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The quest for a functional substrate access tunnel in FeFe hydrogenase†

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Thomas Lautier,abc Pierre Ezanno,de Carole Baffert,de Vincent Fourmond,de Laurent Cournac,fgh Juan C. Fontecilla-Camps,i Philippe Soucaille,abc Patrick Bertrand,de Isabelle Meynial-Sallesabc and Christophe Leger*de Received 9th March 2010, Accepted 6th April 2010 DOI: 10.1039/c004099c

We investigated di-hydrogen transport between the solvent and the active site of FeFe hydrogenases. Substrate channels supposedly exist and serve various functions in certain redox enzymes which use or produce O2, H2, NO, CO, or N2, but the preferred paths have not always been unambiguously identified, and whether a continuous, permanent channel is an absolute requirement for transporting diatomic molecules is unknown. Here, we review the literature on gas channels in proteins and enzymes and we report on the use of site-directed mutagenesis and various kinetic methods, which proved useful for characterizing substrate access to the active site of NiFe hydrogenase to test the putative ‘‘static’’ H2 channel of FeFe hydrogenases. We designed 8 mutations in attempts to interfere with intramolecular diffusion by remodeling this putative route in Clostridium acetobutylicum FeFe hydrogenase, and we observed that none of them has a strong effect on any of the enzyme’s kinetic properties. We suggest that H2 may diffuse either via transient cavities, or along a conserved water-filled channel. Nitrogenase sets a precedent for the involvement of a hydrophilic channel to conduct hydrophobic molecules.

a Universit e de Toulouse, INSA, UPS, INP, LISBP, 135 Avenue de Rangueil, F-31077 Toulouse, France b INRA, UMR792 Ing enierie des Syst emes Biologiques et des Proc ed es, F-31400 Toulouse, France c CNRS, UMR5504, F-31400 Toulouse, France d CNRS, Laboratoire de Bio energ etique et Ing enierie des Prot eines, UPR 9036, Institut de Microbiologie de la M editerran ee, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20. E-mail: [email protected] e Aix-Marseille Universit e, 3 Place Victor Hugo, 1333 Marseille Cedex 3, France; Web: http://bip. cnrs-mrs.fr/bip06 f CEA, Institut de Biologie Environnementale et Biotechnologie, Laboratoire de Bio energ etique et Biotechnologie des Bact eries et Microalgues, F-13108 Saint-Paul-lez-Durance, France g CNRS UMR Biologie V eg etale et Microbiologie Environnementales, F-13108 Saint-Paul-lezDurance, France h Aix-Marseille Universit e, 3 Place Victor Hugo, 1333 Marseille Cedex 3, France i Laboratoire de Cristallographie et Cristallogen ese des Prot eines, Institut de Biologie Structurale Jean-Pierre Ebel, CEA, CNRS, Universit e Joseph Fourier, 41 Rue Jules Horowitz, F-38027 Grenoble, France

† Electronic supplementary information (ESI) available: Complete sequences alignment and further experimental details. See DOI: 10.1039/c004099c This journal is ª The Royal Society of Chemistry 2010

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1. Introduction Intramolecular mass transport is an essential aspect of biological catalysis. This is because many enzymes are large molecules, whose ‘‘business end’’ is buried in the protein interior, rather than exposed to the solvent. In some cases, the protein that surrounds the active site has well defined cavities (which we will call tunnels or channels herein) whose role is to select the right substrate and/or guide it toward the active site.1,2 There are also multi-functional enzymes in which catalytic intermediates are transferred between active sites without escaping to the solvent:3,4 the  long,5,6 whereas carbamoyl channel that guides indole in tryptophan synthase is 25 A phosphate synthetase (CPS) transfers ammonia and carbamate along two tunnel  7–9 segments that traverse a distance of nearly 100 A. The enzyme tunnels that transport indole or ammonia are rather large (about  in CPS9), which facilitated their identification in crystal structures,5 but the 3.3 A situation is less clear regarding biological intramolecular transport of small, diatomic molecules, like O2 and CO. Small heme proteins, like myoglobins, have been extensively studied in this context, both experimentally and theoretically, because time-resolved methods for characterizing the kinetics of binding of these ligands were already available in the mid 1920s (cf. ref. 10 and references therein), and because the molecular weight of these proteins makes computational investigations affordable. In the most common experiment, a CO molecule is photo-dissociated from the heme and wanders in the globin interior for tens to hundreds of nanoseconds before it either recombines or escapes to the solvent, and UV-vis spectroscopy is used to monitor the kinetics of rebinding. In combination with sitedirected mutagenesis,11,12 these experiments have led to a clear picture of the ligand pathways. The protein does not harbor a permanent tunnel. Instead, it has several cavities, identified as empty spaces or Xe-binding sites observed by X-ray crystallography. The ligand accesses the distal pocket by a histidine gate, and diffuses within the protein by ‘‘hopping’’ from one cavity to another, taking advantage of the conformational fluctuations of the protein side chains. Myoglobin has also been used as a model system for developing numerical methods. Approximations, known as ‘‘locally enhanced sampling’’ (LES)13 or ‘‘implicit ligand sampling’’ (ILS),14 have been introduced to increase the efficiency of molecular dynamics (MD) simulations; they rely on the hypothesis that the fluctuations of the proteins are either weakly coupled to, or fully independent of, the motion of the ligands. These methods usually predict pathways and activation energies, but not rates (see, however, ref. 15). Only very recently did brute-force MD simulations make it possible to estimate the macroscopic rate constants for both bi-molecular recombination and exit-tosolvent, from a statistics over many individual CO migration trajectories.16 The agreement with reported experimental values was fair, but Elber recently emphasized that no simulation so far could predict the dominant escape pathway though the histidine gate of myoglobin.15 The source of this disagreement between simulations and experiments is unknown. The above methods have been used to study the ins and outs of various enzymes which use or produce O2, CO, NO, N2 or H2. Well-defined diffusion pathways may provide advantages in terms of catalytic efficiency, protection against harmful products or inhibitors, or selectivity. In acetyl-CoA synthase/CO dehydrogenase (ACSCODH), CO is produced from CO2 at the so-called C cluster and transferred to the A cluster, where it is used for acetyl-CoA synthesis, without being released to the solvent;17,18 CO is not toxic for several microorganisms that use ACS-CODH, and the function of the tunnel identified by X-ray crystallography19,20 may only be to ensure that no CO molecule goes to waste.21 In NiFe hydrogenase, there is experimental evidence that the shape of the tunnel, near the active site, partly restricts access of the inhibitor O2,22–25 and an active area of research concerns the putative O2 egress tunnel in Photosystem II, which is supposed to prevent singlet oxygen production at the P680+Pheo pair by directing away the O2 produced.26 In soybean Faraday Discuss.

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lipoxygenase-1, a tunnel directs O2 towards a certain carbon of the substrate (an Ile to Phe substitution in the channel affects this selectivity27–29), just like in certain flavoproteins it appears to determine how O2 reacts with the active site.30,31 Putative tunnels are most easily identified as hydrophobic cavities in (static) X-ray structures, either by visual inspection or by using software packages (such as CAVER,32 MOLAXIS33 or CASTP34) which perform automatic searches. Xenon is often used as a probe in crystallographic studies, because it is supposed to prefer hydrophobic environments, like O2; it is of a similar size to O2 but it is more electron-rich, thereby facilitating its detection with X-rays.35 Indeed, crystals of NiFe hydrogenase and ACS-CODH flash-cooled after exposure to pressurized xenon house a number of Xe atoms which lie along the predicted channels;20,21,36 this confirms the accessibility of these cavities from the solvent, and suggests that sites that stabilize and sequester Xe atoms in the crystal also facilitate the transit of other small hydrophobic molecules at room temperature. However, the ideal situation encountered with NiFe hydrogenase and ACS-CODH is rare, and Xe-binding experiments sometimes gave puzzling results. For example, in Photosystem II, the numerous Xe binding sites do not match the putative O2 channels,37,38 and the Xe sites are not conserved in four structures of copper-containing amine oxidases.39,40 MD simulations have been employed to probe ligand transport within enzymes (see ref. 41 for a recent review focusing on O2-biocatalysis). The permanent channel observed in the structure of NiFe hydrogenase,36,42 which binds xenon, is indeed used ‘‘in silico’’ by H2.36,43 Its functional character was recently demonstrated using site-directed mutagenesis and various kinetic measurements.24,44 Regarding hemecopper oxidases, the results of MD simulations,45 Xe-binding46 and mutagenesis studies47 also agree with the existence of a conserved,48 permanent O2 tunnel. In other cases, the results of MD simulations supported the idea that small ligands may use transient pathways, rather than permanent channels. For example, from the results of LES calculations and the experimental finding that most of the mutations intended to fill a Xe cavity near the active site of copper amine oxidase do not affect the bimolecular rate of reaction with O2, it has been inferred that O2 uses multiple dynamic pathways.40 In the case of FeFe hydrogenases, a conserved permanent tunnel was identified by analysing the structure of the enzyme from Desulfovibrio desulfuricans,49 and Cohen and coworkers suggested that H2 may also transit via a distinct path, predicted from the protein’s dynamics, which may actually be the dominant route for O2.50,51 The wetlab approach for testing putative diffusion pathways, which we favor, uses site-directed mutagenesis to try to alter the main routes. Most commonly, this consists in increasing the bulk of the side chains that point in the channels. In bifunctional enzymes, mutations in the channel may uncouple the two active sites,7,52–55 but information about the kinetics of channeling is rare.56 When the tunnel connects the active site to the solvent, determining how the mutations affect the rates of transport proved challenging. The kinetics of oxygen binding to cytochrome c oxidase mutants has been studied using the flash flow method, whereby the reduced enzyme inhibited by CO is mixed with O2 in the dark and the reaction is initiated by photo-dissociating the bound ligand.47,57 Time-resolved FTIR has also been used in this respect.58 We recently proposed two quantitative methods for probing the rate of intramolecular diffusion in hydrogenases.24,44 One uses protein film voltammetry (PFV)59–61 to resolve the kinetics of binding and release of the competitive inhibitor CO;62,63 the other is based on interpreting the yield in the isotope exchange assay. Indeed, HD is an intermediate along the reaction pathway from D2 to H2, and because the egress of HD competes with its transformation into H2, the slower intramolecular transport, the less HD dissociates from the enzyme and the less it can be detected in the solvent. Modeling the change in HD concentration against time returns the ratio of rate of HD dissociation over H+/D+ exchange at the active site.44 We also demonstrated a quantitative relation between the rate of diffusion within NiFe hydrogenase and the Michaelis constant for H2.24 This result This journal is ª The Royal Society of Chemistry 2010

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has broad relevance because a variation of Km or kcat/Km is often the main—and sometimes the only—indication that intramolecular transport is impaired in channel mutants. We used an expression of Km in terms of the maximal turnover rate at infinite concentration of H2 (kcat), the second order rate of substrate transport to the active site (k1H ), and the 1st order rate of H2 release (k1H ): 2

2

Km ¼ (kcat + k1H )/k1H 2

2

(1)

This equation shows that a change in Km reveals the reciprocal variation of k1H provided intramolecular transport is slow (k1H  kcat) and on condition that the mutation does not affect kcat. If k1H [ kcat, a mutation that slows diffusion in both directions (to and from the active site) has no effect on Km. The rate constant k1H cannot be measured directly, but in a series of NiFe hydrogenase mutants whose channel is obstructed, we observed that Km was proportional to the reciprocal of the rate of inhibition by CO, showing that the rates of diffusion of H2 and CO are proportional to each other (we found that the ratio of the two approximately equates 30).24 We also showed that O2 and CO diffuse within NiFe hydrogenase at about the same rate, consistent with earlier experiments carried out with myoglobin.64 An increase in Km or a decrease in kcat/Km has been observed in channel mutants of NiFe hydrogenase,24,44,65 nitrogenase,66 and, regarding enzymes that use O2, cytochrome c oxidase,57,67 lipoxygenase,68 copper amine oxidase40 and type-1 cholesterol oxidase.69 Site-directed mutagenesis studies of enzyme channels have given information about structure/function relationships, but the properties of channel mutants cannot always be anticipated: as expected, the width of the hydrophobic channel is an important parameter,70 but hydrophilicity is also very influential, and unexpected structural rearrangements may occur;8 in this respect, it is remarkable that very few structures of channel mutants have been determined by X-ray crystallography.8,44,69,71–73 Regarding nitrogenase, the larger the substrate, the more significant is the increase in Km caused by mutations in the channel (e.g. acetylene > dinitrogen), although the polarity of the substrate also appears to be significant (the greatest effects are observed with azide).66 In NiFe hydrogenase, the sides chains of L122 and V74 define a bottleneck in the channel, near the active site. We initially found no relation between rates of diffusion and the estimated diameters of the channels of NiFe hydrogenase variants (L122F-V74I, L122M-V74M, V74M, PDB entries 3CUR, 3CUS44 and 3H3X74); this may indicate that the limited resolution of the structures makes geometrical characterization inaccurate, or that fluctuations of the side chains of the position-122 and 74 amino acids are significant.44 In contrast, we recently established that increasing the length of the position-74 residue side chain by one CH2 slows the diffusion rate about 30-fold irrespective of the nature of the position-74 amino acid (V74D to V74E, or V74N to V74Q).24 The polarity of this amino acid also matters: replacing a carboxylic acid with an amide, keeping the van der Waals volume constant (V74E to V74Q, or V74D to V74N), slows diffusion about ten-fold.24 This may reveal the stabilization of a water molecule that is part of the barrier to ligand entry, as observed for the V68T mutant of myoglobin.75,76 Such mutation-induced repositioning of water molecules is difficult to predict, and can strongly impact function. For example, certain mutations in the channel of catalase affect the presence of water molecules that are remote from the mutation site, which inactivates the mutants.72,73 On the basis of MD simulations, it has been proposed that a water molecule (rather than a bulky side chain) blocks the channel of the L367F mutant of lipoxygenase.68 The opposite occurs in dehalogenase: MD simulations suggest that a certain polyaromatic replacement (I135F/C176Y/V245F/L246I/Y273F) increases kcat by shielding the active site from bulk solvent, despite the fact that product release is slowed to the point of becoming rate limiting.2 We embarked on a search of the substrate channel in Clostridium acetobutylicum FeFe hydrogenase, by using site-directed mutagenesis and the methods that proved 2

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useful for characterizing H2, O2 and CO transport in NiFe hydrogenase.24,44 We designed 8 mutations that were expected to significantly modify the rate of diffusion along a channel found in the X-ray structure. We purified the mutants and determined their properties by using isotope-exchange assays, by measuring in vitro specific activity for H2 uptake coupled to methyl viologen reduction, by measuring the Michaelis constant relative to H2 and the rates of binding and release of the inhibitors CO and O2. Protein film voltammetry (PFV), which consists in measuring the turnover rate by having the enzyme directly exchanging electrons with the electrode onto which it is adsorbed, proves very useful for precisely characterizing the kinetics of inhibition. We observed that none of the mutations has a strong effect. We discuss the possibility that alternative pathways exist.

2. Results 2.1 The putative channels of FeFe hydrogenase The enzymes that catalyse the oxidation and production of H2 come in two main flavors,77 with an active site consisting of either a dinuclear NiFe cluster or a dinuclear FeFe subsite covalently bound to a 4Fe4S cluster (the so-called ‘‘H cluster’’).78 FeFe hydrogenases are found in anaerobic bacteria, archae and green algae. The algal FeFe hydrogenase from Chlamydomonas reinhardtii (Cr) houses the H cluster and no other cofactor.79 In the enzyme from D. desulfuricans (Dd, PDB code 1HFE49) and Clostridium pasteurianum (Cp, 1FEH80 and 3C8Y81), electrons are transferred to or from the redox partner by 2 or 4 FeS clusters, respectively. Fig. S1† shows a complete alignment of the amino acid sequences of these enzymes, together with those of the catalytic subunits of the complex NADP-dependent hydrogenases of Thermotoga maritima (Tm)82 and Desulfovibrio fructosovorans (Df).83 Fig. 1A shows the overall arrangement of the cofactors in Cp FeFe hydrogenase, which is very similar to the enzymes from C. acetobutylicum (Ca) we study (the percentage of identity equates 71%).

Fig. 1 Side and top views of the structure of C. pasteurianum FeFe hydrogenase (PDB 3C8Y),81 showing the cofactors and the putative channels. The ‘‘static’’ hydrophobic channel, and the water-filled channel are shown as calculated by the program CAVER.32 We indicate the position of the ‘‘dynamic’’ channel by showing as sticks the amino acids that define the corresponding path.50 Red balls are water molecules stabilized in the structure of Cp FeFe hydrogenase; the blue ball indicates the location of the Xe binding site in the homologous enzyme from Dd.77,84 This journal is ª The Royal Society of Chemistry 2010

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Fig. 2 Side view of channel A in the enzyme from Cp (PDB 3C8Y),81 showing the location of the 7 amino acids we targeted. We also indicate the nature of the homologous amino acids in Ca and/or Dd. Red balls are water molecules stabilized in the structure of Cp; those marked with a star are conserved in the structure of Dd FeFe hydrogenase.49 The blue ball indicates the location of the Xe binding site in the enzyme from Dd.77,84

Fig. 1 depicts three putative channels in Cp FeFe hydrogenase. The grids are the surfaces of tunnels calculated by exploring the ‘‘dry’’ (internal water removed) enzyme using the program CAVER.32 Fig. 1B is rotated about 90 . We call ‘‘A’’ the elongated, ‘‘static’’ cavity that was first found by some of us in the structure of Dd hydrogenase.49 It has the same overall shape and position in the enzymes from Cp. In Dd, it stabilizes a single Xe atom,77,84 which appears to be blocked by the site chains of A306, F372 and F160 (A427, F493 and L283 in Cp). Its location is marked by a blue ball on the structure of Cp hydrogenase in Fig. 1B and 2. Channel ‘‘B’’, depicted as a cloud of sticks in Fig. 1, is the ‘‘dynamic’’ path that has been identified from the MD simulations of Cohen and coworkers.50 The two channels meet in a central cavity called ‘‘C’’, slightly closer to the active site than the Xe binding site in the Dd structure. Channel ‘‘W’’ is hydrophilic and stabilizes ten water molecules in both structures of Cp hydrogenase80,81 (red balls in Fig. 1 and 2). Water molecules are also present at the protein surface, near the entrances of channels A and B. The three channels (A, B and W) and the central cavity C extend across the portion of the protein that is common to all FeFe hydrogenases. Tables 1 to 3 show alignments of the amino acids that define cavity C and channels A and B according to the study of Cp FeFe hydrogenase in ref. 50.‡ Table 4 lists the amino acids around channel W.

‡ We list hereafter, using Cp numbering, the amino acids that are close to the pathway A + C calculated with CAVER and shown in Fig. 1 and 2 (they are not exactly those listed by Cohen et al.,50 although some of them, marked with a star hereafter, belong to the list of Cohen et al.): T275, I276, E279, A280*, L283*, V284*, I287*, C299, P324, F417, V423, A427*, L428*, A431*, A435, I461*, N462, N464, F493*. Amino acids F293, M295, M424, V459, Y466, N467, V468, H492, identified by Cohen et al. as part of path A, are remote from the channel identified by CAVER. Faraday Discuss.

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View Online Table 1 Partial alignment of the amino acids that line the central cavity of FeFe hydrogenases. A complete alignment is shown in Fig. S1.† Boldface indicates the amino acids we substituted. Ca ¼ Clostridium acetobutylicum, Cp ¼ Clostridium pasteurianum, Dd ¼ Desulfovibrio desulfuricans, Cr ¼ Chlamydomonas reinhardtii HydA1, Tm ¼ Thermotoga maritima, Df ¼ Desulfovibrio fructosovorans

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Cp Ca Dd Cr Df Tm

A272a A A A A A

T275 T T T T V

I276a I I I I A

E279 E E E E E

T297 T T T T T

C299 C298 C C C C

P324 P323 P P P P

F417 F416 F F F F

V423a V422 V V V V

a

Some of the amino acids whose substitution is proposed in the patent application of King et al.85

Table 2 Partial alignment of the amino acids that line pathway A. Boldface indicates the amino acids we substituted. The superscript ‘‘X’’ marks the xenon binding site Cp Ca Dd Cr Df Tm

A280a A G G G A

L283Xa L282 F L F F

V284c L283 V L L Y

I287ac V286 L L L L

F293c F L L L L

M295 M Q M I Q

M424 M M M I F

A427Xa A426 A A A V

Cp Ca Dd Cr Df Tm

A431a A430 A A A —

V459c V V I V V

I461 I460 V M V L

Y466c L V L L F

N467c N K R V K

V468 V V V I G

H492bc H H D H E

F493Xa F492 F F A I

L428c I L L L L

a Some of the amino acids whose substitution is proposed in the patent application of King et al.85 b An amino acid selected in the study of Tosatto et al.86 c Amino acids which are surface exposed.

Table 3 Partial alignment of the amino acids that line pathway B, which Cohen and Schulten identified from MD simulations using the structure of Cp FeFe hydrogenase.50 Cp Ca Dd Cr Df Tm

M274 M V L L L

E278 E E E E E

A321 A C C A V

I327 I M M M A

T330c T A A A T

A331 A L M I V

Cp Ca Dd Cr Df Tm

Y555c Y Y Y F D

F556c F L L L L

R563bc L K K R T

A564 A S A S H

I567c L L L L Y

L568 L L L L R

T334c T T D T K

M551 M L L L L

Y552 Y Y Y Y Y

b

An amino acid selected in the study of Tosatto et al.86 c Amino acids which are surface exposed.

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Table 4 The ‘‘wet’’ pathway (W)ab Cp Ca Dd Cr Df Tm

S298(4) S S S S S

V304(0) V Q I V V

L315(1)c L L I L L

S319(1) S S S S S

Cp Ca Dd Cr Df Tm

E361(2)h E E E E E

F570(2)hc V T T T T

K571(4)c K H H H T

Y572(3)c Y W Y Y Y

S320 S T S T S

S323(2)h S S S S S

a

The superscript numbers indicate the number of contacts with water molecules in both structures of Cp hydrogenase.80,81 b The superscript ‘‘h’’ marks amino acids that are part of a putative proton-transfer pathway49 that also includes K358 and Q366 (Cp numbering, equivalent to K237 and E245 in Dd). c Amino acids which are surface exposed.

Table 1 shows that the amino acids which line the central cavity are identical in the 6 enzymes, except for the T-to-V and I-to-A substitutions in the sequence of the Tm enzyme. In contrast, the cavities A and B are not fully conserved (cf. Tables 2 and 3), and the buried amino acids do not seem to be more conserved than those that are surface exposed. The amino acids that define the position of the wet channel are very conserved, which suggested that some of them (marked by a superscript h in Table 4) are involved in a proton transport pathway.49 The three water molecules close to the H cluster (marked with a star in Fig. 2) are conserved in the structures of Cp and Dd FeFe hydrogenase; however, CAVER does not find the wet channel in the latter. 2.2 Design of the mutants, purification and solution assays The boldfaced amino acids in Tables 1 and 2 are those we targeted. They line the ‘‘static’’ pathway A and the central cavity C, as shown in Fig. 2. We designed the following mutants (sorted in the order of decreasing distance from the H cluster): I460F and A430I in attempts to narrow the channel at the surface of the enzyme, F492A and A426L to alter the Xe-binding site in the middle of the putative channel, V422W and F416W to obstruct the channel near the active site, and C298A, C298L to respectively enlarge and narrow the entrance of the central hydrophobic cavity (we use Ca numbering hereafter, unless otherwise stated). Site-directed mutagenesis was performed on hydA cloned into the pPHhydA1-LLCstrep-tag vector earlier described by Girbal et al.87 and improved based on the study of Von Abendroth et al.88 to generate the eight HydA mutants for expression in Clostridium acetobutylicum. This expression system allows production and purification of large amounts of highly active FeFe hydrogenase under strict anaerobic conditions. An average amount of 0.8 mg of purified active protein was obtained for both native and mutant enzymes.89 The purity factor, based on the H2 uptake specific activity, is around 60 for most purifications. Since the specific activity of the enzyme decreases over time, the production, purification, H2 uptake measurements and PFV experiments were done promptly, and the enzyme samples were handled and stored at 4  C and never frozen (we observed that the specific activity decreases by a factor of two in each freeze/thaw cycle). The in vitro H2 uptake specific activities were measured in the presence of oxidized methyl viologen. Certain mutations (V422W and F416W) decrease the activity more than 20-fold. All amino acid substitutions affect the activity except those of I460 and A430, which are remote from the active site. The turnover rates are collected in Table 5, with all the other kinetic parameters we determined. Faraday Discuss.

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2.3 Isotope-exchange assays We previously introduced the isotope exchange assay as a mean of probing intramolecular transport in hydrogenases.44 In this non-redox reaction, the enzyme transforms heavy di-hydrogen (D2) into H2 according to Scheme 1. The mechanism is the following: (i) D2 diffuses from the solvent to the active site, where it is heterolytically cleaved; (ii) H+ from the solvent substitutes for D+; and (iii) the D+ of the resulting HD species is eventually replaced with H+, generating H2. The formation of H2 from HD can occur either right away or after HD has been transiently released into the solvent where it can be detected by mass spectrometry. Since HD production depends on the competition between HD-release and H+/D+ exchange at the active site, the ratio of initial rates of production of HD and H2 (v0HD and v0H2, respectively) informs on the rate of intramolecular diffusion. Indeed, it is simply:x v0HD kout ¼2 k v0H2

(2)

where kout is the 1st order rate constant of HD diffusion to the solvent, and k is the rate constant of H+/D+ exchange (Scheme 1). Alternatively the ratio kout/k can be measured from the rates of decrease of [D2] and of the ‘‘itotopic content’’ ([D2] + [HD]/2) and eqn (3) in ref. 44 (method 3 in the supplementary information therein); we used this method because we found that the result is less dependent on the initial values of the concentrations of H2 and HD when the assay is started. To assay isotope exchange activity, we inject the enzyme in a solution saturated with D2, and we use mass spectrometry to monitor the changes in H2 and HD concentrations.44 Fig. 3A shows that the WT enzyme produces HD slightly more quickly than H2, whereas the difference is less pronounced for the A426L mutant (Fig. 3B). We collected in Table 5 the values of kout/k for the WT enzyme and the 8 mutants. Only the A426L mutation decreases this parameter; the effect is small (0.61 versus 0.75), but the results were very reproducible. Since A426 is remote from the active site, it is unlikely that its substitution affects the properties of the H cluster, and the observation that kout/k decreases a little suggests that the A426L mutation slightly hinders intramolecular diffusion.

2.4 Michaelis constants for H2 We discussed in the introduction of this paper the relation between the Michaelis constant and diffusion kinetics. There are several methods for measuring the Michaelis constant of hydrogenase. In one method, used by Hagen and coworkers,90 the enzyme oxidizes H2 with oxidized methyl viologen as the redox partner, the decrease in H2 against time is monitored polarographically using a Clark electrode, and the progress of the reaction is simulated by numerically integrating the one-

Scheme 1 Mechanism of isotope exchange, and definition of the rate constants used in the text. The assay is performed in the absence of a redox partner.

x This relation is simply obtained from the kinetic model which we introduced in the supplementary information section of ref. 44, by noting that v0HD ¼ 2c0(kD  kT), v0H2 ¼ c0(2kT  kD), and kT/kD ¼ (1 + k/kout)/(2 + k/kout), using the notations defined therein. This journal is ª The Royal Society of Chemistry 2010

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12 500  4000 0.75 1.1 9.7 1.5 19 4.7 0.45 6.9

14 000 0.73 1.1 7.7 1.3 15 4.6 0.47 8.3

I460FA 6200  100 0.73 0.9 9.9 1.5 21 6.0 0.58 6.1

A430IAg 1800  600 0.61 2.6 15 1.6 22 3.0 0.34 8.3

A426LAg 11 000  1000 0.68 0.9 8.4 1.4 18 4.2 0.43 16

F492AAg 350 0.72 1.0 9.4 1.4 19 4.0 0.46 8.1

V422WCg 450  100 0.74 0.8 7.4 1.4 16 3.7 0.38 8.0

F416WC

2000  700 0.75 1.1 8.7 1.5 17 4.1 0.43 8.2

C298AC

2100  500 0.75 1.0 7.7 1.4 15 3.7 0.34 9.6

C298LC

a Turnover rate in the H2-oxidized methyl viologen assay. pH 7.2, 37  C. b Value of the ratio kout/k obtained by interpreting the results of isotope exchange experiments using eqn (3) in ref. 44. pH 7.2, 30  C. c Michaelis constant for H2, in units of atm. of H2, determined electrochemically by interpreting the results of experiments such as those in fig 4, with eqn (3); pH 7, 30  C. The value of Km in units of atm. of H2 can be converted to a concentration of H2 using the Henry constant of 7.8 104M/atm. d Rate constants defined in Scheme 2, and measured by fitting data such as those in fig. 5, at pH 7, 30  C, 1 atm of H2. e Rate constant of CO binding extrapolated to zero concentration of H2 using eqn (5) and the value of Km. f Rate constants defined in Scheme 3, and measured by fitting data such as those in fig. 6B, at pH 7, 30  C, 1 atm of H2. g Amino acids targeted in the patent of King et al.85

kcata kout/kb Km (atm. H2)c kin,appCO d/s1atm(CO)1 koutCO d/102 s1 kinCO e/s1atm(CO)1 kinO2 f/s1atm(O2)1 koutO2 f/s1 kdead f/103 s1

WT

Table 5 Summary of the kinetic properties of Ca hydrogenase mutants (sorted in the order of decreasing distance from the H cluster) at 30  C, pH 7 (unless otherwise stated). Boldface indicates values of the parameters which we think are significantly affected by the mutations. Superscripts A or C indicate whether the amino acid lines path A or the central cavity

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Fig. 3 Isotope exchange assay of the WT enzyme (A) and the A426L variant (B). Initial production of HD (black lines) and H2 (red lines) after an aliquot of stock solution of enzyme is injected in a solution saturated with D2. pH 7.2, T ¼ 30  C.

substrate Michaelis–Menten equation. This is more convenient than the conventional use of initial rates because the latter are dependent on the activity of the sample, and because it is difficult to measure initial rates in solution assays for discrete values of the hydrogen concentration. The method which we use consists of measuring electrochemically the rate of H2 oxidation with the enzyme adsorbed onto, and exchanging electrons directly with, an electrode, in a solution initially saturated with H2, and varying the concentration of H2 by flushing the solution with argon. The H2 concentration decreases exponentially with time,62 with a time constant s of about 20 to 30 s, and the change in activity (current, i) against time can be modelled by using the Michaelis–Menten equation in which we introduce a time-dependent concentration of H2: iðtÞ ¼

imax Km t 1þ es ½H2 0

(3)

The time t is counted from the moment the cell is flushed with argon, [H2]0 is the initial concentration of H2. Note that if Km is already larger than [H2] before the latter starts to decrease, the change in current merely follows the change in H2 concentration, and decreases exponentially with time according to: iðtÞ ¼

imax ½H2 0  t e s Km

(4)

In this unfavorable situation, the value of Km cannot be evaluated. However, this is not a limitation of the electrochemical method, since any evaluation of a Michaelis constant requires that the rate be measured at a concentration of substrate that is of This journal is ª The Royal Society of Chemistry 2010

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Fig. 4 Measurement of the Michaelis constant relative to H2 of the WT enzyme (A) and the A426L variant (B). The activity is electrochemically measured while the solution, initially saturated with H2, is flushed by a stream of Ar at t > 0. The data are shown as black lines. The green dashed lines are the best fit to eqn (3), which returned Km ¼ 1.1 and 2.5 atm. of H2 (A and B, respectively), and the red dashed lines illustrate the exponential decay that would be obtained if Km were much greater than the initial concentration of H2, extrapolated from the end of the relaxation of the current using eqn (4). E ¼ 160 mV, 30  C, pH 7, electrode rotation rate u ¼ 3 krpm.

the order of Km or greater, and the solubility of H2 (780 mM at 25  C) sets an intrinsic limit to the greatest measurable value of Km. Fig. 4 shows a typical result obtained with WT Ca FeFe hydrogenase adsorbed onto an electrode whose fast rotation minimises mass transport control. The H2saturated solution was flushed with Ar at t > 0. The perfect fit of the data to eqn (3) (dashed green line) proves that the activity follows Michaelis–Menten kinetics. The fact that the activity starts to decrease as soon as the concentration of H2 decreases shows that the Michaelis constant is relatively high, which is typical of FeFe hydrogenases.90 However, the data significantly depart from the exponential decay extrapolated from the end of the relaxation of the current (dashed red line), and the ratio Km/[H2]0 can be determined: we found Km ¼ 1.1 atm. of H2. The accuracy of this measurement is intrinsically low; we estimate that the error on the value of Km is about 30%. The experiment in Fig. 4B was carried out with the A426L mutant. In this case, the data hardly deviate from an exponential decay, showing that the Michaelis constant is larger, of the order of 2.5 atm of H2 (the error on this parameter is large). Table 5 collects the Michaelis constants of the WT enzyme and the 8 mutants we tested. Each value is the average of at least four measurements. Only the Km of the A426L mutant significantly differs from that of the WT enzyme (2.6 versus 1.1 atm of H2). 2.5 Kinetics of inhibition by CO We used the electrochemical method described previously44,62,63 to measure the rates of binding and release of the competitive inhibitor CO. The H2-oxidation activity is measured as a current, with the enzyme adsorbed onto an electrode immersed and Faraday Discuss.

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rotated in a solution continuously flushed with H2, and small aliquots of a solution saturated with CO are repeatedly injected in the cell. The concentration of CO instantly increases after each injection (the mixing time is about 0.1 s) and the resulting dilution of H2 is negligible. The activity decreases after the addition of CO, and it is fully recovered as CO is flushed away by the stream of H2. Fig. 5 shows the result of this experiment carried out with the WT enzyme. Fig. 5A shows the change in current against time (blue line); the arrows above Fig. 5A indicate the injections of CO. The downward trend is caused by protein desorption; this can be corrected by using a spline function to interpolate the data (black line) and dividing the raw signal by this synthetic curve.89 The result is shown in Fig. 5B (plain line). To deduce the rate constants of CO binding and release from the data, we assume that (i) the inhibition and reactivation are first- and zeroth-order in [CO], respectively, and the concentration of CO decreases exponentially with time, with a time constant s (Scheme 2), and (ii) the current is proportional to the instant coverage of active (CO-free) enzyme.

Fig. 5 Kinetics of inhibition by CO of WT Ca FeFe hydrogenase. The blue line in (A) is the catalytic current resulting from H2-oxidation by the enzyme adsorbed at a rotating graphite disc electrode. The injections of CO are marked by arrows at the top of the figure. The black line is interpolated from the data and shows the current that would have been obtained in the absence of CO; the downward trend is mainly caused by protein desorption. Using this black signal for normalization gives the corrected data shown in black in (B). The dashed green line is a fit using the model depicted in Scheme 2. E ¼ 160 mV, pH 7, 40  C, u ¼ 3 krpm. The current is recorded after a 300 s equilibration period at 160 mV.

Scheme 2 Mechanism of inhibition by CO, and definition of the rate constants used in the text. This journal is ª The Royal Society of Chemistry 2010

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We derived in ref. 63 the analytical equation that can be used to fit the electroCO chemical data recorded after a single injection of CO, to determine kCO in,app[CO]0, kout and s. We now find it more convenient to simulate a single data set obtained by repeatedly adding aliquots of CO-saturated solution (Fig. 5). The green dotted line in Fig. 5B is the best fit calculated by adjusting a single set of rate constants kCO in,app and kCO out , knowing the four values of [CO]0. Since CO is a competitive inhibitor of hydrogenases, H2 protects the active site against CO, all the more that the Michaelis constant is small. Therefore, the rate of inactivation in the absence of H2 must be extrapolated using:63   ½H2  CO CO 1þ (5) kin ¼ kin;app Km We repeated this analysis with the 8 variants, and collected the results in Table 5. The apparent rate of CO binding is greater in the A426L mutant than in the WT enzyme, but this is a consequence of the value of Km being large: none of the mutaCO tions significantly affects kCO in or kout . We note that an alternative strategy for characterizing the kinetics of inhibition by CO (or O2) consists in fitting the exponential relaxation of the catalytic current that follows a step in inhibitor concentration.91 This can be achieved by injecting an aliquot of solution saturated with CO and simultaneously changing the composition of the gas phase above the cell solution. In that case, the rate constant of the relaxCO ation is kCO in,app[CO] + kout . 2.6 Kinetics of inhibition by O2 The procedure we use to quantify the aerobic inactivation of the enzyme24,62,97 resembles the method for studying the inhibition by CO, except that the electrode potential has to be high (greater than about 150 mV vs. SHE), or else O2 is directly reduced on the electrode, which contributes to the current and decreases the concentration of inhibitor that is actually experienced by the enzyme.91 The blue trace in Fig. 6A shows the current recorded with the WT enzyme adsorbed onto a rotating electrode poised at 190 mV, when aliquots of a solution saturated with O2 are repeatedly injected in the cell solution saturated with H2. The effect of film loss and anaerobic inactivation92–96 can be removed by dividing the blue trace by the control signal recorded in an independent experiment carried out at the same electrode potential, but under anaerobic conditions (black trace);89 the result of the division is shown in black in Fig. 6B. This corrected trace is a clear readout of the effect of O2. The current decreases after each injection of O2 and then increases as O2 is flushed by the stream of H2, which reveals the reversible binding of the inhibitor, but the activity is not fully recovered as the solution become anaerobic, because the O2 adduct slowly reacts to form a ‘‘dead’’ enzyme, which cannot be reactivated. The model we use to fit the data (Scheme 3) accounts for the reversible binding of O2 (apparent rates kinO and koutO ), followed by the irreversible reactivation of O2 at the active site (rate kdead). The dashed green line in Fig. 6B shows the best fit of the corrected data, from which the three rate constants in Scheme 3 could be determined. Similar experiments were carried out with all mutants, to obtain the values of the rate constants collected in Table 5. The rates of O2 binding and release decreased slightly in the A426L mutant. Remarkably, the first order reaction of O2 that follows the formation of the adduct is twice as fast in the F492A mutant than in the WT enzyme. 2

2

3. Discussion We have used site-directed mutagenesis and various kinetic methods to probe intramolecular diffusion in Ca FeFe hydrogenase mutants. Putative pathways that may Faraday Discuss.

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Fig. 6 Oxidative inactivation of Ca hydrogenase. The black line in (A) is the decrease in current resulting from film desorption and anaerobic inactiviation recorded under anaerobic conditions at E ¼ 190 mV vs. SHE, 30  C, pH 7, u ¼ 3 krpm. The dashed green line is the best fit to a biexponential decay. The blue line is the result of an independent experiment (with a fresh enzyme film), carried out under identical conditions, except that aliquots of solution saturated with O2 are injected at the times marked by arrows at the top of the figure. The black line in (B) was obtained by dividing the latter signal by the control;89 the dashed green line is the fit to the model depicted in Scheme 3. The data are recorded after a 250 s equilibriation period at 160 mV, followed by 50 s at E ¼ 190 mV.

Scheme 3 Mechanism of aerobic inhibition, and definition of the rate constants used in the text.

guide H2 from the solvent to the active site of FeFe hydrogenases have been previously identified by performing cavity searches,49 looking for Xe-binding sites in the crystals84 or using MD simulations.50,51 The resulting picture was that H2 may use either of two main pathways to enter the enzyme (Fig. 1). Pathway A is called ‘‘static’’ because it is detected as a permanent elongated cavity in the crystal. This channel houses the only observed Xe-binding site.84 Pathway B does not show up using programs which search for cavities in the static structure. It consists of cavities that open transiently as a consequence of the fluctuations of the protein. Molecular dynamics simulations suggested that this transient path is the dominant route for O2.50,51 The amino acids that define pathways A and B are mostly hydrophobic. We have also described a conserved ‘‘wet’’ channel connecting the active site to the solvent; it is lined by the hydrophilic amino acids which interact with water molecules in the structures of Cp hydrogenase (red balls in Fig. 2). These three paths end up in a central cavity called C, which leads to the active site di-iron subcluster. This journal is ª The Royal Society of Chemistry 2010

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The search for a substrate channel is crucial in the case of algal FeFe hydrogenases, because these enzymes could be used for the photosynthetic production of H2 if they resisted inhibition by O2.98,99 We97 and others100 have shown that the competitive inhibitor CO protects FeFe hydrogenases against aerobic inactivation, showing that O2 targets the active site. X-ray absorption spectroscopy measurements100 on the enzyme from Cr suggest that destruction of the 4Fe4S portion of the H cluster follows up this initial attack of the 2Fe subsite; this mechanism has not yet been considered in theoretical studies.101,102 Engineering hydrogenase to slow oxygen access to the H cluster may increase the tolerance of the enzyme to oxygen. A patent application actually protects the redesign of FeFe hydrogenases by increasing the bulk of amino acids that line the putative A–C pathway;85 the authors’ claims were supported by the observation that the V240W mutant of Cr FeFe hydrogenase (V422 in Ca) increases the oxygen tolerance of Cr hydrogenase. We note that this observation is in contrast with the proposal that O2 mainly uses path B in Cp hydrogenase.51 We have previously developed various methods for probing the effects of mutations on the kinetics of diffusion in hydrogenases, which we have recently applied to a number of NiFe hydrogenase mutants.24,44 We showed that certain mutations which obstruct the channel of Df NiFe hydrogenase slow CO and O2 binding, diminish the transient production of HD in the isotope exchange assay and increase the Michaelis constant for H2. Most importantly, we established quantitative relations between the corresponding kinetic parameters, and we demonstrated that O2 and CO diffuse within NiFe hydrogenases at the same rate, but the former reacts slowly at the active site. This explained that the rate of inhibition by CO is always greater than the rate of binding of O2, unless diffusion is so slow that it limits both processes, in which case kinO z kinCO. In WT Ca hydrogenase, kinO and kinCO are of the same order of magnitude (Table 1 in ref. 24 and Table 5 herein), and we therefore concluded that diffusion limits the rates of inhibition by CO and O2 in this enzyme. We emphasized the difference between Ca and Dd FeFe hydrogenases in this respect: transport appears to be faster in the latter (hence kinO  kinCO).24 Similar conclusions were reached by Armstrong and coworkers91 from independent experiments aimed at comparing the kinetic properties of the hydrogenases form Ca, Dd and Cr; the authors also reasoned by examining which rate constants depend on the redox state of the active site, which can be varied by tuning the electrode potential. If true, the above conclusion that diffusion limits the rates of inhibition by CO and O2 in Ca FeFe hydrogenase should have made our investigation very easy, because any variation of the rate of diffusion should result in a proportional variation of the rates of inhibition. And if, as occurs in NiFe hydrogenases,24 the mutations affect the rates of diffusion of all diatomic molecules in the same manner,{ we could also expect an effect of the mutations on the Michaelis constant for H2. The isotopeexchange measurement gives a result that depends only on the ratio of rate of HD release to the solvent over rate of H+/D+ exchange at the active site; we expect a variation of this ratio upon modifying the channel if HD diffusion is slower than HD dissociation from the active site, and if the mutations have little effect on active site chemistry. We have applied these methods to 8 mutants of Ca FeFe hydrogenase, designed in attempts to alter diffusion along the putative A + C pathway (fig. 2). Four of the amino acids we targeted are among the five amino acids mentioned in the patent application of King et al.;85 the latter are marked by a superscript a in 2

2

2

{ The mutations have the same relative effect on the rates of diffusion of O2, CO and H2 in NiFe hydrogenase, although the latter diffuses about 30 times more quickly than do CO and O2.24

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Tables 1 and 2k. Table 5 collects the results; we used boldface for the values of the parameters which we think are significantly affected by the mutations. We designed the I460F and A430I mutants in attempts to obstruct the channel at a distance from the H cluster. The I460F mutant has the same phenotype as the WT enzyme, despite the fact that the lateral chain of I460 points inside the putative channel. Alanine 430 is more conserved than I460 (cf. Table 2). Replacing A430 with isoleucine is also expected to obstruct the channel, but the only effect we observed is an increase (+30%) of the rates of both binding and release of O2, which is difficult to reconcile with the fact that the Michaelis constant for H2 and the kinetics of inhibition by CO are not at all affected. Crystals of Dd hydrogenase exposed to xenon exhibit a single binding site defined by the side chains of A306, F372 and F160 (A426, F492 and L283 in Cp). The mutation A426L should make this binding site smaller, and indeed, we found that it decreases slightly (20%), but significantly, the yield of HD in the isotope exchange assay, suggesting that either HD release (kout) has decreased or that the rate of H+/ D+ exchange at the active site has increased; we consider the first option as more likely, because A426 is remote from the active site. The mutation also increases the Michaelis constant about two-fold, suggesting again that it slows down the diffusion of H2, but the agreement between the two measurements (Km and kout) is only qualitative (ideally, these parameters should be proportional to each other, consistent with our observations with NiFe hydrogenase mutants in ref. 44). The mutation seems to significantly slow O2 binding and release (30 and 20%, respectively), but CO surprisingly it has no effect on the rates of binding and release of CO (kCO in and kout ). The properties of the F492A mutant were unexpected. The mutation has no effect on any of the parameters that depend on intramolecular diffusion kinetics, but it increases about two-fold the 1st order rate of reaction of O2 at the active site (kdead  from the active in Scheme 3); this is so despite the fact that F492 is remote (>10 A) site. The mutations V422W and F416W were designed to block the central cavity but they do not affect the parameters we measured, except the H2-oxidation turnover rate, which is decreased more than 20-fold by either mutation. This substitution of V422 in Ca is equivalent to the V240W mutation that increases the resistance to oxygen of Cr FeFe hydrogenase, defined in ref. 85 as the fraction of H2-production activity that remains after Cr cell extracts have been exposed to small amounts of O2 (1 to 4% O2 for two minutes). We defined and quantified oxygen sensitivity differently, as the rate of inhibition during H2-oxidation. This may be the reason we could detect no improvement of the resistance to O2, although it is possible that the improvement of O2 tolerance observed with the Cr hydrogenase mutant does not result from hindered intramolecular diffusion of O2, or that the equivalent mutations in Cr and Cp do not have the same effects (since the mutant of Cr was not purified, it is unknown whether the mutation in Cr also has a detrimental effect on turnover rate). We have recently emphasized that certain equivalent mutations carried out in homologous NiFe hydrogenases do not have the same effect.25 Phenylalanine 416 is downstream from channels A and B (but not W), and it is therefore most surprising that the F416W mutation has no effect either. This suggests that fluctuations play a major role even at this position, whereas, according to the values of the B-factors in the crystal structure of Cp hydrogenase, the mobility of F417 is low (Fig. 7). Cysteine 298 is a highly conserved residue in the vicinity of the di-iron subsite, whose side chain points inside the cavity, downstream from all paths. We did not attempt to insert bulky residues at this position, fearing that this may destabilize the active site, but we found that this amino acid is not essential for activity. Its substitution does not affect oxygen sensitivity either, whereas in NiFe hydrogenases, k The patent also mentions F252 (A435 in Cp). This journal is ª The Royal Society of Chemistry 2010

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the presence of methionines near the active site greatly improves oxygen resistance.74,103 Overall, we observed that only the A426L mutation has an effect on the rates of intramolecular transport: it slows the production of HD, increases the Michaelis– Menten constant, and decreases the rates of binding and release of O2, although, surprisingly, the mutation has no effect on CO binding and release. It is also remarkable how small these effects are in comparison with those observed in NiFe hydrogenase mutants.24,44 The reason for this is, of course, unclear, but we can propose three distinct explanations. The first option is that the amino acids we targeted are indeed along the main path, but the mutations do not block the channel, possibly because fluctuations are significant. Fig. 7A shows a coloring of the structure of Cp FeFe hydrogenase by B-factor. Channel A is in a region of the protein that is highly disordered in the crystal. The same observation can be made from the structure of Dd hydrogenase (Fig. 7C). The entrance of channel A, near the protein surface, is particularly mobile, and it is may not be so surprising that the channel cannot be blocked here, although Tosatto and coworkers identified one amino acid at the surface of the protein in this region (H492, Cp numbering), which may provide oxygen tolerance to Thermotoga neapolitana FeFe hydrogenase.86 Alternatively, one or several of the mutations we designed may obstruct the channel, but this creates an alternative route that bypasses the blocked channel. In this respect, we note that MD simulations suggest that a mutation that blocks the permanent channel of haloalkane dehalogenase causes the product to use a different (transient) path.71 However, we consider it unlikely that this would have no detectable effect on the kinetics of intramolecular transport. Last, it may be that we did not target the right pathway. Four mutants out of 8 were designed to alter channel A, and two others were expected to modify the central cavity, downstream from paths A and B. We have not yet targeted any amino acid

Fig. 7 Coloring of the structure of Cp FeFe hydrogenase (A and B) and Dd hydrogenase (C) by B-factor (PDB 3C8Y and 1HFE, respectively). (A) The complete structure of Cp hydrogenase. (B) Only the amino acids that line channel A. The orientations are the same as those in Fig. 1A and 2. The B-factors are coded from blue (low) to red (high). Faraday Discuss.

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that is specific to channel W, and it is possible that this channel is actually functional for H2 and O2 transport. Hydrophilic channels are not usually considered for the transport of small hydrophobic ligands. However, several results in the literature suggest that this may be a misconception. For example, hydrophobic ligands are sometimes stabilized in hydrophilic pockets: Prange and coworkers observed that Xe35 or O2104 under pressure can displace water molecules in protein crystals, and in at least one structure of copper amine oxidase, Xe atoms displace two water molecules.40 Conversely, recent MD simulations of myoglobin have shown that two Xe binding sites in Mb transiently house water molecules,105 and the channel of both type-1106 and type-2107 cholesterol oxidase stabilizes water molecules, despite the fact that they are predominantly lined by hydrophobic side chains. In catalase, the results of MD simulations suggest that O2, H2O2 and H2O share the same channel,108 which stabilizes a chain of water molecules in the crystal.72 In ACSCODH, although the channel that transports CO has been identified, the path for CO2 access to the C-cluster is unknown, and, among other options, it has been suggested that a hydrophilic channel is used in this respect.52 Last, there is now evidence that in nitrogenase, a channel that stabilizes a string of water molecules in the crystal is used for transporting the substrates N2,66 whereas the V76I mutation, which was expected to disrupt the putative hydrophobic putative channel, has no effect.109 However, we must note that N2 reduction of ammonia is slow, and therefore a channel that allows fast diffusion of N2 in this enzyme may not be a functional requirement.

4. Conclusion The introduction of this paper made it clear that only in a very few enzymes have substrate channels for small ligands been unambiguously identified. It is unfortunate that the experiments reported here do not clarify the case of FeFe hydrogenase, considering the usefulness of hydrogenases that are engineered to resist inhibition by O2.85,103 Yet, our work illustrates how difficult it may be either to prove the existence of a preferred pathway or to establish that a single pathway does not exist. This is because mutations that do change the rate of diffusion may have no effect on the kinetic properties that can be measured (cf. the discussion of eqn (1) or the discussion in ref. 24). Therefore, the fact that the substitutions we performed did not modify the enzyme’s properties does not imply that the substituted amino acids do not line the preferred gas pathway. In our recent study of NiFe hydrogenase,24 we showed that substitutions that make the channel hydrophilic have a much stronger effect that any attempt to block the channel with a bulky side chain. Therefore, we are now trying to substitute hydrophobic side chains in channel A with charged groups. We now also wonder whether it may be possible to prove that small ligands diffuse within FeFe hydrogenase through transient cavities created by the fluctuations of the protein by designing mutations that make the protein more rigid, possibly by introducing intramolecular disulfide bonds or salt bridges. We also must explore the possibility that, in contrast with previous hypotheses, the wet channel is used for guiding the substrate and inhibitors in FeFe hydrogenases.

Acknowledgements We acknowledge Sebastien Dementin and Emilien Etienne (BIP, Universite de Provence, Marseille), Azat Gabdulkhakov (Institut fuer Kristallographie Freie Universitaet Berlin) and Yvain Nicolet (IBS/LCCP, CEA, CNRS, UJF, Grenoble) for fruitful discussions. Our work is funded by the CNRS, ANR, INSA, CEA, Universite de Provence and City of Marseilles. We also acknowledge support from the ‘‘p^ ole de competitivite Capenergies.’’ This journal is ª The Royal Society of Chemistry 2010

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