(Received for publication, July 17, 1995)
From the
The membrane-anchored thioredoxin-like protein (TlpA) from the
Gram-negative soil bacterium Bradyrhizobium japonicum was
initially discovered due to its essential role in the maturation of
cytochrome aa. A soluble form of TlpA lacking the
N-terminal membrane anchor acts as a protein thiol:disulfide
oxidoreductase. TlpA possesses an active-site disulfide bond common to
all members of the thiol:disulfide oxidoreductase family. In addition,
it contains two non-active-site cysteines that form a structural
disulfide bond (Loferer, H., Bott, M., and Hennecke, H.(1993) EMBO
J. 12, 3373-3383; Loferer, H., and Hennecke, H.(1994) Eur. J. Biochem. 223, 339-344). Here, we compare the
far- and near-UV CD spectra of TlpA before and after reduction of both
disulfides by dithiothreitol and show that the non-active-site
disulfide bond is not required for the integrity of TlpA's native
conformation. In contrast to dithiothreitol, reduced glutathione (GSH)
selectively reduces the active-site disulfide and leaves the
non-active-site disulfide bond intact, even at high molar excess over
TlpA. The selective reduction of the active-site disulfide bond leads
to a 10-fold increase of the intrinsic tryptophan fluorescence of TlpA
at 355 nm, which may be interpreted as a quenching of tryptophan
fluorescence by the active-site disulfide bond. Using the specific
fluorescence of TlpA as a measure of its redox state, a value of 1.9
± 0.2 M was determined for the TlpA:glutathione
equilibrium constant at pH 7.0, demonstrating that TlpA is a reductant,
like cytoplasmic thioredoxins. The DsbA protein, which acts as the
final oxidant of periplasmic secretory proteins in Escherichia
coli, is not capable of oxidizing the active-site cysteines of
TlpA. This suggests that TlpA's primary role in vivo is
keeping the thiols of certain proteins reduced and that TlpA's
active, reduced state may be maintained owing to its kinetically
restricted oxidation by other periplasmic disulfide oxidoreductases
such as DsbA.
Thioredoxins, glutaredoxins, and protein disulfide isomerases
(PDIs) ()catalyze formation and reduction of disulfide bonds
in proteins. The enzymes themselves possess an active-site disulfide
with the conserved sequence
Cys-X-X-Cys(1, 2) . In recent years,
bacterial genes encoding novel periplasmic thiol:disulfide
oxidoreductases have been discovered. These enzymes are involved in
various cellular processes such as disulfide bond formation in
translocated proteins (DsbA, DsbB, and DsbC from Escherichia
coli), cholera toxin maturation (TcpG from Vibrio
cholerae), cytochrome c biogenesis (HelX from Rhodobacter capsulatus, TlpB from Bradyrhizobium
japonicum), and cytochrome aa
maturation
(TlpA from B. japonicum) (reviewed in (3) and (4) ). Of these bacterial proteins, DsbA from E. coli has been most extensively investigated to date. The reduced form
of DsbA is by about 20 kJ/mol more stable than the oxidized
protein(5, 6) . This result quantitatively explains
DsbA's high intrinsic redox potential (E`
)
of -0.089 V, which was calculated from its equilibrium constant
with glutathione(5, 7) . DsbA and eukaryotic PDI, for
which potentials of -0.11 V (8) and -0.175 V (9) have been determined, are believed to preferably transfer
their own disulfide bond to newly secreted
proteins(5, 7) . By contrast, the more reducing
thioredoxins (E`
= -0.23 to
-0.27 V) are presumably involved in keeping cysteines reduced
within the cytoplasm (10, 11, 12) .
The
Gram-negative soil bacterium B. japonicum expresses a
membrane-anchored protein (TlpA) containing a periplasmically oriented
thioredoxin-like domain, which was found to be involved in the
post-translational assembly of the terminal oxidase cytochrome aa(13) . It was demonstrated that the
soluble, thioredoxin-like domain of TlpA has
thiol:disulfide-oxidoreductase activity in vitro, and that two
additional cysteines, which are present outside TlpA's
active-site, form an additional disulfide bond(14) . In this
study, the standard redox potential of TlpA's active-site
disulfide was determined. For this purpose, we used the soluble form of
TlpA (TlpA
) depleted of its N-terminal membrane anchor
(residues 1-37, cf. (14) ). The results are
discussed in the context of the putative in vivo function of
this protein within the periplasm.
An alternative, less expensive way
than the MalE/TlpA fusion strategy was to fuse
TlpA(36-221) to the E. coli OmpA signal
sequence and express it as a soluble, periplasmic protein in E.
coli. For this purpose, the gene coding for TlpA
(codons 36-221) was amplified by the polymerase chain
reaction and cloned into the expression plasmid pRBI (16) via
the StuI and HindIII restriction sites (pRBI contains
a single StuI restriction site at the end of the OmpA signal
sequence). The following primers were used: N-terminal primer (StuI site), 5`-ATACAGGCCTCCCGGGCGCCTACCGGCGATCC-3`;
C-terminal primer (HindIII site),
5`-GCGCGAATTCTTAAAGCGCCGCGGCGGCCTTG-3`.
Cells of E. coli BL21 (15) were transformed with the resulting expression
plasmid pOmpA/TlpA and grown in LB medium containing
ampicillin (100 µg/ml) to an optical density (550 nm) of 0.5 in 10
liters of LB-medium at 25 °C. After induction by
isopropyl-
-D-thiogalactoside (final concentration 1
mM), the cells were grown overnight and harvested by
centrifugation. The cells were suspended in 1/70 volume of cold
extraction buffer (see above), stirred at 4 °C for 1 h, and
centrifuged. The supernatant (periplasmic extract) was extensively
dialyzed against buffer A (10 mM MES/NaOH, pH 6.5).
Precipitated material was removed by centrifugation and the supernatant
was applied to a DE52 cellulose column (50 ml) equilibrated with buffer
A. The eluate containing TlpA
was directly loaded onto a
CM52 cellulose column (15 ml) equilibrated with buffer A. The column
was washed with buffer A and TlpA
was eluted by a linear
gradient (400 ml) from 0 to 500 mM NaCl in buffer A. Fractions
containing TlpA (corresponding to 180-200 mM NaCl) were
pooled and 0.375 volumes of 4 M ammonium sulfate were added
(final ammonium sulfate concentration: 1.5 M). The solution
was loaded onto a phenyl-Sepharose column (15 ml) (Pharmacia)
equilibrated in 20 mM MES/NaOH, pH 6.7, containing 1.5 M ammonium sulfate. An ammonium sulfate gradient (400 ml; 1.5 M to 0 M) in the same buffer led to elution of pure
TlpA
at around 1 M ammonium sulfate. The
TlpA-containing fractions were dialyzed extensively against distilled
water. Typically, 6 mg of homogeneous TlpA
/liter of
bacterial culture were obtained by this procedure. The correct cleavage
of the OmpA signal sequence was verified by N-terminal Edman
sequencing. TlpA
directly secreted into the periplasm of E. coli via the OmpA signal sequence differs from TlpA
obtained by cleavage of the MalE/TlpA
fusion by
having two additional N-terminal residues originating from complete
TlpA. However, both forms of TlpA
were indistinguishable
in their spectroscopic properties and redox behavior.
R, the relative amount of reduced TlpA at
equilibrium, was calculated according to , where F is the measured fluorescence intensity (at 355 nm), and F
and F
are the
fluorescence intensities of completely reduced or oxidized
TlpA
,
respectively.
F was determined both by adding DTT
(final concentration: 1 mM) to the samples containing
0-50 mM GSH
/GSSG and by fitting the measured F values according to , which is obtained by
combining and (see ``Results'').
Both methods led to identical values for F
. The
equilibrium concentration of GSH was determined according to Ellman (18) and the GSSG concentration was determined by the
glutathione reductase assay described below. In all cases, the GSH and
GSSG concentrations were measured after the corresponding equilibration
period in conjunction with the fluorescence measurements. The GSH and
GSSG concentrations were not corrected for reduced and oxidized TlpA,
since the TlpA concentration was negligible compared to the total
concentrations of GSH and GSSG under all conditions.
Fully oxidized
TlpA (1 µM) was equilibrated with a high
molar excess of GSH (0.01 and 0.1 M) or DTT (0.03 M)
at pH 7 as described under ``Experimental Procedures.''
Subsequently, free thiols were alkylated by iodoacetamide and the
proteins were analyzed by reducing and nonreducing SDS-PAGE. Fig. 1demonstrates that the structural disulfide bond was fully
reduced in the presence of DTT, whereas it was not attacked by GSH,
even at concentrations of 0.1 M.
Figure 1:
SDS-PAGE analysis of oxidized,
DTT-reduced and GSH-reduced TlpA. Samples of oxidized
TlpA
(1 µM) were incubated in the presence
of GSH (10 and 100 mM), DTT (30 mM), or in the
absence of any reducing chemicals as described under
``Experimental Procedures.'' After alkylation of free thiols
by iodoactetamide, each sample was analyzed by SDS-PAGE with
(+2-ME) and without (-2-ME) reduction by 2-mercaptoethanol (ME) (see ``Experimental Procedures'' for details).
Oxidized TlpA
which was not incubated with reducing
agents and iodoacetamide was included as an additional standard. S, molecular mass standard. 1, oxidized TlpA
. 2, oxidized TlpA
after incubation with
iodoacetamide. 3, TlpA
reduced by 10 mM GSH and alkylated. 4, TlpA
reduced by 0.1 M GSH and alkylated. 5, TlpA
reduced by
30 mM DTT and alkylated. Proteins were visualized by silver
staining.
Figure 2:
Fluorescence emission spectra of oxidized
TlpA and TlpA
reduced by DTT and GSH. A, comparison of fluorescent properties of GSH-reduced and
DTT-reduced TlpA
. The samples were obtained from the
redox-titration experiment shown in Fig. 4. Reduced TlpA
was obtained either by adding DTT (1 mM final
concentration) to the oxidized sample, or by recording a spectrum of
the sample containing a GSH
/GSSG ratio of 29 M (R = 0.92). Samples were excited at 295 nm using a
light path of 4 mm. Fluorescence spectra of oxidized (
),
DTT-reduced (
) and GSH-reduced TlpA
(
) are
shown. B, fluorescence properties of oxidized and DTT-reduced
TlpA
in the presence or absence of 6 M GdmCl.
Fluorescence emission spectra were recorded at protein concentrations
of 1 µM in 100 mM sodium phosphate, pH 7.0, 1
mM EDTA. Samples of reduced TlpA
contained 1
mM DTT, and samples of unfolded TlpA
contained 6 M GdmCl. Fluorescence spectra of native oxidized (
),
native DTT-reduced (
), unfolded oxidized (
), and unfolded
reduced TlpA
(
) are shown. The excitation
wavelength was 295 nm.
Figure 4:
Redox equilibrium of TlpA
with glutathione. The relative amount of reduced TlpA
at
equilibrium was measured using the specific TlpA
fluorescence at 355 nm (excitation at 295 nm). Oxidized
TlpA
was incubated for 19 h (
), 44 h (
), and 7
days (
) in 100 mM sodium phosphate, pH 7.0, 1 mM EDTA, containing 10 or 5 µM GSSG and different
concentrations of GSH (0.1 to 125 mM, see ``Experimental
Procedures''). The equilibrium constant (K
)
was determined by fitting the data according to ((8) ). After non-linear regression, a value of 1.9
± 0.2 M was obtained (correlation coefficient
>0.997).
The reduction of both
disulfides in TlpA by DTT or the catalytic disulfide by
GSH both led to a strong shift in the emission maximum of the protein
from 335 to 352 nm, a value that is usually observed for unfolded
proteins(22) . Therefore, we compared the fluorescence spectra
of oxidized and reduced TlpA
under native conditions and
of the denatured protein in the presence of 6 M GdmCl (Fig. 2B). At an excitation wavelength of 295 nm,
unfolding resulted in a shift of the emission maxima of both oxidized
and reduced TlpA
to 358 nm. Moreover, the fluorescence
intensities of the oxidized and reduced unfolded proteins were almost
identical and both were about 40% lower than the fluorescence intensity
of native, reduced TlpA
(Fig. 2B). The
minor difference in fluorescence intensity between unfolded oxidized
and reduced TlpA
(Fig. 2B) may reflect
the fact that the quenched tryptophan residues are located in the
immediate vicinity of the active-site disulfide in the primary sequence
of TlpA(13, 20) .
The strongly different
fluorescence spectra for reduced TlpA in the presence and
absence of 6 M GdmCl indicated that TlpA
reduced
at the active-site or at both disulfides still maintained its native
conformation. To confirm this assumption, circular dichroism (CD)
spectra of oxidized and DTT-reduced TlpA
were recorded (Fig. 3, A and B). Far-UV CD measurements
revealed residue ellipticities at 222 nm of -19,500 and
-17,800 deg cm
dmol
for oxidized
and reduced TlpA
(Fig. 3A), respectively,
which is a typical feature of proteins rich in
-helices(22, 23) . In addition, the overall
shapes of the spectra were nearly identical, strongly supporting the
maintenance of native secondary structure in the fully reduced protein.
Figure 3:
Far- and near-UV CD spectra of oxidized
and DTT-reduced TlpA. CD spectra were recorded at protein
concentrations of 28 µM (0.5 mg/ml) in 1 mM sodium phosphate, pH 7.0, 10 µM EDTA at 25 °C as
described under ``Experimental Procedures.'' For complete
reduction of TlpA
, DTT was added to a final concentration
of 0.1 mM, and the spectra were recorded after 3 h. A, far-UV spectra. B, near-UV spectra. Solid line, oxidized TlpA
. Broken line, DTT-reduced
TlpA
.
Comparison of the near-UV CD spectra (Fig. 3B) that
are unique and characteristic for the tertiary structure of a protein,
displayed significant differences between oxidized and DTT-reduced
TlpA and thus demonstrated different, redox
state-dependent conformations and/or environments of at least some of
the aromatic residues in TlpA
. The characteristic near-UV
CD spectra of oxidized and reduced TlpA
are also
consistent with the native state of TlpA
with both
disulfides reduced.
Oxidized TlpA was incubated in the presence of
GSSG and increasing concentrations of GSH (0.1-125 mM,
see ``Experimental Procedures''), and the relative amount of
reduced TlpA
(R) at equilibrium was measured by
the intrinsic TlpA fluorescence (Fig. 4). Incubation periods of
19 h, 44 h, and 7 days yielded identical results, proving that the
equilibrium had been reached. When the same equilibrium measurements
were started from the reduced form of TlpA
, the
equilibrium was reached more slowly, but was almost identical to the
experiments starting from oxidized TlpA
after 7 days
(data not shown).
The high GSH concentrations required to reduce
TlpA provided a clear indication that this protein is far
more reducing than DsbA. By fitting the data by non-linear regression
according to (8) , an equilibrium constant for the
TlpA
/glutathione system of 1.9 ± 0.2 M was determined (correlation coefficient >0.997). A standard
redox potential of -0.213 V for TlpA
's
active-site cysteines at 30 °C and pH 7.0 (E`
) was calculated from the Nernst
equation using a value of -0.205 V (24) for the
glutathione standard potential (E`
, ).
Figure 5:
DsbA is not capable of oxidizing the
active site of TlpA at pH 7. A, overall
fluorescence difference spectrum for the oxidation of
TlpA
by DsbA. Fluorescence spectra of the isolated
reactants (1 µM oxidized and reduced DsbA and 1 µM oxidized and reduced TlpA
in 100 mM sodium
phosphate pH 7.0, 1 mM EDTA) were recorded (excitation
wavelength 280 nm; 30 °C). The spectra of oxidized DsbA and reduced
TlpA
(educts) and reduced DsbA and oxidized TlpA
(products) were added, respectively. The difference spectrum of
the resulting spectra was then calculated.
, DsbA (ox) +
TlpA
(red);
, DsbA (red) + TlpA
(ox);
, overall difference spectrum. B, time
course of the fluorescence signal. Emission was measured at 320 nm
(maximum of the difference spectrum, see A) after mixing
reduced TlpA
with oxidized DsbA (final concentrations: 1
µM) in 100 mM sodium phosphate, pH 7.0, 1 mM EDTA at 30 °C (
). No change in the fluorescence signal
is observed. As a control, the products of the reaction, reduced DsbA
and oxidized TlpA
, were mixed under identical conditions,
and the fluorescence signal was recorded
(
).
As shown for thioredoxin and DsbA, TlpA from B. japonicum exhibits a significant increase in tryptophan
fluorescence upon reduction of the active-site disulfide. In the case
of thioredoxin, this phenomenon is mainly due to the quenching of the
fluorescence of tryptophan 28 by the active-site disulfide
bond(28, 29) . The three-dimensional structure of
oxidized E. coli thioredoxin reveals a direct contact between
tryptophan 28 and the disulfide(30) . TlpA contains 3
tryptophan residues(13) , two of which are at positions
equivalent to the tryptophans 28 and 31 in thioredoxin. Therefore,
assuming a thioredoxin-like fold for TlpA, its redox-dependent
fluorescence behavior is not unexpected. Interestingly, a strong shift
in the emission maximum from 335 to 352 nm occurred in the reduced
protein, which is less pronounced in thioredoxin(20) . The
non-active-site disulfide bond, which was discovered previously in
TlpA(14) , was reduced when native TlpA was
incubated together with DTT. However, far- and near-UV CD spectra as
well as the fluorescence spectra indicated that this cystine bond is
not essential for the maintenance of TlpA's native conformation.
In contrast to the reduction by DTT, TlpA's non-active-site
disulfide bond was not attacked in the presence of a high molar excess
of GSH (Fig. 1).
An indication that the non-active-site
disulfide bond does not contribute to the tryptophan fluorescence
quenching in oxidized TlpA came from the fact that F obtained by fitting the equilibrium data according to was identical to the specific fluorescence of TlpA
reduced by DTT. This allowed us to observe solely the redox state
of the active site via TlpA's intrinsic fluorescence. An
intrinsic standard redox potential of -0.213 V was determined for
TlpA's active site. Thus, the intrinsic redox potential of
TlpA
is comparable with that of cytoplasmic thioredoxins,
which range from -0.23 to -0.27
V(10, 11, 12) , and is therefore
significantly more reducing than eukaryotic PDI (-0.11 to
-0.175 V; (8) and (9) ) and DsbA, whose redox
potential (-0.089 V) was also determined by the equilibrium with
glutathione(5, 7) . However, it is difficult to
compare the published redox potentials of these proteins directly,
because they have been determined under different experimental
conditions such as pH and temperature, and may have been calculated by
using different E`
values for the reference redox
couples. For example, the redox potential of E. coli thioredoxin (-0.27 V) has been determined using
NADPH/NADP
as a reference redox system at 25 °C
and pH 7.0(12) , whereas the value of -0.11 V for bovine
liver PDI was determined using a different reference value for
glutathione (-0.24 V) at 20 °C and pH 7.5(8) . Thus,
there is a need to determine the standard redox potentials of these
proteins in parallel under identical conditions before direct
comparisons can be made. However, the measured values of the
equilibrium constants of TlpA and thioredoxin with glutathione, which
are 1.9 and 2 M(31) , respectively, demonstrate that
both enzymes are reductants and possess nearly identical redox
properties.
It has been suggested that the amino acid residue preceding the C-terminal cysteine residue of the active-site Cys-X-X-Cys is important for redox properties, because a mutation of proline 34 in E. coli thioredoxin to histidine resulted in a higher redox potential (-0.27 to -0.235 V) and in an increase in isomerase activity(29, 32) . In this respect, the active-site sequence of TlpA (Cys-Val-Pro-Cys) is also more related to thioredoxin (Cys-Gly-Pro-Cys), whereas DsbA's active-site Cys-Pro-His-Cys relates to PDI (Cys-Gly-His-Cys).
It was shown for the DsbA protein
from E. coli that it contains a destabilizing disulfide bond
that quantitatively accounts for the oxidative force of the
protein(5, 6) . Therefore, it would have been
interesting to correlate the thermodynamic stabilities of oxidized
TlpA and TlpA
selectively reduced at its
active site with its redox properties. However, severe complications
have to be expected in equilibrium denaturation experiments due to
random disulfide interchange reactions in unfolded TlpA with one
disulfide reduced and one disulfide intact.
The results of the
present study have some interesting implications for the postulated
biological functions of TlpA. In various cellular processes,
thioredoxins (E`
-0.26 V) are involved
in reducing cystine bridges in cytoplasmic proteins(1) . DsbA (E`
= -0.089 V), however, oxidizes
cysteines during folding of proteins after their transport to the
periplasm(3, 25) . TlpA is anchored to the cytoplasmic
membrane, leaving its globular, thioredoxin-like domain exposed to the
periplasm (13) . The reducing properties of TlpA suggest that
it may act as a reductant of protein disulfides within the periplasm.
It seems likely that cysteine residues involved in cofactor binding of
certain periplasmic proteins or periplasmic domains of membrane
proteins have to be kept in a reduced state(33) . Since the
oxidizing conditions of the periplasmic compartment may be deleterious
for certain proteins, it seems reasonable that periplasmically oriented
redox proteins exist, which keep cysteines in those proteins reduced
during protein biogenesis. A reducing function of this type has already
been suggested for the HelX protein of R. capsulatus(33) , a homologue of B. japonicum TlpB(34) . HelX is essential for biogenesis of c-type cytochromes, in which the heme moiety is covalently
bound to cysteine residues in the consensus sequence
Cys-X-X-Cys-His. It has been speculated that HelX
keeps these cysteines in a reduced state as a prerequisite for covalent
heme attachment(33) . However, it has neither been shown that
HelX is indeed a thiol:disulfide oxidoreductase, nor has the redox
potential of HelX been determined.
In E. coli, the
periplasmic DsbA protein has been shown to act as general and efficient
disulfide donor to folding polypeptides (4, 25) . DsbA
specifically transfers its own disulfide to accessible cysteine
residues in polypeptides in an extremely rapid process which is nearly
diffusion-controlled (25) and is recycled as an oxidant by the
DsbB protein, a redox protein of the inner membrane of the
bacterium(27) . Therefore, one would assume that if a
DsbA/DsbB-related system also exists in B. japonicum, it will
maintain the oxidized state of TlpA's active-site disulfide,
which would inactivate TlpA as a reductant. The thermodynamic
equilibrium for the oxidation of TlpA by DsbA lies indeed far on the
side of oxidized TlpA and reduced DsbA. The equilibrium constant for
this reaction is identical to the ratio of the individual equilibrium
constants of the proteins with glutathione (1.9 M and 1.2
10
M(7) , respectively),
and has a value of 1.6
10
.
However, DsbA from E. coli was not capable of oxidizing
TlpAin vitro. Obviously, the reaction is
kinetically restricted, presumably due to steric inaccessibility of
TlpA's active-site dithiol for DsbA. In contrast, DsbA oxidizes
cytoplasmic thioredoxin from E. coli under identical
conditions (k
= 180 ± 20 M
s
at pH 7.0), although
the reaction is significantly slower than the oxidation of organic
dithiols by DsbA (26) . (
)These findings strongly
support the view that TlpA's function in the periplasm is mainly
to act as a reductant. TlpA may thus be fully independent of a
periplasmic oxidation system and may specifically keep certain thiols
of its target proteins in a reduced state. Similar to recycling of
oxidized DsbA by DsbB, TlpA may be specifically kept in a reduced state
by another protein factor or by redox-active membrane components. It is
presently unknown whether the nitrogen-fixing bacterium B.
japonicum contains a periplasmic redox system similar to E.
coli. At present, a few c-type cytochromes are the only
periplasmic proteins of B. japonicum that have been
characterized(34) , and apart from TlpA itself it is not known
whether other disulfide-containing proteins occur in this bacterium. If
an oxidation machinery different from DsbA/DsbB would exist in B.
japonicum, TlpA can presumably also stay reduced due to the low
accessibility of its active-site disulfide. In turn, this suggests that
the number of TlpA's natural targets is limited and that the
enzyme may have developed a high specificity for its substrates.
TlpA is essential for the biogenesis of functional cytochrome aa, because in a tlpA
strain the heme a cofactors are spectroscopically
undetectable, the oxidase is not functional, and subunit I is less
stable(13) . However, subunit I of cytochrome aa
could be excluded from being a direct target protein of TlpA,
since it contains only one cysteine residue that is located in one of
the transmembrane domains(35) . Subunit II of the aa
oxidase contains a characteristic binuclear
copper center (Cu
) for electron transfer, which is located
in a globular, periplasmic domain and ligated by two cysteine thiols,
one methionine and two histidines (36) . The two cysteines must
be in the reduced state to act as ligands. It is tempting to speculate
that TlpA's function is to keep these cysteines reduced during aa
biogenesis.
TlpA is the first example of a
thiol:disulfide oxidoreductase with reducing properties occurring in an
otherwise oxidizing cellular compartment. While this may appear
contradictory, it is plausible that there is a need for a protein
factor in the bacterial cell which guarantees the reduced state and the
cofactor binding properties of certain other proteins, especially in
the case of membrane proteins of respiratory pathways. A prerequisite
for the co-existence of both an oxidizing and a reducing machinery
within the same cellular compartment is that they do not interfere with
each other. The validity of such a model still has to be proven
experimentally, but the kinetically restricted oxidation of
TlpA by DsbA strongly points in this direction.