active site to a region removed 20 Å from the latter. The use of US simulation allows the quantification of the PMF for O2 migration within the channel. Prior to the freeenergy analysis, the reaction coordinate considered for the US was tested against unconstrained and TLES MDs. The resulting O2 pathways (unconstrained, TLES, and US MDs) within the channel are compared in Figure 2 for the V74M mutant. Other mutants’ behaviors are similar (see ref 15 and the Supporting Information). In the TLES MD, we observe the formation of three main O2 clusters superimposed with the channel seen in the X-ray structure (Figure 2a). The lack of O2 density emphasizes the presence of energy barriers dividing the channel into three parts called C3, C2, and C1 (Figure 2b). All three cavities are sampled by O2 copies during the simulation. More details are provided as Supporting Information. During the unconstrained simulations, the O2 behaviors are consistent with those observed in similar systems.7,14 Several O2 molecules enter the main channel and migrate toward the active site, following a pathway similar to that observed in the X-ray structure. Notice that once an O2 has reached the active site, it is able to reversibly reach the bulk solvent through the same pathway. As seen in Figure 2c, a dioxygen molecule crosses the channel from C3 to C2 (red part of the trajectory) and finally reaches the C1 cavity (white part). It then goes back toward bulk solvent (blue part), transiently visiting a vicinal cavity. This alternative cavity is not connected to bulk, and the O2 molecules have to get back into the main channel to leave the protein matrix. The connection between the bulk solvent and the active site only occurs through a crossing of the main channel, as sampled in the TLES MDs. The C3 → C2 → C1 crossing is the most regularly observed during these unconstrained simulations. This unbiased migration provides a direct evaluation of the reaction coordinate that needs to be sampled to compute an accurate PMF. The behavior of the restrained O2 molecules in the US simulations rigorously reproduces those found in the unconstrained simulations, emphasizing that the choice of our restraint allows sampling the lowest free-energy path. Free-Energy Profiles. The PMF for O2 migrations inferred from the US MDs are shown in Figure 3. In all cases, one

Figure 1. O2 inhibition rate (taken from ref 18) as a function of molecular volume of a residue at position 74 in the mutants and the wt (WT enzyme) of D. fructosovorans hydrogenase.

Focusing on the energy required for migration, we showed that the whole energy pathway along the gas diffusion within the channel is affected by mutations at position 74 due to changes in the position of the global energy minimum. Our results suggested that computing explicitly the whole Potential of Mean Force (PMF) along the migration process is a step forward to predicting the experimental behavior of any enzyme inhibition.15 In this article, we use a combination of molecular dynamics and kinetic modeling to provide a predictive model that ranks mutants according to their aero tolerance. Umbrella Sampling (US) Molecular Dynamics are used to compute the PMFs for high (V74Q, V74M) and weak aero-tolerant (V74F and wt) variants. The barrier heights for the forward and backward diffusion processes are converted into chemical rates for each elementary process during the O2 crossing through the channel. A global diffusion rate is then inferred, which is in line with experimental rankings found in the literature. This work suggests that our protocol is sufficiently robust and predictive for further investigations of gas migrations within enzymes.

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METHODS The systems were built from the pdb 1YQW according to our previous protocol.15 The mutants V74M and V74F were obtained using Swiss-PdbViewer (aka DeepView) software23 by changing the valine from the chain Q in methionine and phenylalanine, respectively. The V74M structure obtained does not show any notable deviation from the X-ray structure (pdb id: 3H3X).19 A mutant of sperm whale myoglobin constructed by mutating a residue in the vicinity of the O2 binding pocket revealed that Leu → Ala, Leu → Val, or Leu → Phe mutations had no influence on the tertiary structure of the enzyme, making us confident in our building protocol.24 Molecular dynamics simulations were run for 15 ns using the protocol described in ref 15. The final structures were used as starting points for the US, unrestrained, and Temperature Locally Enhanced Sampling (TLES) simulations. For the US protocol, an O2 molecule was slowly pulled from the vicinity of the active site toward the entry of the channel. The reaction coordinate was set as in ref 15. It is made up of the distance between an atom showing a weak mobility and located in the axis of the channel (Ala 759 N atom) and the O2 center of mass. 677



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associated with the previously identified bottlenecks for migration within the channel. Thermodynamic and kinetic aspects associated with the energy global minimum and barrier heights are related to experimental behaviors.18 Contrarily to the V74F mutant (that favors accumulation of O2 close to the active site), V74Q and V74M mutations appear more aero-tolerant. Both show PMF profiles where O2 is now stabilized far away from the active site. The difference between these mutations lies in the position of the global free-energy minimum (C2 for V74F and wt versus C3 for V74Q and V74M). All PMF curves report two main energy minima (associated with cavities C3 and C2) separated by an energy barrier. For each system, a set of 110 short unconstrained molecular dynamics simulations was performed to assess the presence of a transition state between these cavities. In these simulations, O2 is initially restrained to a position associated with the transition state, and the system is equilibrated. Each system should, in principle, be on a maximum of the unbiased potential energy surface. They should thus, after removing the restraint, randomly relax into a structure associated with either the C2 or C3 cavity depending on subtle changes in the initial conditions. The O2 molecule (initially located at the putative transition state position) is found within the C2 or C3 cavity with statistics as follows: wt 60.9/39.1%, VQ 59.1/40.9%, VM 43.6/ 56.4%, and VF 11.0/89.0%. The location of the transition state does not provide any information on the thermodynamics of the associated products and reactants. The percentages are strongly dependent on the initial conditions. A subtle shift (±0.1 Å) of the O2 position with respect to those shown here lead to a ratio of 100%/0% in favor of either the C2 or C3 cavity, depending on the direction of the shift. Finally, these results demonstrate that C2 and C3 are connected by a single energy maximum. We confirm that when O2 is positioned within the channel, 20 Å away from the active site, the free energy of the system is at a maximum. This transition state then minimizes its potential energy to reach either the C3 or C2 cavity. Kinetic Modeling. O2 migration within this channel is subjected to crossings of energy barriers and also to thermodynamical equilibrium between cavities. A kinetic model considering these features is established to streamline the behavior of the enzymes with respect to O2 from a macroscopic point of view. The model relies on the following steps, associated with distinct in and out rates:

Figure 2. (a) V74M mutant channel. The residues that constitute the enzyme channel wall are shown as a white surface. The accessible volume is shown in red. (b) Density (isovalue = 1) of O2 molecules during the TLES MD. Three cavities can be identified from the TLES analysis. (c) Typical trajectory of an O2 molecule during the unconstrained MD. (d) US trajectory of an O2 molecule. For (c) and (d), the time evolution is shown from red to blue.

Figure 3. PMF for the different systems studied. Error bars are obtained by a Monte Carlo bootstrapping protocol. The wt PMF appears in light gray in each graph. The VQ and wt PMF are taken from ref 15. The position of each cavity is shown in the upper part of the picture.

Transition State Theory26 provides a way to deduce the rate constants, kTST, of a reaction from the change in Gibbs free energy from the reactants to the transition state kTST(T ) = κ

kBT −ΔG† / kBT e h

where ΔG† is the difference in Gibbs free energy between reactants and transition state, kB the Boltzmann constant, h the Planck constant, and κ the transmission coefficient. Here, we infer rate constant ratios through energy barrier differences

observes a free-energy landscape where migration within the channel is thermodynamically favored with respect to bulk solvent. The energy minima correspond to cavities sampled during TLES MDs (C3, C2, C1). The free-energy barriers are 678



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Table 1. Barrier heights in Both Directions and Corresponding Coefficient Rates for the Three Mutants and the wt of D. fructosovoransa ΔG†C3→C2

ΔG†C2→C3

ΔG†C2→C1

k1

k2

k3

k4

6.9 6.5 5.2 2.3

3.4 2.5 7.0 5.3

4.2 1.0 3.0 2.0

1.5 2.9 25.6 3210

513 2300 1.27 21.6

135 28000 1000 5290

0.0001 0.0001 0.0001 0.0001

V74Q V74M wt V74F a

Energies are given in kcal·mol−1, and rates are in s−1.

assuming that the transmission coefficients are similar, both among the different systems and among the various activation barriers within a PMF curve. In the wt, k3 was arbitrarily set to 1000 to model a rapid inhibition of the active site. The other rates for wt were then deduced by † † ki = e(ΔGj −ΔGi )/ kBT kj

with i and j as the steps associated with other energy barriers. For the three mutants, we computed the migration rate ratios associated with crossing from C3 to C2 using the same equation † † k 3WT = e(ΔGmutant −ΔG WT)/ kBT k 3mutant

For each studied system, k2 and k1 were inferred from the corresponding PMF. We assumed, as already reported,18 that the step corresponding to the active site inhibition is irreversible. Accordingly, k4 was arbitrarily attributed as a very low value with respect to k3 (107 in the wt), so that this step thermodynamically strongly favors the C2 → C1 transition. Whatever the value of k4 with respect to k3 (k3/k4= 107−105), the time evolution remains unaffected because the k3/k4 ratio remains very high. Table 1 gathers the (wt k3-relative) rates obtained for the three mutants and the wt. On the basis of these models, the O2 population within the three cavities with respect to time is modeled. The presence of O2 within C1 is associated with active-site inhibition. The time evolution of O2 presence within each cavity is shown in Figure 4. At the beginning of the simulation, O2 molecules are filling the C3 cavity. The model then shows the migration to the other cavities. The C2 cavity is only transiently occupied, and according to our model of irreversible inhibition that hampers a reverse filling of C2, all O2 molecules end within the C1 cavity. We observe a correlation between C1 filling (bold line in Figure 4) and experimental inhibition.18 V74Q and V74M mutants have a slower inhibition compared to the wt. Also in accordance with experimental rankings, the V74F mutant is much less resistant than the wt due to a faster inhibition. The overall inhibition rate is obtained through a fit of [O2] time evolution in C1 assuming a first-order kinetic law (Table 2). The ranking of inhibition rate is recovered from the model. V74Q and V74M are the most aero-tolerant systems, in opposition to the V74F mutant, which is inhibited much more rapidly than the wt. From a quantitative point of view, the model reproduces the experimental rates by less than 2 orders of magnitude. This corresponds to a maximum difference of 2 kcal·mol−1 in the free-energy evaluation of the whole migration process. Such a

Figure 4. Time evolution of the O2 concentration within the three cavities of D. fructosovorans. Bold line: [O2]C1 (related to inhibition); dotted line: [O2]C2; and dashed line: [O2]C3. For better reading, the time scale for the V74F model has been divided by 200.

Table 2. Theoretical and Experimental Rates of Inhibition for the Four Systems Studied system

kin (s−1)

kin (s−1 mM(O2))exp

V74Q V74M wt V74F

0.3 2.7 17.0 1961.0

4 6.5 32 52

difference is close to chemical accuracy. Note also that a fully quantitative approach would require the estimation of the reaction rate at the Ni−Fe site by means of quantum chemical calculations, which is out of the scope of this study. Model for the Impact of Mutations on O2 Diffusion. The nature of the residue found at position 74 regulates the rate of O2 migration. As shown in the Introduction, no direct and simple explanation (such as the volume of the residue) can provide a rationale to this feature. A linear regression between 679



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mutant exhibits a hydrophilic feature together with a medium volume. Both features, associated with an unfavorable interaction with dioxygen, hinder O2 diffusion to the active site. The methionine and phenylalanine residues present equivalent hydrophobicity and volume. The main difference lies in the specific interaction with the gas molecule. The methionine large volume is primarily responsible for the diminution of the O2 diffusion rate, while Phe overcompensates for this feature by a strong interaction with the gas molecule, resulting in a strong stabilization of O2 near the active site. Note that this reasoning applies to the Trp mutant, whose phenotype is reported in Figure 1. The physicochemical features of the Phe residue (hydrophobicity, aromatic cloud for interaction with O2) are partly reproduced, but the larger volume with respect to the phenylalanine would anyway hamper O2 crossing. This would result in a medium aero tolerance of this mutant.

residue 74 hydrophobicity and the O2 diffusion rate in the related mutant leads to a poor correlation (R2 of 0.28; see the Supporting Information). Here, again it demonstrates that such a feature is not, on its own, regulating migration. By looking at the shape of the PMFs, we notice that the region where O2 is in contact with residue 74 is very different among the mutants. The specific interaction between O2 and the considered amino acid at position 74 can be estimated by this difference in the PMFs shape. Glutamine and methionine lead to an increase of energy, although phenylalanine leads to a decrease of energy with respect to the valine found in the wt system. A favorable interaction has been reported between O2 and the aromatic ring of a Tyr residue in human ferritin.27 The electronic cloud of the Tyr residue tends to favor the presence of the dioxygen by π−π interactions. This interaction between O2 and the nearby residue 74 within the enzyme provides additional clues to catch a glimpse of the modulation of the aero tolerance by several features. The aero tolerance trend, within specific series, has partly been discussed earlier.18 For example, in the evolution between Asn and Gln (both sharing amide functions), the addition of a carbon atom within the side chain increases the mutant aero tolerance. The same decrease in O2 diffusion is observed between Asp and Glu (both sharing an acidic function). This suggests that when equivalent chemical functions are considered, the trend can somehow be extrapolated by the size of the side chain. Things are more subtle with totally different chemical functions. Although Met and Phe share similar volume and hydrophobicity, their aero tolerances are much different. We show here that this differential phenotype is rooted in a compensation of the side-chain volume by the interaction with O2. The PMF shape of the V74F mutant clearly shows a decrease of energy when O2 is close to Phe 74. During the US simulations, the interaction between O2 and the Phe side chain is systematically mediated by π−π interactions. We observe an adaptation of the ring position with respect to O2, optimizing the interaction discussed above (see the Supporting Information). This changes the PMF profile, which shows a global energy minimum at position 74, contrarily to other systems. Finally, we can establish a model providing an explanation to the O2 diffusion rate of the studied systems. The O2 rate of diffusion is regulated by a fine-tuned balanced interplay between the volume, the hydrophobic character, and the specific interactions with the dioxygen molecule. This specific interaction is observed in the shape and the barriers heights of the PMF for each system. These features are gathered in Table 3. In the wt system, the valine residue combines a high hydrophobicity with a medium volume. These two features favor O2 presence close to the active site. This results in a weak aero-tolerant enzyme. The glutamine residue in the V74Q

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CONCLUSION Hydrogenases are a promising molecular tool to produce photosynthetic hydrogen. A major impediment in getting these enzymes working in aerobic conditions is their inhibition by molecular oxygen. The design of potential high-aero-tolerant enzymes relies on a thorough understanding of their inhibition mechanism and on the possibility to predict the behavior of many candidates prior to their expression. In this article, we have reported the use of a series of simulations that allow predicting the ranking of mutants of the hydrogenase of D. fructusovorans with respect to their inhibition by O2. The PMF was computed by means of US MD simulations. The pertinence of the chosen reaction coordinate was assessed by TLES and unconstrained MDs. In a multiscale approach, the features of the PMF profiles were converted into chemical rates to build a kinetic model of gas migration within the enzyme channels. We show that the use of state-of-the-art restrained MDs, joint to kinetic modeling, can be considered as a predictive tool for further investigations on gas migration in enzymes. The inhibition is not only governed by the size of the residues within the channel, as shown with the V74F mutant. It is also subjected to a fine-tuned balance between flexibility and physicochemical properties. Taking into account all these features fully justifies the use of elaborate protocols such as molecular modeling, which captures the whole physics of the systems and proposes a full dynamic picture of the process, here fulfilled by a free-energy quantification.

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S Supporting Information *

O2 pathway in the V74F mutant sampled during Temperature Locally Enhanced Sampling and Umbrella Sampling simulations. Protocol for transition state probing. This material is available free of charge via the Internet at http://pubs.acs.org.

Table 3. Qualitative Effect of Each Side Chain at Position 74 with Respect to O2 Interactiona hydrophobicity volume interaction with O2 tolerance a

Val

Gln

Met

Phe

++ − + low

− − −− high

+ −− − high

+ −− +++ low

ASSOCIATED CONTENT

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: 33 4920 76125. Tel: 33 4920 76120 (J.G.). *E-mail: [email protected]. Fax: 33 4920 76125; Tel: 33 4920 76122 (S.A.).

“−” means nonfavorable for O2 diffusion. “+” means favorable. 680



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Author Contributions

(17) Fritsch, J.; Scheerer, P.; Frielingsdorf, S.; Kroschinsky, S.; Friedrich, B.; Lenz, O.; Spahn, C. M. The Crystal Structure of an Oxygen-Tolerant Hydrogenase Uncovers a Novel Iron−Sulphur Centre. Nature 2011, 479, 249−52. (18) Liebgott, P.-P.; Leroux, F.; Burlat, B.; Dementin, S.; Baffert, C.; Lautier, T.; Fourmond, V.; Ceccaldi, P.; Cavazza, C.; Meynial-Salles, I.; et al. Relating Diffusion Along the Substrate Tunnel and Oxygen Sensitivity in Hydrogenase. Nat. Chem. Biol. 2010, 6, 63−70. (19) Dementin, S.; Leroux, F.; Cournac, L.; de Lacey, A. L.; Volbeda, A.; Leger, C.; Burlat, B.; Martinez, N.; Champ, S.; Martin, L.; et al. Introduction of Methionines in the Gas Channel Makes [NiFe] Hydrogenase Aero-Tolerant. J. Am. Chem. Soc. 2009, 131, 10156−64. (20) Leroux, F.; Liebgott, P.-P.; Cournac, L.; Richaud, P.; Kpebe, A.; Burlat, B.; Guigliarelli, B.; Bertrand, P.; Leger, C.; Rousset, M.; et al. Is Engineering O2-Tolerant Hydrogenases Just a Matter of Reproducing the Active Sites of the Naturally Occurring O2-Resistant Enzymes? Int. J. Hydrogen Energy 2010, 35, 10770−10777. (21) Zhang, Y.; Lu, M.; Cheng, Y.; Li, Z. H-NOX Domains Display Different Tunnel Systems for Ligand Migration. J. Mol. Graphics Modell. 2010, 28, 814−819. (22) Abbruzzetti, S.; Spyrakis, F.; Bidon-Chanal, A.; Luque, F. J.; Viappiani, C. Ligand Migration through Hemeprotein Cavities: Insights from Laser Flash Photolysis and Molecular Dynamics Simulations. Phys. Chem. Chem. Phys. 2013, 15, 10686−10701. (23) Guex, N.; Peitsch, M. C. SWISS-MODEL and the SwissPdbViewer: An Environment for Comparative Protein Modeling. Electrophoresis 1997, 18, 2714−23. (24) Carver, T.; Brantley, R., Jr.; Singleton, E.; Arduini, R.; Quillin, M.; Phillips, G., Jr.; Olson, J. A Novel Site-Directed Mutant of Myoglobin with an Unusually High O 2 Affinity and Low Autooxidation Rate. J. Biol. Chem. 1992, 15, 14443−50. (25) Johnson, K.; Simpson, Z.; Blom, T. Global Kinetic Explorer: A New Computer Program for Dynamic Simulation and Fitting of Kinetic Data. Anal. Biochem. 2009, 387, 20−29. (26) Eyring, H. The Activated Complex and the Absolute Rate of Chemical Reactions. Chem. Rev. 1935, 17, 65−77. (27) Ciacchi, L. C.; Payne, M. C. The Entry Pathway of O2 into Human Ferritin. Chem. Phys. Lett. 2004, 390, 491−495.

S.A. ang J.G. designed the study, J.T. and J.D. performed the MD, US, and kinetic calculations, S.F. helped analyze the manuscript, and J.G. and J.T. wrote the paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. C. Meinert is acknowledged for her critical reading of the manuscript. This work was funded by the French National Agency for Research (ANR, PNRB Hyliox).

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REFERENCES

(1) Vignais, P. M.; Billoud, B. Occurrence, Classification, and Biological Function of Hydrogenases: An Overview. Chem. Rev. 2007, 107, 4206−4272. (2) Jones, A. K.; Sillery, E.; Albracht, S. P.; Armstrong, F. A. Direct Comparison of the Electrocatalytic Oxidation of Hydrogen by an Enzyme and a Platinum Catalyst. Chem. Commun. 2002, 866−867. (3) Kim, S.; Lu, D.; Park, S.; Wang, G. Production of Hydrogenases as Biocatalysts. Int. J. Hydrogen Energy 2012, 37, 15833−15840. (4) Ghirardi, M. L.; King, P. W.; Posewitz, M. C.; Maness, P. C.; Fedorov, A.; Kim, K.; Cohen, J.; Schulten, K.; Seibert, M. Approaches to Developing Biological H 2-Photoproducing Organisms and Processes. Biochem. Soc. Trans. 2005, 33, 70−72. (5) Tye, J. W.; Hall, M. B.; Darensbourg, M. Y. Better Than Platinum? Fuel Cells Energized by Enzymes. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 16911−16912. (6) Fontecilla-Camps, J. C.; Volbeda, A.; Cavazza, C.; Nicolet, Y. Structure/Function Relationships of [NiFe]- and [FeFe]-Hydrogenases. Chem. Rev. 2007, 107, 4273−4303. (7) Wang, P.-h.; Best, R. B.; Blumberger, J. A Microscopic Model for Gas Diffusion Dynamics in a [NiFe]-Hydrogenase. Phys. Chem. Chem. Phys. 2011, 13, 7708−7719. (8) Wang, P. H.; Blumberger, J. Mechanistic Insight into the Blocking of CO Diffusion in [NiFe]-Hydrogenase Mutants through Multiscale Simulation. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 6399−6404. (9) Liebgott, P. P.; Dementin, S.; Léger, C.; Rousset, M. Towards Engineering O2-Tolerance in [Ni−Fe] Hydrogenases. Energy Environ. Sci. 2011, 4, 33−41. (10) Liebgott, P. P.; de Lacey, A. L.; Burlat, B.; Cournac, L.; Richaud, P.; Brugna, M.; Fernandez, V. M.; Guigliarelli, B.; Rousset, M.; Leger, C.; et al. Original Design of an Oxygen-Tolerant [NiFe] Hydrogenase: Major Effect of a Valine-to-Cysteine Mutation near the Active Site. J. Am. Chem. Soc. 2011, 133, 986−997. (11) Leroux, F.; Dementin, S.; Burlat, B.; Cournac, L.; Volbeda, A.; Champ, S.; Martin, L.; Guigliarelli, B.; Bertrand, P.; Fontecilla-Camps, J.; et al. Experimental Approaches to Kinetics of Gas Diffusion in Hydrogenase. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 11188−11193. (12) Armstrong, F. A.; Belsey, N. A.; Cracknell, J. A.; Goldet, G.; Parkin, A.; Reisner, E.; Vincent, K. A.; Wait, A. F. Dynamic Electrochemical Investigations of Hydrogen Oxidation and Production by Enzymes and Implications for Future Technology. Chem. Soc. Rev. 2009, 38, 36−51. (13) Hong, G.; Pachter, R. Inhibition of Biocatalysis in [Fe−Fe] Hydrogenase by Oxygen: Molecular Dynamics and Density Functional Theory Calculations. ACS Chem. Biol. 2012, 7, 1268−1275. (14) Wang, P.-h.; Best, R. B.; Blumberger, J. Multiscale Simulation Reveals Multiple Pathways for H2 and O2 Transport in a [NiFe]Hydrogenase. J. Am. Chem. Soc. 2011, 133, 3548−3556. (15) Topin, J.; Rousset, M.; Antonczak, S.; Golebiowski, J. Kinetics and Thermodynamics of Gas Diffusion in a NiFe Hydrogenase. Proteins: Struct., Funct., Bioinf. 2012, 80, 677−682. (16) Shomura, Y.; Yoon, K. S.; Nishihara, H.; Higuchi, Y. Structural Basis for a [4Fe−3S] Cluster in the Oxygen-Tolerant MembraneBound [NiFe]-Hydrogenase. Nature 2011, 479, 253−256. 681


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