From the Botanisches Institut der Universität Bonn, Karlrobert-Kreiten-Strasse 13, 53115 Bonn, Germany
Received for publication, September 15, 2000, and in revised form, November 20, 2000
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ABSTRACT |
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Hydrogen evolution is observed in the green alga
Scenedesmus obliquus after a phase of anaerobic adaptation.
In this study we report the biochemical and genetical characterization
of a new type of iron hydrogenase (HydA) in this photosynthetic
organism. The monomeric enzyme has a molecular mass of 44.5 kDa. The
complete hydA cDNA of 2609 base pairs comprises
an open reading frame encoding a polypeptide of 448 amino acids. The
protein contains a short transit peptide that routes the nucleus
encoded hydrogenase to the chloroplast. Antibodies raised against the
iron hydrogenase from Chlamydomonas reinhardtii react with
both the isolated and in Escherichia coli overexpressed
protein of S. obliquus as shown by Western blotting. By
analyzing 5 kilobases of the genomic DNA, the transcription initiation
site and five introns within hydA were revealed. Northern
experiments suggest that hydA transcription is induced
during anaerobic incubation. Alignments of S. obliquus HydA
with known iron hydrogenases and sequencing of the N terminus of the
purified protein confirm that HydA belongs to the class of iron
hydrogenases. The C terminus of the enzyme including the catalytic site
(H cluster) reveals a high degree of identity to iron hydrogenases.
However, the lack of additional Fe-S clusters in the N-terminal domain
indicates a novel pathway of electron transfer. Inhibitor experiments
show that the ferredoxin PetF functions as natural electron donor
linking the enzyme to the photosynthetic electron transport chain. PetF
probably binds to the hydrogenase through electrostatic interactions.
Many prokaryotes and several eukaryotes have an enzyme complex in
common catalyzing the reversible reduction of protons to molecular
hydrogen. The diverse group of hydrogenases can be divided into three
classes according to their metal composition in the active center (1).
The nickel-iron hydrogenases are widespread among all bacteria families
and have been well characterized during the last 30 years (2). The iron
sulfur proteins consist of one to four subunits and have an additional
nickel atom in the catalytic site (3, 4). In contrast, the iron
hydrogenases possess only [Fe-S] clusters and an iron cofactor with
an unique structure of six iron atoms (5, 6). The third class of
hydrogenases lacks the iron sulfur clusters as well as additional metal
atoms and was found only in methanogenic bacteria (7, 8).
Until now, iron hydrogenases have only been found in hydrogen-producing
anaerobic bacteria and protozoa (9-13). The enzymes allow fermentative
anaerobes to evolve H2 without exogenous electron acceptors
other than protons (14). They show a high specific activity that is
about 100 fold higher compared with the nickel-iron hydrogenases (15).
Furthermore, all iron hydrogenases are extremely sensitive to oxygen
and carbon monoxide. The structures of the iron hydrogenases from
Clostridium pasteurianum and Desulfovibrio desulfuricans have recently been investigated by x-ray
crystallography (16, 17). The proteins consist of one or two subunits
and have a remarkable iron cofactor (H cluster) in the catalytic site. The H cluster contains an unusual supercluster comprising a [4Fe4S] subcluster and a [2Fe] center, which are bridged together by a single
cysteinyl sulfur (18). A number of conserved amino acids forms a
hydrophobic pocket that shields the [2Fe] subcluster from the
solvent. In all known iron hydrogenases at least eight conserved cysteines were found at the N-terminal site of the protein that coordinate two further [4Fe-4S] clusters (F cluster). It is discussed that the F clusters are responsible for the electron transfer from the
surface of the protein to the active site (17, 19).
In green algae, Gaffron (20) discovered a hydrogen metabolism 60 years
ago. After anaerobic adaptation, he observed both H2 uptake
and hydrogen evolution dependent on the CO2 partial pressure (21, 22). After bubbling the cells with an inert gas like
argon, high rates of H2 production can be measured in the
light (23). Electrons are supplied either by photochemical water
splitting at photosystem II, which results in simultaneous production
of hydrogen and oxygen, or by metabolic oxidation of organic compounds
with release of CO2 (24-27). Light-dependent electron transport from organic substrate through the plastoquinone pool to the hydrogenase provides the cells with ATP under anaerobic conditions (28, 29).
From the unicellular green alga Chlamydomonas reinhardtii a
monomeric iron hydrogenase with high specific activity has been isolated (30, 31). In contrast, a nickel-iron hydrogenase was described
for another well examined green alga, Scenedesmus obliquus
(32, 33). The protein consists of two subunits of about 36 and 55 kDa
and might be located in the chloroplast.
To investigate whether hydrogenases of the iron-only type also occur in
green algae other than C. reinhardtii, we decided to look
for the gene of a hydrogenase in S. obliquus. Interestingly, we isolated the protein and the gene encoding a monomeric iron hydrogenase (HydA). Although the H cluster of the HydA protein of
S. obliquus is very conserved, the N-terminal site is
completely different compared with other iron hydrogenases. Further
cysteines are not present. These cysteine residues coordinate the
typical F clusters that are necessary for the electron pathway in other iron hydrogenases. We performed physiological measurements of the
hydrogen evolving activity in the present of chloroplast ferredoxin specific inhibitors as well as antibodies against this protein. The
results clearly indicated that ferredoxin transfers electrons to the
hydrogenase and links the enzyme to photosynthesis. The expression of
the hydA gene is regulated at the transcriptional level. The
mRNA is transcribed very rapidly during the process of anaerobic adaptation.
Algae Strains, Growth, and Anaerobic Conditions--
Wild-type
S. obliquus Kützing 276-6 was obtained originally
from the culture collection of algae at the University of
Göttingen. Cells were cultured photoheterotrophically (34) in
batch cultures at 25 °C under continuous irradiance of 150 µmol
photons × m Hydrogen Evolution Assay--
The in vitro
hydrogenase activity was measured by using a gas chromatograph from
Hewlett Packard (HP 5890, Series II) equipped with a thermal
conductivity detector and a molecular sieve column. Methylviologen
reduced by sodium dithionite was used as electron donor as described
before (30). 1 unit is defined as the amount of hydrogenase
evolving 1 µmol H2 × min
The in vivo activity in the presence of different inhibitors
of the photosynthetic electron flow was determined as described (30).
After anaerobic adaptation, cells were harvested, diluted in fresh Tris
acetate phosphate medium, and transferred to sealed tubes. Inhibitors
were added 1 h before H2 evolving activity was measured. Cells were broken by sonification. Thylakoid membranes and
photosynthetic electron transport chain remained intact as shown by
O2 polarography. Ferredoxin of C. reinhardtii
and S. obliquus was isolated according to the method of
Schmitter et al. (35).
Rapid Amplification of cDNA Ends-Polymerase Chain
Reaction--
RACE-PCR (36)1
was performed with the SMARTTMRACE cDNA Amplification
Kit (CLONTECH Laboratories, Palo Alto, CA)
according to the manufacturer's recommendations except for
modifications of the PCR and hybridization conditions. Starting
material consisted of 1 µg of mRNA from anaerobically adapted
cells. The reverse transcription reaction was carried out with a
Moloney murine leukemia virus reverse transcriptase in two separate
reaction tubes containing either the 5'- or the 3'-RACE-PCR specific
primer from the kit. The cDNA of each sample served as template for
the following PCR. For the 5'-RACE-PCR, Universal Primer Mix
(CLONTECH) and the antisense primer Sc7 were used. The
amplification of the 3'-cDNA end was performed with Universal
Primer Mix and the sense primer Sc6. To obtain more distinct PCR
signals, the PCR was repeated for both reactions with nested universal
primers and designed primers (inverse Sc6 and inverse Sc7,
respectively) using a dilution of the products of the first PCR as template.
Primer Extension--
RACE-PCR was also implemented to map the
transcription initiation site of the hydA mRNA (37). A
gene-specific primer (Sc17) was used to carry out the first strand
cDNA synthesis with the Supercript II reverse transcriptase (Life
Technologies, Inc.) and 200 ng of mRNA as template. PCR was
performed using either Sc12 or Sc27 and the SMARTTM
specific adapter primer Universal Primer Mix. Two different DNA fragments of 234 and 183 bp were amplified under standard PCR conditions. Both fragments were cloned into the pGem®T-Easy vector (Promega, Madison, WI) and sequenced using primers from the polylinker of the vector.
Genome Walking with Genomic DNA--
Applying the
CLONTECH Genome Walker Kit, genomic libraries from
S. obliquus were generated by digestion with different blunt end cutting endonucleases (NaeI, DraI,
PvuII, HincII, and EcoRV) and by
adapter ligation at the ends of the resulting DNA fragments. These
libraries were utilized as independent templates in five different PCR
reactions (38). Two gene-specific primers (Sc27 and Sc35) derived from
the hydA cDNA sequence of S. obliquus were used in combination with a kit adapter primer (AP1) in a first PCR
reaction. Subsequently, 1 µl of the first PCR served as a template in
a secondary PCR, applying two nested gene-specific primers (i-Sc10 and
Sc32) along with a nested kit adapter primer (AP2). The resulting
products were cloned into pGem®T-Easy and sequenced. Sequencing was
performed by the dideoxy chain termination method (39).
Purification of the Iron Hydrogenase--
40-liter cultures of
S. obliquus were grown heterotrophically. After
centrifugation (10 min at 5000 × g), the pellet was
resuspended in 200 ml of Tris acetate phosphate medium. The cells were
anaerobically adapted by flushing the solution with argon for 1 h
in the dark. All further purification steps were performed in an
anaerobic chamber (Coylab, Ann Arbor, MI). The cells were
disrupted in a 50 mM Tris/HCl buffer, pH 8.0, 10 mM sodium dithionite by vortexing 3 min with glass beads.
The further purification steps were made as described earlier for the
isolation of the iron hydrogenase of C. reinhardtii
(30). Automated Edman degradation of the N-terminal site of the protein
was performed with an Applied Biosystem model 477A sequencer with
online analyzer model 120 A.
RNA Blot Hybridization--
Total RNA of S. obliquus
was isolated according to the method described earlier (40). Equal
amounts (20 µg) were separated electrophoretically on 1.2% agarose
gels containing formaldehyde (41). The RNA was transferred onto nylon
membranes (Hybond+, Amersham Pharmacia Biotech) and
hybridized with RNA probes labeled with DIG-dUTP using the in
vitro transcription method. A 1.3-kilobase EcoRI
cDNA fragment was used to detect transcripts of the hydA gene, whereas a DIG-dUTP-labeled cDNA encoding constitutively expressed plastocyanin (42) was used as control. Hybridization reactions were carried out using protocols supplied by the manufacturer (Roche Molecular Biochemicals).
Sequence Analysis Software--
Nucleic acid and protein
sequences were analyzed with the programs Sci Ed Central (Scientific
Educational Software) and ClustalW (43). The Blast server (44) of the
National Center for Biotechnology Information (Bethseda, MD) was used
for data base searches.
Recombinant Expression in Escherichia coli--
The
hydA open reading frame was amplified by PCR using the
primer pair Sc29 and Sc30 containing flanking
NdeI-BamHI sites. The PCR product was cloned into
the pGem®T-Easy vector. After digestion with
NdeI-BamHI, the hydA gene was cloned
into the corresponding site of the pET9a expression vector (Promega)
producing pLF29.2. The insert of pLF29.2 was sequenced confirming that
the fragment contains the exact full coding region of the hydrogenase
without transit peptide. E. coli strain BL21(DE3)pLysS was
transformed with pLF29.2. Expression was induced with 1 mM
isopropyl-thio- Induction of Hydrogenase Activity and Purification of the Iron
Hydrogenase Protein--
Anaerobic adaptation is the most efficient
way to induce hydrogenase activity in S. obliquus. Bubbling
the alga culture in the dark with argon led to a dramatic increase
(10-fold) of hydrogenase activity during the first 2 h. We
purified the enzyme of S. obliquus to homogeneity by
successive column chromatography. Because the enzyme is irreversible
inactivated by lowest oxygen levels, all purification steps were
performed under strictly anaerobic conditions and in the presence of
reducing agents (dithionite). The purification scheme resulted in a
5200-fold purification of HydA with 5% recovery (data not shown). The
most powerful step for purifying the protein was a Q-Sepharose high
performance column chromatography with pH gradient elution. Gel
filtration chromatography of hydrogenase on a calibrated Superdex-75
column resulted in a single activity peak corresponding to a molecular
mass of 45 kDa. The monomeric structure of the enzyme could also be
shown on a SDS-polyacrylamide gel after Coomassie Blue staining (data
not shown). The N-terminal sequence of HydA was determined by Edman
degradation. The protein sequence
(AGPTAECDRPPAPAPKAXHWQ) is, except for two amino
acids, identical to the amino acid sequence deduced from the DNA data
(AGPTAECDCPPAPAPKAPHWQ). In the course of our
purification procedure, we never found a hint for a second hydrogenase
in S. obliquus because the hydrogenase activity was never
separated in several distinct fractions. Biochemical data show a high
similarity of HydA to the iron hydrogenase from C. reinhardtii (Table I). The enzymes
have a high temperature optimum of about 60 °C, are strongly
inhibited by O2 and CO, and catalyze the H2
evolution with a typical high specific activity. Experiments with
inhibitors of translation on ribosomes (data not shown) and analysis of
the gene structure (see below) show that HydA from S. obliquus is translated in the cytoplasm and then transported into
the chloroplast.
Ferredoxin Is the Natural Electron Donor of the Iron
Hydrogenase--
Hydrogenase activity was determined in intact and
broken cells after anaerobic adaptation. The integrity of the
photosynthetic electron transport in the sonified cell preparation was
demonstrated by the rate of oxygen evolution (154 µmol
O2/mg Chl × h). This rate corresponds to 85%
of the oxygen evolution measured with intact Scenedesmus cells.
In S. obliquus, the hydrogen evolution is linked to the
photosynthetic electron transport chain through PSI. As shown in
Table II, the cells were still able to
photoproduce hydrogen when electron flow of PSII was blocked by
3-(3,4-dichlorophenyl)-1,1-dimethylurea. In contrast, addition of
2,5-dibromo-3-methyl-6-isopropyle-p-benzochinone resulted in
inhibition of the H2 production, thus giving evidence of
the involvement of PSI in the supply of electrons to hydrogenase. The
electron transport from PSI to ferredoxin was inhibited using the
artificial electron acceptor 2,6-dichlorophenolindophenol. In this
reaction, 2,6-dichlorophenolindophenol is reduced instead of
ferredoxin, and the electron transfer to hydrogenase is
interrupted.
Hydrogenase activity was dramatically reduced (up to 30-fold) by the
ferredoxin antagonist sulfo-disalicylidinepropandiamine (Table II).
Similar results were achieved with
The electron transfer properties of different plant-type ferredoxins
were measured in vitro with dithionite as reducing reagent. The ferredoxin proteins of spinach, C. reinhardtii, and
S. obliquus were comparable regarding their capability to
reduce purified S. obliquus hydrogenase. In this assay, we
obtained H2-evolving activities of 420, 390, and 350 units/mg protein with S. obliquus, C. reinhardtii, and spinach ferredoxin, respectively. No hydrogen production could be measured with other possible electron donors like
cytochrome and NADPH. In D. desulfuricans the iron
hydrogenase was reported to catalyze both hydrogen production and
uptake with low potential multiheme cytochromes like cytochrome
c3 (17).
Molecular Characterization of hydA Encoding an Iron
Hydrogenase--
To isolate the gene encoding a iron hydrogenase in
S. obliquus, we isolated poly(A)+ RNA from cell
cultures after 1 h of anaerobic adaptation. Isolated RNA was
transcribed and amplified by reverse transcription-PCR using
oligonucleotides derived from conserved regions within the C. reinhardtii hydA gene.2
The complete cDNA clone of 2609 bp was obtained by 5'- and 3'-RACE PCR. It contains an open reading frame of 1344 bp encoding a
polypeptide of 448 amino acids (Fig. 1)
followed by an extensive 3'-untranslated region of about 1100 bp. The
coding region of S. obliquus hydA exhibits features common
to other green algae such as high GC content (64.2%) and a
characteristic putative polyadenylation signal, TGTAA, 15 bp upstream
of the poly(A)+ sequence (45).
In an effort to examine the exon-intron structure and the promoter
region of the hydA gene, about 5 kilobases of the genomic DNA from S. obliquus were sequenced. The gene comprises five
introns with a total size of 1310 bp (Fig. 1) whose 5' and 3' ends
contain typical plant splice donor and acceptor sites that follow the GT/AG rule.
A genomic Southern blot was probed with a 750-bp PCR fragment to
determine the copy number of the hydA gene (Fig.
2). Single bands were observed in lanes
with samples digested with HincII, EcoRV, and
NdeI and a double band in the lane containing genomic DNA
digested with SacI. The band migration positions matched the sizes predicted from the sequence of the hydA gene,
indicating that HydA is encoded by a single copy gene (Fig. 2). The
same hybridization pattern was observed even under low stringency
conditions (hybridization temperature 50 °C; data not shown). The
transcription start position was determined by primer extension using
RACE-PCR and was found 139 bp upstream of the ATG start codon. We
designed several primers within 100 bp of the 5' end of the known
hydA cDNA to confirm the accuracy of the transcription
initiation site. All of the sequenced PCR clones had the same 5' ends
at position +1. As described for other green algae genes, a highly
conserved TATA box element upstream of the transcription starting point is absent (46). However, the TACATAT motive at position HydA Is a Novel Type of Iron Hydrogenase--
The polypeptide
derived from the cDNA sequence has a length of 448 amino acids and
a predicted molecular mass of 48.5 kDa (44.5 kDa without transit
peptide, respectively); consequently, HydA is the smallest hydrogenase
protein known so far. The N terminus of HydA is basic and contains
numerous hydroxylated amino acids and an Val-Xaa-Ala motive at position
35, a characteristic feature of chloroplast transit peptides (47,
48).
The processed HydA protein is compared with four bacterial and two
eukaryotic iron hydrogenases as shown in Fig.
3. The homology in the C-terminal region
of all proteins is quite striking. For example, the S. obliquus HydA protein shows 44% identity and 57% similarity to
the C. pasteurianum iron hydrogenase (9). The H cluster in
S. obliquus might be coordinated by four cysteine residues
at positions 120, 175, 335, and 340. Other strictly conserved amino
acid structures like FTSCCPGW (343), TGGVMEAALR (474), and MACPGGCXXGGGQP () probably define a pocket
surrounding the active center as shown by the structural data of
C. pasteurianum and D. desulfuricans (16, 17). On
the other hand, the N-terminal region is completely different from all
other iron hydrogenases. The protein sequences of the other enzymes
comprise at least two [4Fe-4S] ferredoxin-like domains (called F
cluster) that are necessary for the electron transport from the
electron donor to the catalytic center. The iron hydrogenases of
C. pasteurianum, Thermotoga maritima, and Nyctotherus
ovalis (9, 12, 11) contain an extra [4Fe-4S] cluster and one
[2Fe-2S] center. This N-terminal domain with the F cluster or other
[Fe-S] centers is completely lacking in HydA of S. obliquus. This indicates that there is a direct electron transport
pathway from the exogenous donor to the H cluster.
To verify that the isolated cDNA encodes a iron hydrogenase, the
hydA clone was expressed in the heterologous system
E. coli. One band of recombinant HydA protein was
observed on SDS-PAGE at ~44 kDa, in agreement with the molecular mass
of the polypeptide predicted from the cDNA sequence. Antibodies
raised against the HydA protein of C. reinhardtii, which
cross-react with other iron hydrogenases but not with nickel-iron
hydrogenases (data not shown), were applied in Western blot analysis.
One distinct signal with the overexpressed HydA protein of S. obliquus was obtained (Fig. 4).
The lysate of induced E. coli cells exhibited no hydrogenase
activity. This result corresponds to observations by Voordouw et
al. (50) and Stokkermans et al. (51), who also detected no H2 production of recombinant iron hydrogenases in
E. coli cells. The reason for that might be that the
bacterial cells do not have the ability to assemble the special H
cluster of iron hydrogenases.
Rapid Induction of hydA mRNA during Anaerobic
Adaptation--
The regulation of the hydA gene expression
was examined by Northern blot analysis and reverse transcription-PCR.
Aerobically grown cells of S. obliquus did not show a
hydrogenase activity (Fig.
5A). Total RNA and also
mRNA were isolated from cells that were induced by argon bubbling
for 0, 1, and 4 h. Northern blot analysis and reverse
transcription-PCR demonstrated that the hydA gene is
expressed after anaerobic adaptation. There is a very weak signal
without adaptation (t = 0), but strong signals of the
transcript could be detected after anaerobic induction (Fig. 5,
B and C). The full length of the hydA
cDNA clone was confirmed by the transcript signal (2.6 kilobases)
on the Northern blot.
In green algae, the occurrence of a hydrogen metabolism induced by
anaerobic conditions is well established. Despite the great interest in
hydrogen evolution for practical applications ("biophotolysis"), the hydrogenase genes from green algae have not yet been isolated. The
hydA gene and the isolated HydA protein of S. obliquus that we present in this work belong to the class of iron hydrogenases.
Iron hydrogenases have been isolated only from certain anaerobic
bacteria and some anaerobic eukaryotes as well as from the anaerobically adapted green alga C. reinhardtii (30). The
enzymes are found to exist in monomeric (9, 13, 53, 54), dimeric (17),
and multimeric (12) forms; however, in eukaryotes only monomeric
proteins have been isolated (10, 11).
The HydA protein of S. obliquus is synthesized in the
cytoplasm. The first 35 residues (Met1-Ala35)
of the amino acid sequence derived from the cDNA sequence are supposed to function as a short transit peptide that routes the nuclear
encoded protein to the chloroplast. Several positively charged amino
acids that describe a typical feature for algal transit peptides (47)
are found in HydA. The three terminal residues of the signal sequence,
Val-Xaa-Ala, constitute the consensus sequence for stromal peptidases
(48).
The hydrogenase of S. obliquus represents a novel type of
iron hydrogenase. The monomeric enzyme of 448 amino acids and a calculated molecular mass of 44.5 kDa for the processed protein is the
smallest iron hydrogenase isolated so far. The protein sequence
consists of an unusual N-terminal domain and a large C-terminal domain
containing the catalytic site. The structurally important C terminus of
the S. obliquus HydA sequence is very similar to that of
other iron hydrogenases. Four cysteine residues at positions 120, 175, 336, and 340 coordinate the special [6Fe] cluster (H cluster) of the
active site (Fig. 6). A number of
additional residues define the environment of the catalytic center.
Peters et al. (16) postulated 12 amino acids in C. pasteurianum to form a hydrophobic pocket around the cofactor. Ten
residues are strictly conserved, while two amino acids vary within the
iron hydrogenase family (Ser232 and Ile268 in
C. pasteurianum, Ala119 and Thr155
in T. vaginalis, and Ala44 and
Thr80 in S. obliquus). A small insertion of 16 amino acids is noted in S. obliquus, but this addition
occurs in an external loop of the protein and probably has no special
function (Fig. 6).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 s
1. For anaerobic
adaptation, 4-liter cultures were bubbled vigorously with air
supplemented with 5% CO2. After harvesting the cells in
the mid exponential stage of growth, the pellet was resuspended in
fresh Tris acetate phosphate medium. The algae were anaerobically adapted by flushing the culture with argon in the dark.
1 at 25 °C.
-D-galactoside at an
A600 of 0.3. Pelleted cells were resuspended in
lysis buffer (100 mM Tris/HCl, 4 mM EDTA, 16%
glycine, 2% SDS, 2% mercaptoethanol, 0.05% bromphenol blue, 8 M urea). After heating, the protein extract was separated
by 10% SDS-PAGE and blotted onto a polyvinylidene difluoride membrane.
Western blot analyses were performed using antiserum against the
iron hydrogenase of C. reinhardtii at 1:1000 dilution as
described earlier (31).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Biochemical data comparison of purified iron hydrogenases from C. reinhardtii and S. obliquus
Effects of different photosynthetical inhibitors on hydrogenase
activity
-PetF-antibody was raised against
spinach ferredoxin. DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea;
DBMIB, 2,5-dibromo-3-methyl-6-isopropyle-p-benzochinone;
Sulfo-DSPD, sulfo-disalicylidinepropanediamin; DCPIP,
2,6-dichlorophenolindophenol.
-PetF-antibodies that
specifically recognize the ferredoxin protein. In both cases, the
hydrogenase enzyme can not evolve hydrogen, thus demonstrating the role
of ferredoxin as the obligatory electron donor for the hydrogenase reaction.
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Fig. 1.
Schematic representation of S. obliquus
hydA genomic and cDNA structures. A, the
coding region of the hydA cDNA is illustrated as a
large arrow with the transit peptide shown in
black. The untranslated 5' and 3' sequences are marked as
lines. The arrows below indicate the sequencing
strategy; each arrow represents an independent sequence
determination. TSP, transcription start point;
ATG, start codon. B, the mosaic structure of
hydA is indicated by gray (exons) and white
boxes (introns). The S2 probe and different restriction enzymes
that were used in the Southern blot experiments are mentioned.
25 in a
GC-rich region shows similarities to other TATA motives in C. reinhardtii and therefore might be involved in gene
expression.
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Fig. 2.
The hydA gene is located in
the genome of S. obliquus as a single copy gene.
Southern analysis was carried out by digesting genomic DNA of S. obliquus with four different restriction endonucleases
(SacI, HincII, EcoRV, and
NdeI). 10 µg of DNA was loaded per lane. The S2 DNA-Probe
(750 bp) was used for the hybridization as indicated in Fig. 1.
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Fig. 3.
Comparison of the deduced HydA protein
sequence with other iron hydrogenases. The protein alignment was
done by using the Vector NTI program (InforMax). White
letters with black background indicate amino acids
identical to the HydA protein. Black letters with gray
background indicate conserved changes of the amino acids. S. o., S. obliquus (this work); M. e., Megasphaera
elsdenii (13); D. d., D. desulfuricans (17);
T. v., Trichomonas vaginalis (10); C. p., C. pasteurianum (9); T. m., T. maritima (12); N. o.,
N. ovalis (11).
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Fig. 4.
Recombinant expressed HydA reacts with
antibodies raised against C. reinhardtii iron
hydrogenase. The hydA gene coding region corresponding
to amino acid 36 to residue 448 was cloned
NdeI-BamHI into pET9a. HydA protein was expressed
upon induction with IPTG. Lane 1, recombinant expressed HydA
protein from S. obliquus; lane 2,
recombinant expressed iron hydrogenase from C. reinhardtii; lane 3, purified hydrogenase from C. reinhardtii; lane 4, total proteins from induced
E. coli cells without plasmid. A, SDS-PAGE.
Lane M, molecular mass marker (Bio-Rad) indicating relative
molecular masses in kDa. SDS-PAGE stained with Coomassie Blue is shown.
B, Western blot probed with HydA antibody. The recombinant
proteins of lanes 1 and 2 from A were diluted
1:10.
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Fig. 5.
Induction of the hydrogenase activity and
differential expression of the hydA gene during
anaerobic adaptation. A, S. obliquus
cells were anaerobically adapted by flushing the culture with argon in
the dark. After removing cell samples at the indicated times, the algae
were broken by Triton X-100 treatment. The in vitro
hydrogenase activity was measured as described under "Experimental
Procedures." B, Northern hybridization was performed with
the hydA-specific probe. Adapted cells were harvested at 0, 1, and 4 h, and the RNA was isolated. 20 µg of total RNA was
loaded per lane. C, the same RNA was hybridized with a
constitutive expressed gene (plastocyanin) as control.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Schematic alignment of the conserved cysteine
residues and other important amino acids of the H cluster. The
protein is illustrated as a large gray arrow. Small
arrows indicate parallelograms which demonstrate conserved amino
acids in the protein. Cysteines participating at the coordination of
the [Fe-S] clusters are gray, whereas identical amino
acids are black. An insertion of 16 amino acids in the
S. obliquus protein is illustrated as a spotted
bar. FS4, [4Fe-4S] cluster; FS2,
[2Fe-2S] cluster.
Until now, all iron hydrogenases possessed a ferredoxin-like domain in the N terminus coordinating two [4Fe4S] clusters (FS4A and FS4B; Refs. 10 and 13 and Fig. 6). The iron sulfur clusters facilitate the transfer of electrons between external electron donors or acceptors and the H cluster. The N terminus of the S. obliquus protein is strongly reduced compared with other iron hydrogenases, and no conserved cysteines are found. Therefore, we postulate that all accessory Fe-S clusters (FS2, FS4A, FS4B, and FS4C) are missing. No hints for a second subunit have been observed during purification of the protein.
In contrast to earlier observations in S. obliquus (32), we could neither detect the postulated two subunits of a potential nickel-iron hydrogenase nor find a nickel dependence related to the hydrogenase activity. Francis reported about two forms of hydrogenases in S. obliquus (52), but although we used the same alga strain and identical adaptation conditions, we were not able to detect a second hydrogenase activity during the purification steps.
Physiological studies have shown that the hydrogen evolution is coupled to the light reaction of the photosynthesis (24-26). In contrast to earlier observations in S. obliquus (25, 26), we measured PSII independent H2 production that is not influenced by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The electrons required for H2 evolution come from redox equivalents of the fermentative metabolism and are supplied into the photosynthetic electron transport chain via the plastochinone pool.
For the first time we demonstrate that the ferredoxin PetF functions as the in vivo electron donor of the iron hydrogenase from S. obliquus. Hydrogenase activity can be specifically blocked by addition of the ferredoxin antagonist sulfo-disalicylidinepropandiamine (55) and antibodies raised against the PetF protein. In vitro, a hydrogen evolution by HydA was only measured with plant-type [2Fe-2S] ferredoxins like PetF of S. obliquus, C. reinhardtii, and spinach as electron mediators. Bacterial iron hydrogenases are known to be reduced by [4Fe-4S] ferredoxins and do not accept electrons from plant-type proteins (56).
The analysis of the three-dimensional structure of the iron hydrogenase
from C. pasteurianum (CpI) gave evidence that the interaction with external electron donors might occur at the accessory [Fe-S] clusters in the N-terminal domain (14). Based on the x-ray
structure of CpI, we modeled the iron hydrogenase of S. obliquus (57). As shown in Fig. 7, a
region of positive surface potential is observed within HydA based on a
local concentration of basic residues. In contrast to the docking
position of ferredoxin in CpI, these charged amino acids in the
S. obliquus iron hydrogenase are located within the
C-terminal domain, forming a niche for electron donor fixation.
|
The known algal ferredoxin proteins exhibit high degrees of sequence identity (over 85%), and the charged amino acids are strictly conserved. The petF sequence of S. obliquus is unknown, but very recently the x-ray model of the ferredoxin from another Scenedesmus species (Scenedesmus vacuolatus; Ref. 58) was published. The structure revealed negatively charged amino acids like aspartate and glutamate near the [2Fe-2S] cluster. The [Fe-S] center and the H cluster of the hydrogenase probably come into close proximity through electrostatic interactions. This geometry is consistent with efficient electron transfer among these prosthetic groups.
As already shown in various studies, a correlation exists between the
duration of time of the anaerobic adaptation and the increase of
hydrogen production (30, 32). Reverse transcription-PCR and Northern
blot analyses with mRNA of aerobic and anaerobically adapted cells
from S. obliquus showed an increased level of
hydA transcript after 1 h of induction.
Correspondingly, hydrogen evolution was only measured after a short
time of anaerobic adaptation. These results suggest that the expression
of the hydA gene is regulated at the transcriptional level.
The small amount of transcript that was detected at t = 0 may be due to transcript synthesis induced by microanaerobic
conditions during the RNA isolation procedure. Alternatively, a low
level of hydA transcript might be constitutively present in
the cell and is only drastically increased after anaerobic adaptation.
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ACKNOWLEDGEMENTS |
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We thank Dr. W. Hachtel for critical reading and A. Kaminski for providing us with sequence information before publication.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grant Ha2555/1-1.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This paper is dedicated to Jan Vlcek who found evidence for an iron hydrogenase in S. obliquus for the first time. Unfortunately, he died 4 years ago in a tragic car accident.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ271546.
To whom correspondence should be addressed: E-mail:
t.happe@ uni-bonn.de.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M008470200
2 T. Happe, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction; bp, base pair(s); PAGE, polyacrylamide gel electrophoresis; PSI, photosystem I.
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