Tài liệu Báo cáo khoa học: Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O2 sensitive without affecting its transductory activity docx

Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O 2 sensitive without affecting its transductory activity Ophe ´ lie Duche ´ 1 , Sylvie Elsen 1 , Laurent Cournac 2 and Annette Colbeau 1 1 Laboratoire de Biochimie et Biophysique des Syste ` mes Inte ´ gre ´ s (UMR 5092 CNRS-CEA-UJF), De ´ partement Re ´ ponse et Dynamique Cellulaires, Grenoble, France 2 CEA Cadarache, De ´ partement des Sciences du Vivant, De ´ partement d’Ecophysiologie Ve ´ ge ´ tale et de Microbiologie, Laboratoire d’Ecophysiologie de la Photosynthe ` se, UMR 6191 CNRS-CEA-Aix Marseille II, Saint Paul-lez-Durance, France Hydrogenases are enzymes involved in H 2 metabolism. They occur widely in bacteria and in some eukaryotes [1]. The various hydrogenases differ in their metal con- tent (FeFe, NiFe), their localization in the cell, their relationship with metabolism, and the way their synthe- sis is regulated [2]. They catalyze the reversible reaction H 2 « 2H + +2e ) and are known to be O 2 sensitive. In general, iron hydrogenases, which actively evolve H 2 , are quickly and irreversibly inactivated in the presence of O 2 [3]. In contrast, most [NiFe] hydrogenases are only reversibly inhibited by O 2 . The structure of the bimetallic active site and the mechanisms of hydrogen oxidation in [NiFe] hydro- genases have been thoroughly studied by various bio- physical methods (reviewed in [4,5]). The information obtained has given clues to the inactivation of the enzyme by O 2 .InDesulfovibrio hydrogenases, it has been shown that the Fe atom is linked to three non- protein ligands: 1 CO and 2 CN – [6]. The Ni and Fe ions are asymmetrically bridged by two cysteine sulfur atoms and one oxygenic species (O 2 – or OH – ), which appears in the oxidized enzyme [7–9]. The catalytic Keywords gas access channel; hydrogenases; oxygen sensitivity; Rhodobacter capsulatus Correspondence A. Colbeau, Laboratoire de Biochimie et Biophysique des Syste ` mes Inte ´ gre ´ s, DRDC, CEA ⁄ Grenoble, 17 rue des martyrs, 38054 Grenoble Cedex 9, France Fax: +33 4 38 78 51 85 Tel: +33 4 38 78 30 74 E-mail: umr5092@dsvsud.cea.fr Website: http://www-dsv.cea.fr/bbsi (Received 13 May 2005, revised 26 May 2005, accepted 6 June 2005) doi:10.1111/j.1742-4658.2005.04806.x In the photosynthetic bacterium Rhodobacter capsulatus, the synthesis of the energy-producing hydrogenase, HupSL, is regulated by the substrate H 2 , which is detected by a regulatory hydrogenase, HupUV. The HupUV protein exhibits typical features of [NiFe] hydrogenases but, interestingly, is resistant to inactivation by O 2 . Understanding the O 2 resistance of HupUV will help in the design of hydrogenases with high potential for bio- technological applications. To test whether this property results from O 2 inaccessibility to the active site, we introduced two mutations in order to enlarge the gas access channel in the HupUV protein. We showed that such mutations (Ile65 fi Val and Phe113 fi Leu in HupV) rendered HupUV sensitive to O 2 inactivation. Also, in contrast with the wild-type protein, the mutated protein exhibited an increase in hydrogenase activity after reductive activation in the presence of reduced methyl viologen (up to 30% of the activity of the wild-type). The H 2 -sensing HupUV protein is the first component of the H 2 -transduction cascade, which, together with the two- component system HupT ⁄ HupR, regulates HupSL synthesis in response to H 2 availability. In vitro, the purified mutant HupUV protein was able to interact with the histidine kinase HupT. In vivo, the mutant protein exhib- ited the same hydrogenase activity as the wild-type enzyme and was equally able to repress HupSL synthesis in the absence of H 2 . Abbreviations MG medium, malate ⁄ glutamate medium; MN medium, malate ⁄ ammonia medium; RH, regulatory hydrogenase; SH, soluble NAD-linked hydrogenase. FEBS Journal 272 (2005) 3899–3908 ª 2005 FEBS 3899 activity of [NiFe] hydrogenases, i.e. binding and oxidation of H 2 , is preceded by an activation step in the presence of H 2 or a reductant. During this reduc- tive activation, the oxygenic species is lost and reap- pears during reoxidation, as shown by X-ray analyses. This ligand is thus a signature of the inactive, unready state [10,11]. The regulatory hydrogenases (RHs) form a subclass of [NiFe] hydrogenases, identified in Rhodobacter cap- sulatus and Bradyrhizobium japonicum (HupUV) [12– 15] and in Ralstonia eutropha (HoxBC) [16,17]. They are able to catalyze the three typical reactions of hydrogenases (H 2 uptake, H 2 evolution and H–D exchange) [14,18] but are unable to sustain growth [16,19]. These hydrogenases are the first element of a multicomponent system that regulates the synthesis of the energy-linked hydrogenase in response to H 2 ; their role is to detect the availability of H 2 . In addition to the H 2 sensor protein, this system comprises a histidine kinase and a response regulator (HupT and HupR respectively in R. capsulatus), which form a two- component regulatory system functioning by phosphate transfer [20]. We have demonstrated that the H 2 sen- sor, HupUV, interacts directly with the histidine kinase HupT [13], thus promoting its autophosphorylation in the absence of H 2 . The phosphate is then transfered to the response regulator HupR, which, in contrast with most response regulators, is active in the unphosphory- lated state [20]. Consequently, this phosphorylation leads to the inactivation of the transcriptional factor HupR and to the decrease in the synthesis of HupSL hydrogenase in the absence of H 2 . A homologous system has been found in R. eutropha, namely the HoxBC ⁄ HoxJ ⁄ HoxA system [21]. Compared with standard hydrogenases, RHs from R. capsulatus and R. eutropha exhibit unusual bio- chemical features. The most interesting feature is that they are O 2 insensitive [14,16,18,22], and thus could offer an attractive option for applications in a future hydrogen economy. However, the hydrogenase activity of RHs is low, and the reason for the O 2 insensitivity is not well understood. It has been suggested that this insensitivity results from limited O 2 access to the active site [16]. Indeed, hydrophobic channels have been iden- tified that may serve as pathways for gas access to the deeply buried active site [23–25]. As both molecular H 2 and O 2 are hydrophobic gases, they probably use the same access pathway to the hydrogenase active site. The amino-acid sequences of the O 2 -resistant RHs have been compared with those of the O 2 -sensitive hydrogenases from Desulfovibrio species [25]; five of the six amino acids lining the putative channel were found to be different in the H 2 sensors. In a mutated model of Desulfovibrio fructosovorans hydrogenase with two of these amino acids, Val74 and Leu122, replaced by Ile and Phe, respectively, the accessibility of the active site was predicted to be significantly decreased, suggesting that a partial blocking of the gas channel by the presence of bulky residues may indeed explain the O 2 insensitivity of the sensor enzymes [25]. In this study, we replaced Ile65 and Phe113 (corres- ponding to amino acids 74 and 122 in the large sub- unit of D. fructosovorans hydrogenase) of the large subunit (HupV) of HupUV with Val and Leu, respect- ively, and showed that these amino acids are indeed involved in the O 2 insensitivity of the isolated protein. We have also shown that the mutated HupUV protein is as active in vivo as wild-type HupUV and is func- tional in the H 2 -transduction system. Results Overproduction of mutated HupUV proteins in R. capsulatus We used site-directed mutagenesis to modify two bulky residues lining the putative gas access channel in the large subunit HupV (Ile65 and Phe113 replaced by Val and Leu, respectively) After mutagenesis, the hupUV genes were cloned into the expression vector pSE102. In pSE103 and pOD7, the wild-type and mutated hupUV genes, respectively, are expressed from the strong nif promoter. To assess H 2 -uptake activity catalyzed by these pro- teins in whole cells, the plasmids pSE103 and pOD7 were introduced into R. capsulatus JP91 cells devoid of HupSL enzyme. When grown under conditions that promote nitrogenase synthesis (under light and in the absence of oxygen and ammonia), the two strains exhibited similar hydrogenase activity, assayed by reduction of methylene blue in the presence of H 2 [spe- cific activity in whole cells ranging from 0.08 to 0.15 lmol reduced methylene blueÆmin )1 Æ(mg pro- tein) )1 , compared with 0.01–0.02 in the JP91 strain without any plasmid]. The production level of the two proteins was also similar, as shown by western immuno- blotting of entire cells revealed by antibodies against His 6 tag (not shown). For the purification of the HupUV proteins, the two plasmids were introduced into a HupUV – strain of R. capsulatus, BSE16, which was grown under light and in the absence of oxygen and ammonia. As the HupU subunit was produced as a fusion protein with an N-terminal His 6 tag, we were able to purify the complex His 6 HupUHupV by affinity chromatography on a Ni 2+ -charged column. Figure 1A shows the last O 2 sensitivity of the regulatory hydrogenase HupUV O. Duche ´ et al. 3900 FEBS Journal 272 (2005) 3899–3908 ª 2005 FEBS step of the purification of wild-type and mutated pro- teins, with the two subunits in a stoichiometric ratio. Under native conditions (Fig. 1B), the two proteins displayed the same pattern. The molecular masses of the two bands ( 80 and 170 kDa) were estimated by runs in native gels with different acrylamide concentra- tions [26], as previously described [13]. They corres- pond, respectively, to the dimeric form, HupUV, and the tetrameric form, Hup(UV) 2 , both of which exhibit hydrogenase activity (see below). These results suggest that the quaternary structure of the mutated protein was well conserved. Hydrogenase activity and stability of purified mutated HupUV protein We observed that, after breakage in a French Press of BSE16 cells producing mutated protein, hydrogenase activity decreased noticeably in the soluble extracts (which contained only HupUV, HupSL being retained in the membrane fraction), suggesting inactivation by air. The hydrogenase activity of the purified proteins, assayed by H 2 uptake in the presence of benzyl violo- gen, was about fivefold lower in the mutant protein OD7 than that of wild-type HupUV [9.2 vs. 2.0 lmol reduced benzyl viologenÆmin )1 Æ(mg protein) )1 ). To check the stability of the proteins in the presence of O 2 , soluble extracts and purified proteins were stored in air at 4 °C for several days, and, each day, H 2 -uptake activity was determined by measuring ben- zyl viologen reduction in an aliquot. As shown in Fig. 2, the mutant OD7 had lost 80% of its activity after 3 days under air in soluble extracts, and in  1.5 days when purified. The mutant protein was par- tially protected when stored under N 2 ; it exhibited  50% activity during the same time (as compared with 20% under air) (not shown). Thus in the mutant protein, there was specific inactivation of the catalytic activity by O 2 , but the mutation could also modify the conformation of the protein, rendering it unstable. H–D exchange activity catalysed by wild-type and mutated HupUV proteins The effect of O 2 on the activity of aerobically purified HupUV proteins was then assessed directly by a MS method monitoring continuously the H–D exchange in either the absence or presence of O 2 . The results are given in Table 1. In the wild-type HupUV protein, the activity and the rate of HD and H 2 formation were similar under aerobic and anaerobic conditions. These results are in agreement with a previous study reporting that the H–D exchange reaction catalyzed by the HupUV protein was high in the presence of O 2 [18]. In this study, we observed that the activity of the mutant OD7 was repro- ducibly twofold higher in the absence of O 2 than under aerobiosis [1.3 ± 0.3 vs. 0.7 ± 0.1 lmolÆmin )1 Æ(mg protein) )1 , respectively]. In all cases, the rate of HD formation was twice that of H 2 formation. To check whether the low activity of the mutant protein OD7 was due to the fact that O 2 could now reach the active site and partially inactivate it, we repeated the assays in the presence of reduced methyl viologen. It is well known that standard hydrogenases need to be activated by reduction to become catalyti- cally competent [27]. The activities of the aerobically purified proteins were assayed under anaerobiosis by H–D exchange, as described above, and then 0.16 mm MV + was added. Table 1 shows that addition of MV + did not further activate the HupUV protein, whereas, interestingly, the activity of the OD7 protein AB Fig. 1. SDS ⁄ polyacrylamide gel (A) and native gel (B) of wild-type and mutated HupUV proteins. (A) Cell extracts from 5 L were puri- fied on two successive Ni 2+ -charged columns. Then 10 lL of the pools purified on the second HiTrap column and eluted with 250 m M imidazole were loaded on to an SDS ⁄ 12% polyacrylamide gel. Lane 1, wild-type; lane 2, mutant. (B) An 8-lg sample of each protein was run on a native polyacrylamide gel and stained with Coomassie Brilliant Blue. Lane 1, wild-type; lane 2, mutant. Fig. 2. Inactivation of wild-type and mutated HupUV proteins in air. Soluble extracts obtained after centrifugation of sonicated cells at 50 000 r.p.m. for 1 h (A) and purified proteins (B) were kept at 4 °C under air, and H 2 -uptake hydrogenase activity was assayed every day during 1 week. Wild-type, diamonds; mutant, circles. Data rep- resent the mean results from two or three independent assays. O. Duche ´ et al. O 2 sensitivity of the regulatory hydrogenase HupUV FEBS Journal 272 (2005) 3899–3908 ª 2005 FEBS 3901 was fourfold higher after reduction by MV + . This suggests that the mutated HupUV protein was inacti- vated during aerobic purification, but partial activity could be recovered under reducing conditions, this activity remaining threefold lower than that of the wild-type. Figure 3 illustrates the effect of reduced MV + on H–D exchange activity catalysed by the puri- fied HupUV proteins (note the difference in the scale). In vitro interaction of the mutated HupUV protein with HupT In a previous study, we showed that HupUV (probably the HupU subunit) interacts with the N-terminal domain of the histidine kinase HupT to transduce the signal of H 2 availability [13]. We therefore addressed the question of whether the mutation of some amino acids of HupUV modifies the conformation of the pro- tein and consequently its interaction with the histidine kinase. Mutated and wild-type HupUV proteins were incubated with HupT, and their interactions visualized directly on native acrylamide gels (Fig. 4). When HupT was incubated with any one of the HupUV proteins, a new active band appeared with a higher molecular mass, representing a Hup(UV) 2 –HupT 2 complex, as previously determined for wild-type HupUV [13], and the amount of free HupUV decreas- ed. There was no difference in migration between the two HupUV–HupT complexes, suggesting that the mutations did not substantially modify the interaction. Table 1. H–D exchange activity and rate of H 2 and HD formation by wild-type and mutated HupUV proteins of R. capsulatus. The values are initial rates corrected for gas consumption by the mass spectrometer. Activity and H 2 or HD rate of formation are expressed as lmol formedÆ min )1 Æ(mg protein) )1 as described [44]. Assays under aerobiosis and anaerobiosis were performed separately. When noted, reduced methyl viologen (MV + ) was present at 0.16 mm. Data are means from two or three independent experiments, with variation of less than 15%. Proteins Aerobiosis Anaerobiosis Anaerobiosis + MV + Activity HD formation H 2 formation Activity HD formation H 2 formation Activity HD formation H 2 formation HupUV (wild-type) 18.5 ± 0.6 8.0 ± 0.8 4.7 ± 0.1 15.6 ± 1.2 7.0 ± 0.5 4.0 ± 0.2 15.5 ± 1.7 7.0 ± 0.7 3.7 ± 0.5 OD7 (mutant) 0.7 ± 0.1 0.3 ± 0.1 0.2 ± 0.1 1.3 ± 0.3 0.7 ± 0.1 0.3 ± 0.1 5.2 ± 1.3 2.5 ± 0.6 1.2 ± 0.5 Fig. 3. Reductive activation by reduced MV + in HupUV proteins assayed by MS. The vessel containing 1.5 mL Mes buffer was saturated with D 2 and made anaerobic as explained in Experimental procedures. Then 3 lg wild-type or mutated HupUV protein was added. Exchange activity was assayed under anaerobiosis for 1–2 min, then Zn-reduced MV + was added and the activity followed for 2 or more minutes. (A) Wild-type HupUV protein; (B) mutant OD7 protein. Fig. 4. Interaction between HupT and HupUV proteins. Lanes 1 and 2, wild-type HupUV; lanes 3 and 4, mutant OD7. HupT was present in lanes 2 and 4. O 2 sensitivity of the regulatory hydrogenase HupUV O. Duche ´ et al. 3902 FEBS Journal 272 (2005) 3899–3908 ª 2005 FEBS The mutated HupUV protein can regulate the synthesis of HupSL hydrogenase The next question we addressed was to check whether the O 2 -sensitive mutated protein was able to function in vivo, i.e. to transduce the H 2 signal, and in the absence of H 2 , to repress hydrogenase synthesis, even in presence of O 2 . The plasmids pSE103 and pOD7, which expressed hupUV genes from the nif promoter, were not suitable for in vivo experiments, because this promoter is not active under aerobiosis [28]. For this reason, the plasmids pSE60 and pOD15, in which the hupUV genes were cloned under control of the fruc- tose-induced fru promoter, were constructed and used to complement the hupUV mutant strain BSE16. The complemented cells were grown under aerobiosis or anaerobiosis in the presence of 3 mm fructose and in either the presence (derepressing conditions) or absence (repressing conditions) of H 2 .H 2 was produced endo- genously as a by-product of nitrogenase activity during anaerobic growth in malate ⁄ glutamate (MG) medium. The presence of O 2 and ammonia inhibits activity and synthesis of nitrogenase; when indicated, H 2 was added externally during aerobic growth in malate ⁄ ammonia (MN) medium. Table 2 summarizes the results. In all conditions tested, the BSE16 mutant strain exhibited a high level of hydrogenase activity compared with the wild-type B10, because, in the absence of the HupUV protein, which is part of the repressing system, hupSL gene expression remains fully activated [12]. As shown in Table 2, mutated HupUV protein, produced from plasmid pOD15, was able to repress hydrogenase syn- thesis in the absence of H 2 to the same extent as the wild-type protein. Thus, the availability of H 2 was still detected even when growth was carried out in the pres- ence of O 2 . Discussion In regulatory hydrogenases, it has been hypothesized that bulky residues lining the gas channel participate in O 2 resistance by blocking O 2 access to the active site [25]. To check this hypothesis, we replaced, by site- directed mutagenesis, two amino acids that line the gas access channel, Ile65 and Phe113, with Val and Leu, respectively. Interestingly, these replacements rendered the protein O 2 sensitive, demonstrating that these resi- dues are involved in O 2 sensitivity of the RH. This was corroborated by experiments showing that the H–D exchange activity of the mutant protein increased greatly in the presence of reduced MV, at variance with that of the wild-type protein. However, even after reductive activation, the hydrogenase activity of puri- fied mutated HupUV protein remained twice as low as that of the wild-type, suggesting that O 2 may also irre- versibly inactivate the active site. Another explanation is that the mutations could also modify the structure around the active site and⁄ or the binding of ligands, thus decreasing the catalytic efficiency of the enzyme. Our results suggest that, in vivo, the mutated HupUV protein is protected from O 2 inactivation, as it exhibited about the same hydrogenase activity as the wild-type one. This was further corroborated by complementation experiments, which showed that the OD7 mutated pro- tein produced in a hupUV mutant was able to restore the regulation of HupSL synthesis. This implies that it was able to transmit the information about the availab- ility of H 2 to the histidine kinase, HupT. Indeed, we showed that the mutated protein was able to interact with HupT in vitro at the same HupUV ⁄ HupT ratios and under the same conditions as the wild-type one [13]. ‘Standard’ [NiFe] hydrogenases are known to be reversibly inactivated by O 2 .O 2 could affect either the enzyme during the activation step and ⁄ or the active enzyme in the catalytic cycle. For instance, in the hydrogenase from Allochromatium vinosum, it has been observed that O 2 added during the activation step of the ready enzyme increases the lag phase without preventing the activation [29]. On the other hand, when added to the active enzyme, O 2 would react directly with the act- ive NiFe site, thus inactivating the reaction with H 2 [30]. It should be noted that the occurrence of direct binding of O 2 to the active NiFe site is under debate and was not observed for hydrogenase from Desulfovibrio gigas [8]. Some hydrogenases, however, are able to consume H 2 in the presence of O 2 , and exhibit noticeable resist- ance to this gas. The best-known enzyme is the soluble Table 2. Hydrogenase activities of the wild-type B10 and hupUV BSE16 strains from R. capsulatus, complemented with wild-type and mutated hupUV genes. Cells were grown overnight at 30 °C anaerobically in the light (MN or MG medium) or aerobically in the dark (MN or MN medium + 10% H 2 )toanA 660 of  1.5. In MG medium, H 2 was evolved from nitrogenase activity. Fructose (3 m M) was added at the beginning of growth at an A 660 of  0.6. Hydrogenase activity was assayed with methylene blue and was expressed as lmol reduced MBÆh )1 Æ(mg protein) )1 . The values are the means from at least three independent experiments. Strains Hydrogenase activity Anaerobiosis Aerobiosis MN MG (H 2 )MN MN+H 2 B10 19 ± 10 44 ± 3 24 ± 1 80 ± 17 BSE16 92 ± 21 36 ± 10 82 ± 8 116 ± 14 BSE16 (pSE60) 16 ± 13 52 ± 12 18 ± 3 65 ± 16 BSE16 (pOD15) 17 ± 2 68 ± 9 16 ± 1 64 ± 17 O. Duche ´ et al. O 2 sensitivity of the regulatory hydrogenase HupUV FEBS Journal 272 (2005) 3899–3908 ª 2005 FEBS 3903 NAD-linked hydrogenase (SH) from the strictly aero- bic bacterium R. eutropha [31]. This hydrogenase har- bours an active site different from that of ‘standard’ hydrogenases with two additional CN – groups, tenta- tively assigned to the Fe atom and the Ni atom [6,32]. It has been hypothesized that these two CN – groups may shield the active site from O 2 attack by steric hin- drance [32,33]. The Ni-bound CN – seems to be respon- sible for the O 2 insensitivity of the enzyme, and is linked to the presence of the hypX gene [34,35], found also in other aerobic bacteria such as Rhizobium [36]. Indeed, in SH purified from an HypX – strain, the cata- lytic turnover (the hydrogenase activity) was shown to be independent of the presence of O 2 , but the enzyme was irreversibly inactivated if O 2 was present during the autocatalytic activation [35], probably because of formation of some peroxide or superoxide. In a recent study, a mutant of HoxH, the active-site-containing subunit of the SH, was constructed by replacement of Leu118 with Phe; this mutation led to an O 2 -sensitive phenotype, and it was postulated that this bulky resi- due impaired the incorporation of the Ni-linked CN – , thus conferring O 2 sensitivity [37]. Interestingly, this mutated HoxH subunit contains the two bulky residues corresponding to Phe113 and Ile65 of the wild-type HupUV proteins, and conserved in the other H 2 -sensing RHs, such as R. eutropha HoxBC [21] and B. japonicum HupUV [15]. Therefore, and paradoxic- ally, the presence of these residues, which seem in our study to confer O 2 resistance on HupUV, did not pro- tect the protein against O 2 in the HypX – mutant. Although the active site of HupUV from R. capsula- tus has not yet been studied, that of the homologous protein, HoxBC, of R. eutropha was shown to be very similar to that of standard hydrogenases, with a Fe atom liganded by 1 CO and 2 CN – [16], and the binding of an hydride to Ni and Fe after H 2 reduction has recently been demonstrated [38]. However, in contrast with standard hydrogenases, the RH exists only as two redox forms, i.e. ready oxidized and reduced. The O 2 and MV + responses observed in the mutant HupUV protein suggest that it has reached unready states, and further studies will be needed to determine which ones. In a recent study using X-ray absorption spectroscopy, Haumann et al. [39] suggested that the specific Ni co-ordination may also be crucial to the O 2 insensitivity of the R. eutropha RH. In particular, the number of S ligands was decreased by one upon formation of the active state, but binding of O 2 to the active site was pre- vented because an O ⁄ N ligand from an amino acid was already bound at the free position at the Ni site. In any case, it appears that in the O 2 -resistant hydrogenases, O 2 is prevented from contacting the active site, even if various mechanisms are certainly involved. In the case of the regulatory HupUV protein, our results favour the hypothesis of Volbeda et al. [25], which explains the O 2 resistance of RHs by limited accessibility of the active site to O 2 . In the mutated protein, O 2 has access to the active bimetallic site, which would remain in the inactive form, and, consequently, this protein exhibits some features of the standard hydrogenases that must be activated in the presence of H 2 or a reductant [27]. Our conclusions are strengthened by a recent paper from Friedrich’s group [40], which shows the O 2 sensitivity of HoxBC proteins mutated in residues lining the gas access chan- nel in R. eutropha. In this respect, the comparative analysis of wild-type, O 2 -resistant and mutated, O 2 -sensitive HupUV proteins by biophysical methods may lead to the improved understanding of the mecha- nisms of O 2 resistance ⁄ sensitivity in [NiFe] hydrogen- ases in general. RHs that are insensitive to O 2 and, as isolated, ready to function are potentially of great bio- technological interest, but their activity is low. When the basis of their O 2 resistance is understood, it will be possible to design a hydrogenase that exhibits high activity together with O 2 insensitivity. Experimental procedures Bacterial strains and plasmids The strains and plasmids used in this study are listed in Table 3. R. capsulatus strains were grown heterotrophically at 30 °C under anaerobiosis in the light or under aerobiosis in the dark with shaking, in MG medium (7 mm glutamate, 30 mmdl-malate) or MN medium (7 mm ammonium sul- fate, 30 mmdl-malate) [19]. Escherichia coli strains were grown at 37 °C in Luria–Bertani medium. Antibiotics were used at the following concentrations: 100 (ampicillin) and 10 (tetracycline) mgÆL )1 for E. coli and 1 (tetracycline) mg ⁄ L )1 for R. capsulatus. DNA manipulation and bacterial mating Standard recombinant DNA techniques were performed as described by Sambrook et al. [41]. Restriction enzymes were used as indicated by the manufacturers. Triparental matings were performed with the plasmid helper pRK2013 as des- cribed previously [42]. Construction of plasmids with mutations in the hupV gene A 3.2-kb fragment bearing hupUV genes cloned into pUC18 was used to modify two amino acids with the QuikChange O 2 sensitivity of the regulatory hydrogenase HupUV O. Duche ´ et al. 3904 FEBS Journal 272 (2005) 3899–3908 ª 2005 FEBS Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA). The mutagenesis leading to plasmid pOD585 was carried out in two successive steps with the following sets of oligonucleotides as primers: UV5 (5¢-CGCGGATCTGCG TCTGCTCGATCTCGC-3¢) and UV6 (5¢-GCGAGATCG AGCAGACGCCGCAGATCCGCG-3¢) to replace Ile65 from HupV with Val; UV7 (5¢-GCATTTCAACCTCCT GTTCATGCCCGATTTC-3¢) and UV8 (5¢-GAAATCG GGCATGAACAGGAGGTTGAAATGC-3¢) to replace Phe113 from HupV with Leu. The 3.2-kb fragment corres- ponding to the mutated hupUV genes was excised from plas- mid pOD585 with NdeI–BamHI and cloned into pSE50 digested with the same enzymes in place of the wild-type hupUV genes, leading to plasmid pSE504. Plasmid pSE102 was cleaved with NcoI–BamHI, to clone 3.2-kb fragments from pSE50 and pSE504 digested with the same enzymes, leading to plasmids pSE103 and pOD7, respectively, which were introduced into R. capsulatus hupUV mutant BSE16 or hupSL mutant JP91 by conjugation. From these plasmids, the HupU subunit will carry an N-terminal His 6 tag for easy purification of the HupUV complex. Purification of the His 6 -HupUV proteins In the plasmids pSE103 and pOD7, wild-type and mutated hupUV genes were expressed from the nifHDK promoter. For this reason, cells (from 5 L culture) were grown under conditions allowing strong expression of the nif promoter (MG medium, anaerobiosis, under light). Proteins were purified on a HiTrap chelating column (Amersham Pharmacia Biotech, Piscataway, NJ, USA) as described previously [13]. Elution of the 5-mL column with buffer containing 100 mm imidazole gave an active pool, which was concentrated on a 1-mL column by elu- tion with 250 mm imidazole in the buffer. The pools were dialyzed three times in 25 mm Tris ⁄ HCl (pH 8) contain- ing 10% (v ⁄ v) glycerol and 150 mm NaCl, at 4 °C. The purified proteins were divided into aliquots and stored at )80 °C. Enzyme assays Hydrogenase activity was assayed by the rate of H 2 uptake or H–D exchange. H 2 uptake was determined spec- trophotometrically in 20 mm Tris ⁄ HCl buffer (pH 8), either in whole cells with 0.15 mm methylene blue (MB) as artificial electron acceptor, at A 565 , or in cell extracts and purified proteins with 2 mm benzyl viologen (BV), at A 555 [43]. In native gels, hydrogenase activity was revealed by incubating the gels under H 2 for 10–40 min in 20 mm Tris ⁄ HCl buffer (pH 8), containing 2 mm BV. The reac- tion was stabilized by adding 1 mm triphenyltetrazolium chloride. The H–D exchange reaction was measured at 30 °C and determined by a MS method as previously des- cribed in detail [18,4]. Briefly, the reaction vessel was filled with 1.5 mL Mes buffer (50 mm, pH 6) and then sparged with D 2 until saturation, and the vessel was closed. Then 3 lg purified wild-type or mutated HupUV proteins were introduced into the vessel, and the changes in D 2 ,HD and H 2 were monitored by scanning masses 4, 3 and 2, Table 3. Bacterial strains and plasmids used in this study. Strain or plasmid Relevant characteristics Source or reference Strains R. capsulatus B10 Wild-type [45] BSE16 hupUV Hup c [12] JP91 hupSL Hup – [46] Plasmids pUC18 Ap r [47] pFRK-I Ap r Ble r Gm r Km r ; fruP fusion vector [48] pRK2013 Km r ; plasmid helper [49] pSE50 Ap r ; pET-15b with 3.2 kb NdeI-SalI insert containing hupUV [12] pSE102 Tc r ; pnif expression vector [13] pPHU231 Tc r ; pRK290 with a 388-bp HaeII insert containing pUC18 polylinker P. Hu ¨ bner, unpublished observations pOD585 Ap r ; pUC18 with mutated hupUV genes This work pSE504 Ap r ; pSE50 with 3.2-kb NdeI-BamHI from pOD585 This work pSE103 Tc r ; pSE102 with 3.2-kb NcoI-BamHI from pSE50 This work pOD7 Tc r ; pSE102 with 3.2-kb NcoI-BamHI from pSE504 This work pOD12 Ap r Gm r ; pFRK-I with a 3.2-kb NdeI-BamHI from pOD585 This work pSE60 Tc r ; pPHU234 with 6.2-kb HindIII containing hupUV [12] pOD15 Tc r ; pPHU231 with 6-kb HindIII-BamHI from pOD12 This work O. Duche ´ et al. O 2 sensitivity of the regulatory hydrogenase HupUV FEBS Journal 272 (2005) 3899–3908 ª 2005 FEBS 3905 respectively. When required, the medium was made aero- bic by the addition of H 2 O 2 (5 lL 0.3% H 2 O 2 ) decom- posed by the addition of catalase (500 U) thus liberating O 2 , or was made anaerobic by the addition of catalase (500 U), glucose (5 mm) and glucose oxidase (40 U). Zn- reduced methyl viologen (MV + 0.16 mm) was added to the anaerobic medium in some experiments. The rates of D 2 consumption and H 2 and HD production were correc- ted for simultaneous consumption by the spectrometer. This consumption, which showed first-order kinetics, was assayed in the absence of protein. In vitro interaction of HupUV and HupT The proteins (50 pmol HupUV and 250 pmol HupT) were incubated for 10 min at 30 °C in buffer containing 10 mm Tris ⁄ HCl (pH 8), 20 mm NaCl, 10% (v ⁄ v) glycerol, 1 mm EDTA and 1 mm dithiothreitol as previously described [13]. Proteins were then run on a native acrylamide gel in 0.5 · Laemmli buffer, and the gel was revealed by hydro- genase activity staining in the presence of BV. Complementation of hupUV mutant with mutated hupUV genes The mutated hupUV genes excised from plasmid pOD585 by NdeI–BamHI digestion were used to replace a 1.7 kb NdeI–BamHI fragment (deletion of the Ble r Km r cartridge) of the plasmid pFRK-I, leading to plasmid pOD12. pFRK-I contains a fructose-activated promoter, pfru, from R. capsulatus. 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Gene 33, 103–119. 48 Duport C, Meyer C, Naud I & Jouanneau Y (1994) A new gene expression system based on a fructose-depen- dent promoter from Rhodobacter capsulatus. Gene 145, 103–108. 49 Ditta G, Stanfield S, Corbin D & Helinski DP (1980) Broad-host range DNA cloning system for gram- negative bacteria: construction of a gene bank of Rhizobium meliloti . Proc Natl Acad Sci USA 77, 7347–7351. O 2 sensitivity of the regulatory hydrogenase HupUV O. Duche ´ et al. 3908 FEBS Journal 272 (2005) 3899–3908 ª 2005 FEBS . Enlarging the gas access channel to the active site renders the regulatory hydrogenase HupUV of Rhodobacter capsulatus O 2 sensitive without affecting. by limited accessibility of the active site to O 2 . In the mutated protein, O 2 has access to the active bimetallic site, which would remain in the inactive