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J. Biol. Chem., Vol. 275, Issue 28, 21114-21120, July 14, 2000
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andFrom the Lehrstuhl für Mikrobiologie der Universität München, Maria-Ward-Strasse 1a, 80638 München, Germany
Received for publication, February 7, 2000, and in revised form, April 26, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The formation of a complex between the specific
chaperone-type protein HypC and the precursor form of the large subunit
HycE in the maturation pathway of hydrogenase 3 from Escherichia
coli has been studied by targeted replacement of amino acids in
both proteins. HypC and its homologs contain the motif
MC(L/I/V)(G/A)(L/I/V)P at the amino terminus, from which the methionine
residue is post-translationally removed. The exchange of the cysteine
residue led to complete loss of the ability to interact with the
precursor form of HycE, but replacement of the proline residue had no
effect. Site-directed replacement of the conserved cysteine residues in
HycE involved in nickel binding was also performed. Exchange of
Cys241 resulted in the inability of the HycE variant to
interact with HypC and to incorporate nickel. The variants of HycE in
which Cys244 and Cys531 were replaced by
alanine residues were unable to incorporate nickel, although the
mutated proteins could interact with HypC. Intriguingly, the precursor
of HycE in which the Cys534 residue was exchanged could
form the complex with HypC, could incorporate nickel, and was
C-terminally processed, but it delivered an inactive enzyme. Our
findings are in favor of a model in which binding of HypC masks
Cys241; Cys244 and Cys531 bind the
iron and nickel moieties, respectively; and C534 closes the bridge
between the two metals after C-terminal processing has taken place.
Despite the growing knowledge about the structures and catalytic
or other functional roles for metal centers in many metalloproteins, information as to how metal centers are biosynthetically assembled is
scarce. Recent studies on several systems (1-5) revealed the involvement of a surprisingly complex cascade of reactions in the
maturation process in which numerous auxiliary proteins are implicated.
One class of them was found to participate in metal center assembly by
stabilizing a partially unfolded apoprotein conformation that is
competent for the specific binding of a metal ion or a metal cofactor
(1-5). This mechanism is reminiscent of the role postulated for
molecular chaperones, which facilitate the correct folding of nascent
polypeptide chains in vivo but are not components of the
final structures. Accordingly, these particular auxiliary proteins were
designated specific chaperone-type proteins (1-5), their mode of
action and the basis of their specificity being not yet fully understood.
One type of enzymes whose metal center formation has been studied in
some detail are hydrogenases. They play a central role in the
metabolism of many microorganisms by catalyzing the production or
consumption of molecular hydrogen according to the reaction, H2 Here we report on the interaction between the precursor form (pre-HycE)
of the large subunit of the hydrogenase 3 from E. coli and
the chaperone-type protein HypC. We identified cysteines both in HypC
and pre-HycE as being essential for the interaction. A model is
presented for the role of the cysteine residues of pre-HycE in the
metal incorporation process.
Bacterial Strains--
E. coli DH5 Site-directed Mutagenesis--
Site-directed mutagenesis was
performed by recombinant polymerase chain reaction either on the pACE1
plasmid (18) to yield hycE mutants or on the pJA1021 plasmid
(17) to yield hypC mutants. The Expand High Fidelity
polymerase chain reaction system (Roche Molecular Biochemicals) was
used. This approach is based on the polymerase chain reaction
amplification of an entire plasmid DNA by mutagenic primers divergently
oriented but overlapping in their 5'-ends (19-21). The resulting
linear DNA molecules are then transformed into the
recA Growth Conditions and Preparation of Extracts--
Strains were
cultivated anaerobically in TGYEP medium containing 30 mM
sodium formate as described previously (5) and supplemented with a 1 µM concentration each of sodium molybdate and sodium selenite and 5 µM nickel chloride. When required,
chloramphenicol was added at a final concentration of 30 µg/ml.
Following the addition of a 1% inoculum, cultures were grown at
37 °C until they reached an optical density of 1.0 at 600 nm
(A600). Cells were harvested by centrifugation
and washed in 50 mM Tris-HCl, pH 7.4. Cytoplasmic fractions
obtained by ultracentrifugation at 100,000 × g (named
S100 extracts), and crude extracts were prepared as described
previously (5).
Enzyme Assays--
Total hydrogenase activity of crude extracts
was assayed by H2-dependent reduction of benzyl
viologen as described by Ballantine and Boxer (22) and expressed in
µmol of H2/min/mg of total protein.
Polyacrylamide Gel Electrophoresis and Western
Blotting--
Proteins separated on a 10% SDS polyacrylamide gel by
using the discontinuous buffer system of Laemmli (23) were transferred onto nitrocellulose membranes and blotted with anti-HycE antibody (1:1000) or anti-HypC antibody (1:500). For antibody detection, the
protein A-horseradish peroxidase conjugate (Bio-Rad, Munich, Germany)
and the Chemiluminescent Lumilight kit from Roche Molecular Biochemicals were used. Electrophoresis on nondenaturing gels was
conducted as described previously (5), employing 40 µg of total
protein from S100 extracts.
Nickel Incorporation Experiments--
Strains were cultivated at
37 °C in 250 ml of TGYEP medium supplemented with 30 mM
formate, 1 µM sodium molybdate, 1 µM sodium selenite, and 150 nM 63Ni (specific
radioactivity, 190 µCi/µmol). At an A600 of
1.0-1.2, the cells were harvested by centrifugation, washed, and
resuspended in 50 mM Tris-HCl, pH 7.4, containing a 30 µg/ml concentration each of phenylmethylsulfonyl fluoride and DNase
I. S100 extracts were then prepared and applied onto a 10%
nondenaturing gel. After electrophoresis, the gel was dried and exposed
for autoradiography for 1 week.
Covalent Modification of Protein Thiols and Nondenaturing
Polyacrylamide Gel Electrophoresis
(ND-PAGE)1--
40 µg of
total protein from S100 extracts were incubated for 15 min with or
without 15 mM dithiothreitol followed by the addition of
one-fourth volume of 0.5 M iodoacetamide or potassium
iodoacetate in 1 M Tris-HCl, pH 8.7. After 5 min or 30 min
at room temperature, the samples were mixed with the sample buffer and
analyzed by electrophoresis on a 10% nondenaturing polyacrylamide gel.
Identification of Key Residues in HypC for the Maturation of the
Hydrogenase 3--
Among the auxiliary proteins required for
hydrogenase maturation is a family of small proteins from 80 to 110 residues in size with an acidic character (pI ~4-5). They are
products of hypC, hybG, or hupF genes.
It was shown that HypC from E. coli is able to form a stable
complex with the immature form of the large subunit of the hydrogenase
3, HycE (5). A comparison of the amino acid-derived sequence of HypC
with those of homologs from other organisms is given in Fig.
1. Noteworthy is the presence of a
conserved motif at the amino-terminal part of the protein, namely
MC(L/I/V)(G/A)(L/I/V)P. Amino-terminal sequencing of the purified HypC
protein indicated that the methionine residue is post-translationally
removed (data not shown), yielding an N terminus with a reactive
thiolate. To examine its possible role in the maturation process, this
cysteine residue (Cys2) and Pro6 from HypC were
chosen as candidates for site-directed mutagenesis and were replaced
individually by either neutral (alanine and serine) or charged
(arginine) amino acids and by alanine and threonine, respectively. The
resulting pJA1021 derivatives carrying the mutated genes were used to
transform strain DHP-C, which lacks the chromosomal hypC
gene.
The transformants were grown under fermentative conditions, and the
cells were analyzed for H2-dependent benzyl
viologen reduction (Table II) and for
processing of the large subunit HycE (Fig. 2). Transformants carrying plasmids in
which Cys2 of HypC was replaced were devoid of hydrogenase
3 activity, supporting the idea that the maturation of the enzyme may
be blocked. In contrast, replacement of Pro6 had no effect.
To ascertain that the cellular level of the mutant proteins was not
changed, the transformants were analyzed by immunoblotting. It was
found that under fermentative conditions they produced the same amount
of HypC as present in the strain transformed with the plasmid carrying
the wild-type hypC gene (data not shown). Fig. 2 displays
the immunoblotting analysis of the maturation state of HycE in the
transformants carrying the different hypC alleles. HycE was
present in the parental strain MC4100 in the mature form (Fig. 2,
lane 1) and in the DHP-C strain in the precursor form (Fig. 2, lane 3). DHP-C transformants with
the pJA1021 plasmid (Fig. 2, lane 4) or that
coding for the Pro6 variant (data not shown) exhibited a
partial restoration of the maturation of HycE, confirming that these
substitutions did not abolish the HypC function. Laser densitometry
analysis indicated that the processing level was approximately 30%,
which agrees with previous findings (17) that complementation of a
DHP-C strain by the respective gene in trans restores
maturation of hydrogenase 3 only to a limited extent. The lack of full
complementation has been interpreted previously by the assumption that
maturation follows a channeled pathway that requires the stoichiometric
amount and activity of the components involved (17, 24-26). Any effect of overexpression of the hypC gene in trans on
the maturation process can be ruled out (Table II). In agreement with
the inability of the Cys2 mutant proteins to support the
generation of hydrogenase activity, HycE is not processed in these
strains (Fig. 2, lanes 5-7).
To gain further insight into the impairment of the HypC function, the
ability of the HypC variants to form a complex with the precursor form
of HycE during the maturation process was examined. A ND-PAGE system
was employed to study in vivo complex formation between HypC
and pre-HycE (5). S100 extracts from strains transformed with the
different plasmids were separated by ND-PAGE, and Western blot
experiments were performed using antisera directed against HypC. The
results show that the Cys2 mutants were no longer able to
form a complex with the immature form of HycE (Fig.
3). They migrate in the position of free
HypC with the interesting exception of the C2S variant. There is no obvious explanation for its retarded migration, but it could be the
consequence of some modification at the hydroxyl or at the amino group
of the amino-terminal serine. Again, the variant in which
Pro6 was replaced showed the same pattern as the wild-type
protein (data not shown).
Site-directed Mutagenesis of the Cysteine Residues Involved in the
Coordination of the [NiFe] Center--
The drastic effect of the
cysteine substitutions in HypC raised the question of the nature of its
interaction with pre-HycE. In view of the specificity of the
interaction and of the deleterious effect of the C2 replacement in
HypC, a plausible assumption was that one of the conserved cysteine
residues in HycE may be also involved. Hence, each of the cysteine
residues present in the two nickel-binding motifs of HycE were
substituted by an alanine residue via site-directed mutagenesis, which
led to genes coding for the C241A, C244A, C531A, and C534A mutant HycE
proteins. Under fermentative conditions, none of the mutants exhibited
hydrogenase 3 activity (Table II). These observations were corroborated
by the results of the immunoblotting analysis of HycE. As already noted
previously for other auxiliary proteins involved in hydrogenase maturation (17, 18, 24, 26), the deletion of the chromosomal hycE gene cannot be fully complemented by expression of
hycE in trans (Fig.
4, lane 3),
possibly because of some imbalance in stoichiometry between pre-HycE
and the components of the maturation machinery. Despite a low
processing level, the expression of hycE in trans
yielded hydrogenase 3 activity nearly identical to that of the parental
strain (Table II). With the striking exception of the C534A mutant,
none of the other mutant proteins were subjected to HycI-mediated
proteolysis (Fig. 4). Intriguingly, therefore, replacement of
Cys534 by alanine allows C-terminal processing but prevents
development of hydrogenase activity (Table II).
We then investigated whether the mutated HycE polypeptides, notably the
C534A variant, could incorporate nickel. To this end, cultures were
grown in presence of 63Ni, S100 extracts were separated by
ND-PAGE, and 63Ni-labeled proteins were identified by
autoradiography (Fig. 5) and Western blot
analysis (Fig. 6). In the case of the
parental strain MC4100 (Fig. 5, lane 1,
band 1), a major 63Ni signal
comigrated with material immunoreacting with antiserum directed against
HycE. It corresponds to the mature form of HycE as displayed by ND-PAGE
(Fig. 6A, lane 1). In addition, a
slower migrating 63Ni-labeled protein band also present in
the HD705 extract (which is devoid of the hycE gene) can be
seen (Fig. 5, lane 2). It may represent the large
subunit of either hydrogenase 1 or 2. In the case of the HD709 extract,
two major pre-HycE conformers produced in absence of the HycI protease
and identified by Western blot analysis (Fig. 6A,
lane 3) contained 63Ni (Fig. 5,
lane 3, bands 2 and
3), one of them being associated with HypC (band
2) (see Fig. 6). Finally, from the four HycE variants, only
that carrying the C534A replacement gave a 63Ni-labeled
protein band that immunoreacted with anti-HycE antiserum and was
equivalent to the signal obtained with the transformant carrying the
plasmid with the wild-type hycE gene (Fig. 5,
lanes 5 and 9, band
1). These results support the notion that nickel incorporation in the C534A variant is not accompanied by the generation of hydrogenase 3 activity.
In order to check if any of the cysteine substitutions in HycE impaired
the ability for complex formation with HypC during the maturation
process, each of the transformants was analyzed with the procedure
described previously (5). As expected, the C534A protein that underwent
HycI-mediated processing was able to specifically interact with HypC
(Fig. 6B, lane 7). The same result was
obtained for the transformants possessing the HycE variants C244A and
C531A. On the other hand, the C241A variant was unable to interact with
HypC (Fig. 6B). It was previously pointed out (5) that only
a certain conformer of pre-HycE is competent to bind HypC (Fig.
6A, lane 3, band
2), whereas the majority of the immature polypeptide cannot
participate in this interaction (Fig. 6A, lane
3, band 3), at least under the
experimental conditions employed. Indeed, the absence of the
characteristic fastest migrating immunoreactive form of HycE in the
C241A mutant (Fig. 6A, lane 4,
band 2) is paralleled by the absence of a
HypC-pre-HycE complex (Fig. 6B, lane
4).
Effect of Reducing Agents and Alkylating Agents on the Stability of
the HypC-Pre-HycE Complex--
The results described thus far had
indicated that cysteine residues both in HypC and HycE are essential
for the complex formation between the two proteins. This prompted the
analysis of whether the formation of a disulfide bridge could be
responsible for the protein-protein interaction. For this purpose, S100
extracts of strain HD709, which contain large amount of the complex
(Fig. 6B, lane 3), were analyzed under
both reducing and nonreducing conditions by SDS-PAGE followed by
immunoblotting analysis using antiserum directed against HycE. There
was no alteration of the migration of the HycE polypeptide under
nonreducing conditions (Fig. 7,
lanes 2 and 5). As a control, extracts
from strain DHP-C (
To further characterize the interaction, HD709 extracts were treated
with different alkylating or reducing reagents and then separated by
ND-PAGE. Although some dissociation occurred as indicated by the
appearance of free HypC, the presence of 10 or 25 mM
dithiothreitol was not sufficient to fully resolve the complex (data
not shown). In the absence of reducing agents, the complex is extremely
stable, since no dissociation is detectable after 2 h of incubation.
The amenability of the cysteine residues within the complex to
alkylation was then determined by their reaction with iodoacetic acid
or iodoacetamide. Thiols potentially masked in disulfide bonds under
these native conditions were reduced by preincubation with
dithiothreitol, and the electrophoretic behavior of HypC associated or
not with pre-HycE was examined by ND-PAGE. The reaction of such S100
extracts from strain HD709 treated with each of the alkylating agents
either under reducing or nonreducing conditions resulted in the
complete dissociation of the complex after only 5 min of incubation
(Fig. 8). Any accessible thiolate group
not involved in disulfide bond formation should react and subsequently carry one negative charge after modification with iodoacetic acid. The
different electrophoretic mobility of the liberated HypC revealed that
alkylation of the protein had occurred.
A key role in the maturation process of [NiFe]-hydrogenases has
been demonstrated recently for the chaperone-type protein HypC and its
interaction with the HycE apoprotein of the E. coli hydrogenase 3 (5). This complex has been identified in each of the
mutants with a lesion in hyp genes, supporting the idea that
its formation may constitute an early step in the maturation process
(5).
In the present work, it was shown that cysteine residues both from HypC
and the precursor form from the large subunit are crucial for the
interaction. Fig. 9 displays the function
of the thiolates in liganding the [NiFe] cluster in the mature
subunit (Fig. 9A) as delineated from the three-dimensional
structure of the Desulfovibrio gigas enzyme (8) and compares
it with the role postulated for these thiolates during the maturation
process on the basis of the results reported (Fig. 9B). The
proposed model implies that (i) Cys241 is masked in
pre-HycE by the interaction with HypC, (ii) Cys244
coordinates the iron, (iii) Cys531 coordinates the nickel,
and (iv) Cys534 provides a free thiolate attacking the
nickel and iron atoms after proteolytic removal of the carboxyl
terminus and thereby closes the bridge. Below we will discuss the
evidence available that supports this model.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2H+ + 2e
. These enzymes
can be divided into two major families: iron-only hydrogenases
([Fe]-hydrogenases) and nickel-iron hydrogenases ([NiFe]-hydrogenases) (6, 7). They are composed of a small electron
transfer subunit (28-35 kDa) and a large catalytic subunit (45-65
kDa). In [NiFe]-hydrogenases, the large subunit harbors the metal
center ligated to the protein by one motif in the amino-terminal portion of the polypeptide
(RXCX2CX3H)
and a second one at the carboxyl-terminal region
(DPCX2CX2(H/R)) (6, 8). A
major and so far highly conserved characteristic of
[NiFe]-hydrogenases is their complex biogenesis, which involves at
least seven accessory gene products (9). Although this pathway is far
from being understood, substantial progress has been made regarding the
maturation of the catalytic subunit. A current model suggests that
maturation is initiated by the binding of iron together with the
diatomic ligands followed by nickel insertion. Finally, the
metal-containing precursor is converted to the mature form by
proteolytic removal of a C-terminal extension, mediated by a specific
protease (10-13). The three-dimensional structure of one of these
proteases, namely the hydrogenase 2-specific protease of
Escherichia coli, HybD (14), has been resolved recently by
x-ray crystallography. Another accessory protein, HypC, is a specific
chaperone-type protein required for the maturation of the catalytic
subunit of the hydrogenase 3 (5). Such genes coding for auxiliary
proteins have been found in all organisms synthesizing
[NiFe]-hydrogenases.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was used as
host for plasmid construction and maintenance. The other strains
employed in this study were derivatives of E. coli MC4100
(15), namely HD705 (MC4100, 
hycE) (16), HD709 (MC4100,
hycI) (10), and DHP-C (MC4100, hypC)
(17).
DH5 strain and replicating circles are
subsequently recovered by recombination between the homologous ends of
the linear template. The cysteine residues at positions 241, 244, 531, and 534 of HycE were replaced by alanine via this procedure using the
following primer pairs: EC241A-EC241Arev, EC244A-EC244Arev,
EC531A-EC531Arev, and EC534A-EC534Arev (Table I). The Cys2
and the Pro6 residues of HypC were replaced using the
following primer pairs: HypC2rev-HypC2A, HypC2rev-HypC2S,
HypC2rev-HypC2R, and HypCP6rev-HypCP6 (Table
I). All mutations were confirmed by DNA
sequencing. The resulting mutated plasmids were used to transform
E. coli HD705 in the case of the pACE1 derivatives or DHP-C
in the case of the pJA1021 plasmid series.
Oligonucleotide primers used in this work
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sequence comparison of the HypC protein
family. The HypC sequence of E. coli (30) is compared
with those of HybG from E. coli (31), HypC from A. eutrophus (32), Rhodobacter capsulatus (33),
Azotobacter vinelandii (34), Rhizobium
leguminosarum bv. viciae (35), Synechocystis
PCC6803 (36), Methanococcus jannaschii MJ0200 (37),
Archaeoglobus fulgidus (38), Methanobacterium
thermoautotrophicum (39), and Helicobacter pylori J99
(40). The ClustalW algorithm was used with a PAM250 weight table, and
the consensus sequence is indicated above the sequence alignment.
Total hydrogenase activity of wild-type and mutant E. coli
strains
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Fig. 2.
Immunoblotting analysis of the precursor and
mature forms of HycE in transformants expressing different HypC
variants. Crude extracts (30 µg of total protein) were subjected
to SDS gel electrophoresis and reacted with antibodies raised against
HycE. Extracts from the following strains were applied on the gel:
MC4100 (lane 1), HD705 ( hycE)
(lane 2), DHP-C (hypC)
(lane 3), DHP-C/pJA1021 (lane
4), DHP-C/pJA1021-C2A (lane 5),
DHP-C/pJA1021-C2R (lane 6), and DHP-C/pJA1021-C2S
(lane 7). Precursor and mature forms of HycE are
indicated by arrows.
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Fig. 3.
Immunoblotting analysis of HypC-pre-HycE
complex formation in transformants carrying mutant hypC
genes on a plasmid. S100 extracts (40 µg of total
proteins) were subjected to nondenaturing gel electrophoresis and
reacted with anti-HypC antibodies as previously reported (5).
Lane 1, MC4100; lane 2,
HD705 ( hycE); lane 3, DHP-C
(hypC); lane 4, HD709
(hycI); lane 5, DHP-C/pJA1021;
lane 6, DHP-C/pJA1021-C2A; lane
7, DHP-C/pJA1021-C2R; lane 8,
DHP-C/pJA1021-C2S.
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Fig. 4.
Immunoblotting analysis of HycE precursor and
mature forms in transformants expressing HycE mutant forms. Crude
extracts (30 µg of total proteins) were subjected to SDS-PAGE as
indicated in Fig. 2. Lane 1, MC4100;
lane 2, HD705 ( hycE);
lane 3, HD705/pACE1; lane
4, HD705/pACE1-C241A; lane 5,
HD705/pACE1-C244A; lane 6, HD705/pACE1-C531A;
lane 7, HD705/pACE1-C534A. Precursor and mature
forms of HycE are indicated by arrows.
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Fig. 5.
In vivo incorporation
of 63Ni by transformants carrying mutated hycE
genes. S100 extracts (70 µg of total proteins) were
subjected to ND-PAGE and autoradiographed. Lane 1, MC4100;
lane 2, HD705 ( hycE);
lane 3, HD709 (hycI);
lane 4, DHP-C (hypC);
lane 5, HD705/pACE1; lane
6, HD705/pACE1-C241A; lane 7,
HD705/pACE1-C244A; lane 8, HD705/pACE1-C531A;
lane 9, HD705/pACE1-C534A. Characteristic bands
are marked by arrows.
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Fig. 6.
Immunoblotting analysis of HypC-pre-HycE
complex formation in transformants carrying mutated hycE
alleles on a plasmid. S100 extracts (40 µg of total
proteins) were subjected to ND-PAGE and reacted using anti-HycE
antibodies (A) or anti-HypC antibodies (B).
Lane 1, MC4100; lane 2,
HD705 ( hycE); lane 3, HD709
(hycI); lane 4, HD705/pACE1-C241A;
lane 5, HD705/pACE1-C244A; lane
6, HD705/pACE1-C531A; lane 7,
HD705/pACE1-C534A.
hypC), which contain the precursor form of HycE,
were analyzed under identical conditions, and an identical pattern was
observed. These observations provide circumstantial evidence that HypC
does not interact with pre-HycE via the formation of a disulfide bridge that under nonreducing conditions should give rise to a slower migrating species.
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Fig. 7.
Effect of reducing or nonreducing conditions
on the migration of the HypC-pre-HycE complex upon SDS-PAGE. After
incubation with (lanes 1-3) or without
(lanes 4-6) 
-mercaptoethanol in the Laemmli
sample buffer, S100 extracts (40 µg of total proteins) of different
strains were separated by SDS-PAGE and reacted using antiserum directed
against HycE. Lanes 1 and 4, MC4100;
lanes 2 and 5, HD709
(hycI); lanes 3 and 6,
DHP-C (hypC).
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Fig. 8.
Reactivity of the sulfhydryl groups in the
HypC-pre-HycE complex with alkylating agents. HD709 extracts that
had been incubated with or without dithiothreitol were alkylated with
iodoacetic acid (IAA) or iodoacetamide (IAM) and
separated on a ND-PAGE. HypC-pre-HycE complex and alkylated HypC are
marked by arrows.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Model of the functional role of the conserved
cysteines in the HycE polypeptide. A, in the mature
large subunit according to the crystal structure of the D. gigas enzyme (41); B, in the precursor form of the
large subunit.
Masking of Cys241 by HypC-- When each of the four cysteine residues playing a role in the ligation of the [NiFe] cluster of the mature subunit was replaced by an alanine, the C241A variant was the only one that was no longer able to interact with HypC. It is assumed that the effect is specific, since an identical replacement of Cys244 only 3 residues away allowed the interaction to take place. In addition, exchange of the well conserved Arg239 residue by leucine also in close vicinity of Cys241 did not prevent complex formation.2
It is assumed that Cys241 of pre-HycE directly or indirectly interacts with Cys2 from the HypC protein. The chemical nature of the interaction is unknown at present and requires the purification of substantial amounts of the maturation intermediate. It could consist in the interaction of each thiol as hydrogen donor with a strong acceptor group from the other partner. Alternatives would be that the thiols of Cys2 of HypC and Cys241 of HycE could be sandwiched by some metal ion, like iron or zinc. However, the in vitro addition of chelating agents as EDTA or 2,2'dipyridyl has no influence on the complex stability (data not shown). Interestingly, once the complex is formed it cannot be titrated by a surplus of a HypC variant in which the N-terminal cysteine was replaced by an alanine or serine residue.2 If the interaction were noncovalent and assuming that the specificity of recognition is not only determined by the two cysteine residues, this should have been the case. Disulfide bonding as a possibility can be ruled out because the complex is readily dissociated by iodoacetic acid or iodoacetamide and because no migration difference of the complex under SDS-PAGE was observed under oxidative compared with reducing conditions.
Coordination of the Iron by Cys244 and of Nickel by Cys531-- There are substantial number of arguments that the two metals are incorporated separately into the precursor of HycE. The most convincing ones are that the precursor formed in a strain grown in absence of nickel can be matured in vitro by the addition of nickel alone (11). This provides support for the contention that iron has already been incorporated and that nickel is inserted independently as a late step. Furthermore, the maturation endopeptidase scans the precursor for the presence of nickel by binding to the metal, which excludes the existence of the bridged cluster (14, 27). Evidence that Cys244 carries the iron in the precursor can only be deduced from the structure of the mature cluster (8). The direct interaction between Cys541 and nickel, however, has been proven in the case of [NiFeSe]-hydrogenase from Desulfovibrio baculatus in which the cysteine is naturally substituted by a selenocysteine whose selenium atom has been shown to directly interact with nickel (28, 29).
Cys534 of Pre-HycE Contains a Free Thiol-- An intriguing finding was also that Cys534 of HycE is not essential for maturation. The C534A variant forms a complex with HypC, accepts nickel, and is proteolytically processed but does not yield an active enzyme. Incorporation of nickel and processing, although not interaction with HypC, was recently demonstrated for an analogous mutant in the large subunit of the NAD-reducing hydrogenase of Alcaligenes eutrophus (26). Cys534, which provides the thiolate for bridging the iron and the nickel in the mature cluster, does not interact with the metals during the maturation process.
Working Model-- Based on these results, a working model on the sequence of events in the assembly of the [NiFe] center is proposed (Fig. 9). First, the thiolate of Cys241 is masked by interaction with HypC. Since nickel can be incorporated into this complex, Cys241 cannot be involved in the initial ligation of the metals of the center. Second, iron binding takes place at Cys244, and nickel is coordinated by Cys531.
The key intermediate for the assembly of metal center, therefore, is
the complex between pre-HycE carrying the iron at Cys244
and the chaperone-type protein HypC. It is assumed that the C terminus
of HycE sticks out of the complex so that nickel can be added to
Cys531. By an unknown reaction, the linkage between
pre-HycE and HypC is then cleaved, rendering pre-HycE a substrate for
the endopeptidase. Only nickel-containing pre-HycE from which HypC has
been released can be cleaved by the endopeptidase.2 This
cleavage triggers a conformational switch feeding the C terminus toward
the two metals and closing the bridge between them by reaction with the
free thiol of Cys534. For a proof of this model, a number
of questions have to be answered. The most relevant ones are the
disclosure of the chemical nature of the interaction of pre-HycE with
HypC and the determination of the reaction that leads to the release of HypC.
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ACKNOWLEDGEMENTS |
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We thank Dr. F. Lottspeich for the amino-terminal sequencing analysis of HypC and valuable discussions and Dr. R. Glass for helpful discussions and advice.
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FOOTNOTES |
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* This work was funded by grants from the Alexander von Humboldt Foundation (to A. M.) and from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to A. B.).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.
To whom correspondence should be addressed. Tel.: 89-2180-6134;
Fax: 89-2180-6122; E-mail: Axel.Magalon@lrz.uni-muenchen.de.
Published, JBC Papers in Press, April 26, 2000, DOI 10.1074/jbc.M000987200
2 A. Magalon and A. Böck, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: ND, nondenaturing; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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