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 t
Trang 1the regulatory hydrogenase HupUV of Rhodobacter
activity
Ophe´lie Duche´1, Sylvie Elsen1, Laurent Cournac2 and Annette Colbeau1
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 H2metabolism
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
H2« 2H++2e)and are known to be O2sensitive In
general, iron hydrogenases, which actively evolve H2,
are quickly and irreversibly inactivated in the presence
of O2 [3] In contrast, most [NiFe] hydrogenases are
only reversibly inhibited by O2
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 O2 In Desulfovibrio 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 (O2 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
H2, which is detected by a regulatory hydrogenase, HupUV The HupUV protein exhibits typical features of [NiFe] hydrogenases but, interestingly,
is resistant to inactivation by O2 Understanding the O2 resistance of HupUV will help in the design of hydrogenases with high potential for bio-technological applications To test whether this property results from O2 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 (Ile65fi Val and Phe113 fi Leu in HupV) rendered HupUV sensitive to O2 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 H2-sensing HupUV protein is the first component of the H2-transduction cascade, which, together with the two-component system HupT⁄ HupR, regulates HupSL synthesis in response to
H2 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 H2
Abbreviations
MG medium, malate⁄ glutamate medium; MN medium, malate ⁄ ammonia medium; RH, regulatory hydrogenase; SH, soluble NAD-linked hydrogenase.
Trang 2activity of [NiFe] hydrogenases, i.e binding and
oxidation of H2, is preceded by an activation step in
the presence of H2 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 (H2 uptake, H2 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 H2; their
role is to detect the availability of H2 In addition to
the H2sensor 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 H2
sen-sor, HupUV, interacts directly with the histidine kinase
HupT [13], thus promoting its autophosphorylation in
the absence of H2 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 H2 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 O2 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 O2 insensitivity
is not well understood It has been suggested that this
insensitivity results from limited O2access 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 H2
and O2 are hydrophobic gases, they probably use the
same access pathway to the hydrogenase active site
The amino-acid sequences of the O2-resistant RHs
have been compared with those of the O2-sensitive
hydrogenases from Desulfovibrio species [25]; five of
the six amino acids lining the putative channel were
found to be different in the H2 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 O2insensitivity 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 O2 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 H2-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 H2-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 H2 [spe-cific activity in whole cells ranging from 0.08 to 0.15 lmol reduced methylene blueÆmin)1Æ(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 His6 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 His6 tag, we were able to purify the complex His6HupUHupV by affinity chromatography
on a Ni2+-charged column Figure 1A shows the last
Trang 3step 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 H2 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)1Æ(mg protein))1) To
check the stability of the proteins in the presence of
O2, soluble extracts and purified proteins were stored
in air at 4C for several days, and, each day,
H2-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 N2; 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 O2, 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 O2 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 O2 The results are given in Table 1
In the wild-type HupUV protein, the activity and the rate of HD and H2 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 O2 [18] In this study, we observed that the activity of the mutant OD7 was repro-ducibly twofold higher in the absence of O2than under aerobiosis [1.3 ± 0.3 vs 0.7 ± 0.1 lmolÆmin)1Æ(mg protein))1, respectively] In all cases, the rate of HD formation was twice that of H2formation
To check whether the low activity of the mutant protein OD7 was due to the fact that O2 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 aerobicatalyti-cally 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
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 H2-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.
Trang 4was 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 H2 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–HupT2 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 H2and 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 H2or HD rate of formation are expressed as lmol formedÆ min)1Æ(mg protein))1as 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
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 D2and 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.
Trang 5The mutated HupUV protein can regulate the
synthesis of HupSL hydrogenase
The next question we addressed was to check whether
the O2-sensitive mutated protein was able to function
in vivo, i.e to transduce the H2 signal, and in the
absence of H2, to repress hydrogenase synthesis, even
in presence of O2 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 H2 H2 was produced
endo-genously as a by-product of nitrogenase activity during
anaerobic growth in malate⁄ glutamate (MG) medium
The presence of O2 and ammonia inhibits activity and
synthesis of nitrogenase; when indicated, H2was 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 H2 to the same extent as the
wild-type protein Thus, the availability of H2 was still
detected even when growth was carried out in the pres-ence of O2
Discussion
In regulatory hydrogenases, it has been hypothesized that bulky residues lining the gas channel participate
in O2resistance by blocking O2access 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 O2 sensitive, demonstrating that these resi-dues are involved in O2sensitivity 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 O2may 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 O2inactivation, 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 H2 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 O2 O2 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 O2added 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, O2would react directly with the act-ive NiFe site, thus inactivating the reaction with H2[30]
It should be noted that the occurrence of direct binding
of O2to the active NiFe site is under debate and was not observed for hydrogenase from Desulfovibrio gigas [8] Some hydrogenases, however, are able to consume
H2 in the presence of O2, 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% H2) to an A660of 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 A660of 0.6.
Hydrogenase activity was assayed with methylene blue and was
expressed as lmol reduced MBÆh)1Æ(mg protein))1 The values are
the means from at least three independent experiments.
Strains
Hydrogenase activity
BSE16 (pSE60) 16 ± 13 52 ± 12 18 ± 3 65 ± 16
Trang 6NAD-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 O2 attack by steric
hin-drance [32,33] The Ni-bound CN–seems to be
respon-sible for the O2 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 O2, but the enzyme
was irreversibly inactivated if O2 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 O2-sensitive
phenotype, and it was postulated that this bulky
resi-due impaired the incorporation of the Ni-linked CN–,
thus conferring O2 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
H2-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 O2 resistance on HupUV, did not
pro-tect the protein against O2in 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 H2 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 O2
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 O2insensitivity
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 O2to 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 O2-resistant
hydrogenases, O2 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 O2 resistance of RHs by limited accessibility of the active site to O2 In the mutated protein, O2 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 H2 or a reductant [27] Our conclusions are strengthened by a recent paper from Friedrich’s group [40], which shows the O2 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, O2-resistant and mutated,
O2-sensitive HupUV proteins by biophysical methods may lead to the improved understanding of the mecha-nisms of O2 resistance⁄ sensitivity in [NiFe] hydrogen-ases in general RHs that are insensitive to O2and, as isolated, ready to function are potentially of great bio-technological interest, but their activity is low When the basis of their O2resistance is understood, it will be possible to design a hydrogenase that exhibits high activity together with O2insensitivity
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 30C under anaerobiosis in the light or under aerobiosis
in the dark with shaking, in MG medium (7 mm glutamate,
30 mm dl-malate) or MN medium (7 mm ammonium sul-fate, 30 mm dl-malate) [19] Escherichia coli strains were grown at 37C 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)1for 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
Trang 7Site-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
hupUVgenes, 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 His6tag for easy
purification of the HupUV complex
Purification of the His6-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 H2 uptake or H–D exchange H2 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 A565, or in cell extracts and purified proteins with 2 mm benzyl viologen (BV), at
A555[43] In native gels, hydrogenase activity was revealed
by incubating the gels under H2 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
30C 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 D2 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 D2, HD and H2 were monitored by scanning masses 4, 3 and 2,
Table 3 Bacterial strains and plasmids used in this study.
Strains
R capsulatus
Plasmids
containing hupUV
[12]
containing pUC18 polylinker
P Hu¨bner, unpublished observations
Trang 8respectively When required, the medium was made
aero-bic by the addition of H2O2 (5 lL 0.3% H2O2)
decom-posed by the addition of catalase (500 U) thus liberating
O2, 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
D2consumption and H2 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 30C 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 BlerKmrcartridge)
of the plasmid pFRK-I, leading to plasmid pOD12
pFRK-I contains a fructose-activated promoter, pfru, from
R capsulatus From pOD12, the HindIII–BamHI fragment
containing mutated hupUV genes downstream of pfru was
cloned into the broad host range plasmid pPHU231
diges-ted with the same enzymes The resulting plasmid, pOD15,
was introduced into the R capsulatus hupUV mutant
BSE16 by triparental conjugation [13]
Acknowledgements
We thank P M Vignais and M Satre for critical
read-ing of the manuscript We also thank P Carrier for
excellent technical assistance O.D was supported by a
two-year postdoctoral grant from the Commissariat a`
l’Energie Atomique (CEA) The work was supported
by research grants from the CEA, the Centre National
de la Recherche Scientifique(CNRS: ACI ‘Energie
Con-ception Durable’) and the Universite´ Joseph Fourier
(UJF) de Grenoble
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