©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Bacterial Thioredoxin-like Protein That Is Exposed to the Periplasm Has Redox Properties Comparable with Those of Cytoplasmic Thioredoxins (*)

(Received for publication, July 17, 1995)

Hannes Loferer (1) Martina Wunderlich (2) Hauke Hennecke (1) Rudi Glockshuber (2)(§)

From the  (1)Mikrobiologisches Institut and (2)Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule, Zürich, Switzerland

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(3). 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.


INTRODUCTION

Thioredoxins, glutaredoxins, and protein disulfide isomerases (PDIs) (^1)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(3) 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`(0)) 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) = -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(3)(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.


EXPERIMENTAL PROCEDURES

Materials

1,4-Dithio-DL-threitol (DTT), reduced glutathione (GSH), oxidized glutathione (GSSG), and polymyxin B were purchased from Sigma. Yeast glutathione reductase and NADPH were from Boehringer (Mannheim, Germany), isopropyl-beta-D-thiogalactoside was obtained from Biomol (Hamburg, Germany), DE52- and CM52-cellulose were purchased from Whatman (Maidstone, United Kingdom), and phenyl-Sepharose was obtained from Pharmacia (Uppsala, Sweden). All other chemicals were from Merck (Darmstadt, Germany) and of highest purity available. The DsbA protein was purified from an overproducing E. coli strain as described previously(7) .

Expression and Purification of TlpA

The soluble form of TlpA (TlpA, residues 38-221), lacking its N-terminal membrane anchor (residues 1-37) was expressed as a MalE fusion protein in E. coli BL21 (15) and purified after cleavage by factor Xa as described previously(14) , except that the periplasmic extract was prepared as follows. After centrifugation, cells were resuspended in cold extraction buffer (20 mM Tris/HCl, pH 8.5, 1 mM EDTA, 1 mg/ml polymyxin B; 2 ml/g wet cells). The suspension was gently stirred at 4 °C for 1 h and centrifuged at 35,000 times g for 30 min at 4 °C. The MalE/TlpA fusion protein was purified from the supernatant as described(14) . The fully oxidized state of purified TlpA was ascertained by demonstrating the lack of reaction of free thiol groups with dithionitrobenzoic acid(18) . For this purpose TlpA was added at a final concentration of 10 µM into 1 ml of 6 M guanidinium chloride in 0.2 M Tris/HCl, pH 8.0, containing 1 mM dithionitrobenzoic acid(14, 18) . The absorbance at 412 nm was measured against a control containing the same solution without protein ( = 13.6 mM cm for p-nitrothiophenol)(18) . As a control of the reactivity of thiol groups in this concentration range, a calibration curve was established with reduced glutathione (1-50 µM).

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-beta-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.

Reduction of TlpAby GSH

One µM TlpA was incubated in degassed 100 mM sodium phosphate, pH 7.0, 1 mM EDTA with GSH (0.1 or 10 mM) or DTT (30 mM) under a nitrogen atmosphere at 30 °C for 16 h in a volume of 50 µl. Oxidized TlpA alone was incubated for the same period as a control. Afterwards, 50 µl of 0.4 M iodoacetamide were added to block free thiols, and the reactions were incubated for 20 min at room temperature. Each sample was split into two aliquots and then mixed with 15 µl of SDS-PAGE sample buffer either containing or lacking 20% (v/v) 2-mercaptoethanol. After a 5-min incubation at room temperature the samples were loaded onto a 15% polyacrylamide-SDS gel(17) . Proteins were visualized by silver staining.

Circular Dichroism Spectra

Far- and near-UV circular dichroism spectra were monitored in a JASCO J710 CD spectropolarimeter at a protein concentration of 28 µM (0.5 mg/ml) in 1 mM sodium phosphate, pH 7.0, 10 µM EDTA at 25 °C. Sixteen spectra were averaged for each sample at a band width of 1 nm and a scan speed of 20 nm/min. 0.1-mm cuvettes were used for the far-UV spectra (270 to 180 nm) and 10-mm cuvettes for recording the near-UV spectra (330 to 250 nm). For reduction of all cysteines of TlpA, DTT was added to a final concentration of 0.1 mM. After incubation for 3 h at room temperature, the spectrum of DTT-reduced TlpA was recorded. The fully reduced state of TlpA was demonstrated both by SDS-PAGE analysis and fluorescence spectroscopy.

Fluorescence Measurements

All fluorescence measurements were carried out at 30 °C on a HITACHI F-4000 spectrofluorimeter. All buffers used were filtered (pore size 0.2 µm). To avoid air oxidation, the solutions were degassed and subsequently flushed with nitrogen. Fluorescence spectra of oxidized and reduced TlpA were measured in 100 mM sodium phosphate, pH 7.0, 1 mM EDTA at a scan speed of 60 nm/min. The excitation wavelength was 280 or 295 nm at a slit width of 5 nm, whereas a slit width of 10 nm was chosen for the emission beam. The protein concentration was 0.6-1 µM. Complete reduction of TlpA was achieved by addition of DTT to 1 mM. For the fluorescence measurement of TlpA with the catalytic disulfide selectively reduced by GSH, the sample from the redox-titration experiment at a GSH^2/GSSG ratio of 29 M was analyzed.

Determination of the Redox Potential of the Active-site Cysteines of TlpA

For determination of the equilibrium constant between TlpA and glutathione, oxidized TlpA (0.5 µM) was incubated in 100 mM sodium phosphate, pH 7.0, 1 mM EDTA at 30 °C containing different concentrations of GSH (100 µM to 125 mM). Ten µM GSSG was added to the reaction mixtures having GSH^2/GSSG ratios from 2 mM to 1 M, whereas the mixtures of GSH^2/GSSG from 2 to 40 M were supplied with 5 µM GSSG. To reach equilibrium, the redox mixtures were incubated under a nitrogen atmosphere for 19 h, 44 h and 7 days. The samples were removed immediately before fluorimetric measurements. To minimize influences of glutathione absorption, an excitation wavelength of 295 nm was used.

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^2/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.

Determination of GSSG Concentration

The GSSG concentration present in the reaction mixtures after equilibration was quantified enzymatically using yeast glutathione reductase (EC 1.6.4.2; Boehringer), in order to account for air oxidation of reduced glutathione during the incubation period and the GSSG contaminations present in the GSH stock solution. Reactions were carried out in degassed 100 mM sodium phosphate, pH 7.0, 1 mM EDTA at 25 °C. NADPH was added to a final concentration of 200 µM. Reactions were started by addition of 1 unit of enzyme. After 10 min the decrease in absorption at 340 nm was recorded ( = 6220 M cm). A linear calibration curve was obtained with GSSG concentrations ranging from 1 to 200 µM. The GSSG contamination in the GSH stock solution was reproducibly found to be 0.4% (w/w).

Protein Concentration

The molar extinction coefficient of unfolded, oxidized TlpA ( = 18,590 M cm) was calculated from its amino acid sequence as described by Gill and von Hippel(19) . From the comparison of the absorbance of native and denatured TlpA (6.5 M GdmCl) at 280 nm, an extinction coefficient of = 17,270 M cm was determined for the native, oxidized protein. Protein absorption spectra were recorded on a CARY 3A UV/VIS spectrophotometer.


RESULTS

Stability of the Non-active-site Disulfide Bond against GSH and DTT

TlpA differs from DsbA and thioredoxin in that it contains a structural disulfide bond in addition to the active-site disulfide. Since this structural disulfide may interfere with the redox analysis of the active-site disulfide in the presence of other thiol compounds, we first investigated its stability against DTT and GSH. As shown previously, the redox state of the structural disulfide can be identified by different migration of the oxidized and reduced form in nonreducing SDS gels(14) .

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.



Spectral Properties of TlpA

We then analyzed the influence of GSH and DTT on the catalytic disulfide bond of TlpA by fluorescence spectroscopy. It was previously observed that DsbA (7) and thioredoxin from E. coli(20) , as well as calf liver PDI (21) show an increase in fluorescence intensity upon reduction of the active-site cysteines. This can be explained by the quenching effect of the active-site disulfide on the fluorescence of an adjacent tryptophan. In the case of DsbA, this feature was used to determine the redox potential of its active site(7) . At an excitation wavelength of 295 nm (selective excitation of tryptophans), a 9.5-fold increase in the fluorescence of TlpA at 355 nm after equilibration with a [GSH]^2/[GSSG] ratio of 29 M was detected (Fig. 2A). Therefore, GSH exclusively reacts with the catalytic disulfide bond in TlpA. As shown below (cf. Fig. 4and ``Discussion''), about 94% of all TlpA molecules are reduced at the active-site at equilibrium under the above conditions. Reduction of TlpA by DTT (1 mM) resulted in a 10-fold increase in fluorescence at 355 nm. This corresponds exactly to the different relative increase in fluorescence between TlpA reduced by DTT and by a [GSH]^2/[GSSG] ratio of 29 M. Therefore, the fluorescence spectra of TlpA reduced at both disulfide bonds and TlpA reduced solely at the active-site are virtually identical.


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^2/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 (bullet), DTT-reduced (circle) 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 (bullet), native DTT-reduced (circle), 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 (bullet), 44 h (circle), 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^2 dmol for oxidized and reduced TlpA (Fig. 3A), respectively, which is a typical feature of proteins rich in alpha-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.

Determination of the Redox Potential of TlpA's Active-site Cysteines

The fluorescence properties of TlpA and the selective reduction of its active-site disulfide by GSH were then used to measure the equilibrium constant between TlpA and glutathione. The redox equilibrium of TlpA with glutathione is given by and .

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`(0)(T)) was calculated from the Nernst equation using a value of -0.205 V (24) for the glutathione standard potential (E`, ).

Attempts to Oxidize the Reduced Active-site of TlpAby DsbA from E. coli

The equilibrium constant of TlpA with glutathione of 1.9 ± 0.2 M indicates that TlpA is a reductant similar to thioredoxin. The bacterial periplasm, however, is considered to be an oxidizing environment, where disulfide bonds are readily formed. In E. coli, the DsbA protein has been shown to be the final oxidant of protein thiols in the periplasm. DsbA is a very efficient oxidant of thiols in folding secretory proteins and is recycled as an oxidant by DsbB, an integral inner membrane protein of E. coli(25, 26, 27) . Since oxidation of TlpA by a DsbA/B-related oxidative system in the periplasm of B. japonicum would not be consistent with a reducing function of TlpA in vivo, we analyzed the kinetics of oxidation of the active site of TlpA by DsbA from E. coli. We determined the overall fluorescence difference spectrum for the oxidation of TlpA selectively reduced at its active site by DsbA, which demonstrates that the reaction must result in a more than 2-fold increase in the overall fluorescence emission at 320 nm (Fig. 5A). However, no change in the fluorescence signal could be observed after the onset of the reaction at pH 7 (Fig. 5B). Since the thermodynamic equilibrium of the reaction lies far on the side of reduced DsbA and oxidized TlpA, the reaction must be restricted kinetically, presumably due to steric inaccessibility of TlpA's active site.


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. bullet, DsbA (ox) + TlpA (red); circle, 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 (bullet). 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 (circle).




DISCUSSION

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`(0) 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) approx -0.26 V) are involved in reducing cystine bridges in cytoplasmic proteins(1) . DsbA (E`(0) = -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 times 10M(7) , respectively), and has a value of 1.6 times 10^4.

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(2) = 180 ± 20 M s at pH 7.0), although the reaction is significantly slower than the oxidation of organic dithiols by DsbA (26) . (^2)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(3), 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(3) 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(3) oxidase contains a characteristic binuclear copper center (Cu(A)) 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(3) 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.


FOOTNOTES

*
This work was supported by grants from the Federal Institute of Technology, Zürich, and the Swiss National Foundation for Scientific Research (to H. H. and R. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule, ETH-Hönggerberg, CH-8093 Zürich, Switzerland. Tel.: 41-1-633-6819; Fax: 41-1-633-1036; RUDI@MOL.BIOL.ETHZ.CH.

(^1)
The abbreviations used are: PDI, protein disulfide isomerase; CD, circular dichroism; DTT, 1,4-dithio-DL-threitol; GdmCl, guanidinium chloride; GSH, reduced glutathione; GSSG, oxidized glutathione; MES, 2-(N-morpholino)ethanesulfonic acid; NADPH/NADP, reduced and oxidized form of nicotinamide adenine dinucleotide phosphate; PAGE, polyacrylamide gel electrophoresis; TlpA, soluble form of TlpA (see (14) ).

(^2)
M. Wunderlich and R. Glockshuber, unpublished results.


ACKNOWLEDGEMENTS

We thank our colleagues Heinrich Fischer and Klaus Maskos for fruitful discussions, Dr. Gerhard Frank for N-terminal sequencing of TlpA, and Mike Ibba for critically reading the manuscript.


REFERENCES

  1. Holmgren, A. (1989) J. Biol. Chem. 264, 13963-13966 [Free Full Text]
  2. Gilbert, H. F. (1994) in Mechanisms of Protein Folding (Pain, R. H., ed) pp. 104-136, IRL Press, Oxford
  3. Loferer, H., and Hennecke, H. (1994) Trends Biochem. Sci. 19, 169-171 [CrossRef][Medline] [Order article via Infotrieve]
  4. Bardwell, J. C. A. (1994) Mol. Microbiol. 14, 199-205 [Medline] [Order article via Infotrieve]
  5. Zapun, A., Bardwell, J. C. A., and Creighton, T. E (1993) Biochemistry 32, 5083-5092 [Medline] [Order article via Infotrieve]
  6. Wunderlich, M., Jaenicke, R., and Glockshuber, R. (1993) J. Mol. Biol. 233, 559-566 [CrossRef][Medline] [Order article via Infotrieve]
  7. Wunderlich, M., and Glockshuber, R. (1993) Protein Sci. 2, 717-726 [Abstract/Free Full Text]
  8. Hawkins, H. C., de Nardi, M., and Freedman, R. B. (1991) Biochem. J. 275, 341-348 [Medline] [Order article via Infotrieve]
  9. Lundström, J., and Holmgren, A. (1993) Biochemistry 32, 6649-6655 [Medline] [Order article via Infotrieve]
  10. Berglund, O., and Sjöberg, B.-M. (1970) J. Biol. Chem. 245, 6030-6035 [Abstract/Free Full Text]
  11. Gleason, F. K. (1992) Protein Sci. 1, 609-616 [Abstract/Free Full Text]
  12. Krause, G., Lundström, J., Barea, J. L., de la Cuesta, C. P., and Holmgren, A. (1991) J. Biol. Chem. 266, 9494-9500 [Abstract/Free Full Text]
  13. Loferer, H., Bott, M., and Hennecke, H. (1993) EMBO J. 12, 3373-3383 [Abstract]
  14. Loferer, H., and Hennecke, H. (1994) Eur. J. Biochem. 223, 339-344 [Abstract]
  15. Studier, F. W., and Moffatt, B. A. (1986) J. Mol. Biol. 189, 113-130 [Medline] [Order article via Infotrieve]
  16. Wunderlich, M., and Glockshuber, R. (1993) J. Biol. Chem. 268, 24547-24550 [Abstract/Free Full Text]
  17. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  18. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77 [Medline] [Order article via Infotrieve]
  19. Gill, S. C., and von Hippel, P. H. (1989) Anal. Biochem. 182, 319-326 [Medline] [Order article via Infotrieve]
  20. Holmgren, A. (1972) J. Biol. Chem. 247, 1992-1998 [Abstract/Free Full Text]
  21. Lundström, J., and Holmgren, A. (1990) J. Biol. Chem. 265, 9114-9120 [Abstract/Free Full Text]
  22. Schmid, F. X. (1989) in Protein Structure, A Practical Approach (Creighton, T. E., ed) pp. 251-285, IRL Press, Oxford
  23. Johnson, W. C., Jr. (1988) Annu. Rev. Biophys. Biophys. Chem. 17, 145-166 [CrossRef][Medline] [Order article via Infotrieve]
  24. Szajewski, R. P., and Whitesides, G. M. (1980) J. Am. Chem. Soc. 102, 2011-2026
  25. Bardwell, J. C. A., McGovern, K., and Beckwith, J. (1991) Cell 67, 581-589 [Medline] [Order article via Infotrieve]
  26. Wunderlich, M., Otto, A., Seckler, R., and Glockshuber, R. (1993) Biochemistry 32, 12251-12256 [Medline] [Order article via Infotrieve]
  27. Bardwell, J. C. A., Lee, J.-O., Jander, G., and Beckwith, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1038-1042 [Abstract]
  28. Holmgren, A. (1980) Biochemistry 20, 3204-3207
  29. Krause, G., and Holmgren, A. (1991) J. Biol. Chem. 266, 4056-4066 [Abstract/Free Full Text]
  30. Katty, S. K., LeMaster, D. M., and Eklund, H. (1990) J. Mol. Biol. 212, 167-184 [Medline] [Order article via Infotrieve]
  31. Holmgren, A. (1984) Methods Enzymol. 107, 295-300 [Medline] [Order article via Infotrieve]
  32. Lundström, J., Krause, G., and Holmgren, A. (1992) J. Biol. Chem. 267, 9047-9052 [Abstract/Free Full Text]
  33. Beckman, D. L., and Kranz, R. G. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2179-2183 [Abstract]
  34. Thöny-Meyer, L., Ritz, D., and Hennecke, H. (1994) Mol. Microbiol. 12, 1-9 [Medline] [Order article via Infotrieve]
  35. Bott, M., Bolliger, M., and Hennecke, H. (1990) Mol. Microbiol. 4, 2147-2157 [Medline] [Order article via Infotrieve]
  36. Kelly, M., Lappalainen, P., Talbo, G., Haltia, T., van der Oost, J., and Saraste, M. (1993) J. Biol. Chem. 268, 16781-16787 [Abstract/Free Full Text]

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