YcdB from Escherichia coli Reveals a Novel Class of Tat-dependently Translocated Hemoproteins*

The Tat (twin-arginine translocation) system of Escherichia coli serves to translocate folded proteins across the cytoplasmic membrane. The reasons established so far for the Tat dependence are cytoplasmic cofactor assembly and/or heterodimerization of the respective proteins. We were interested in the reasons for the Tat dependence of novel Tat substrates and focused on two uncharacterized proteins, YcdO and YcdB. Both proteins contain predicted Tat signal sequences. However, we found that only YcdB was indeed Tat-dependently translocated, whereas YcdO was equally well translocated in a Tat-deficient strain. YcdB is a dimeric protein and contains a heme cofactor that was identified to be a high-spin FeIII-protoporphyrin IX complex. In contrast to all other periplasmic hemoproteins analyzed so far, heme was assembled into YcdB in the cytoplasm, suggesting that heme assembly could take place prior to translocation. The function of YcdB in the periplasm may be related to a detoxification reaction under specific conditions because YcdB had peroxidase activity at acidic pH, which coincides well with the known acid-induced expression of the gene. The data demonstrate the existence of a class of heme-containing Tat substrates, the first member of which is YcdB.

The Tat (twin-arginine translocation) system of Escherichia coli serves to translocate folded proteins across the cytoplasmic membrane. The reasons established so far for the Tat dependence are cytoplasmic cofactor assembly and/or heterodimerization of the respective proteins. We were interested in the reasons for the Tat dependence of novel Tat substrates and focused on two uncharacterized proteins, YcdO and YcdB. Both proteins contain predicted Tat signal sequences. However, we found that only YcdB was indeed Tat-dependently translocated, whereas YcdO was equally well translocated in a Tat-deficient strain. YcdB is a dimeric protein and contains a heme cofactor that was identified to be a high-spin Fe IIIprotoporphyrin IX complex. In contrast to all other periplasmic hemoproteins analyzed so far, heme was assembled into YcdB in the cytoplasm, suggesting that heme assembly could take place prior to translocation. The function of YcdB in the periplasm may be related to a detoxification reaction under specific conditions because YcdB had peroxidase activity at acidic pH, which coincides well with the known acid-induced expression of the gene. The data demonstrate the existence of a class of heme-containing Tat substrates, the first member of which is YcdB.
The Tat (twin-arginine translocation) system delivers folded proteins across biological membranes (1). So far, the functional Tat systems from bacterial cytoplasmic membranes and plant plastid thylakoid membranes have been characterized (1). Transport is driven by the membrane potential, and the costs for translocation appear to be very high (2), which could explain why many organisms prefer the less energyconsuming translocation of unfolded proteins via the Sec apparatus. The most complete Tat substrate analyses have been reported for Escherichia coli (3). Many Tat substrates from E. coli are translocated as heterodimers or after cytoplasmic cofactor assembly; and therefore, they obviously need to be translocated in a folded conformation. However, even in this model organism, several postulated Tat substrates exist, the Tat dependence of which is neither established nor understood. Among these are YcdO and YcdB, two uncharacterized proteins with genes that appear to be organized in one operon with the gene ycdN, which encodes a putative metal ion transporter (4). In the E. coli K12 genome, ycdN seems to be largely truncated and may therefore form no functional gene product (5). YcdO and YcdB are proteins of unknown function. In proteomic studies, YcdO has been shown to be periplasmic and more abundant at acidic pH (6). In agreement with this, studies on global gene regulation have shown that the expression of the whole ycdNOB gene cluster is induced in response to acidic conditions (7). No further experimental work on YcdO or YcdB has been reported, but a similarity of YcdB to a dye-decolorizing peroxidase of the fungus Geotrichum candidum has been recognized by certain algorithms created at The Institute of Genomic Research (accession number TIGR01412).
In this study, we aimed to analyze biochemically the uncharacterized putative E. coli Tat substrates YcdB and YcdO. While YcdO turned out not to be a Tat substrate, we found that YcdB is indeed translocated by the Tat system. YcdB contains a noncovalent heme cofactor, and this cofactor is assembled in the cytoplasm. The data demonstrate that YcdB is the first member of a new class of hemecontaining Tat substrates with homologs in many bacterial species.
Biochemical Methods-YcdO and YcdB were purified by affinity chromatography using Strep-Tactin Superflow TM resin (IBA GmbH) following the manufacturer's protocol, but EDTA was generally omitted from buffers. Protein concentrations were determined according to Lowry et al. (12), and SDS-PAGE analyses were carried out according to Laemmli (13). For native gels, SDS was omitted from all buffers, and samples were not reduced. The YcdO or YcdB constructs were detected by Western blot analyses after SDS-PAGE as described previously (14) using either Strep-Tactin-horseradish peroxidase conjugate (IBA GmbH) or anti-high potential iron-sulfur protein-His 6 polyclonal antibodies (14). Subcellular fractionations were carried out as described previously (15). The molecular masses of purified proteins were determined by electrospray mass spectrometry. The spectra were acquired with an ESI-Q-Tof 2 mass spectrometer (Waters Corp., Manchester, UK) equipped with a nanospray ion source. The samples were injected via a PicoTip (New Objective, Inc., Cambridge, MA) with a syringe pump (Harvard Apparatus, Holliston, MA) at a flow rate of 300 nl/min. The MaxEnt TM 1 algorithm was used for deconvoluting the data to a single charge state. Analytical ultracentrifugation was carried out with YcdB at 0.4 mg/ml in 50 mM Tris (pH 8.0) and with YcdO at 0.2 mg/ml in 20 mM Tris (pH 7.4) in a Beckman Optima XL-A centrifuge equipped with an An50Ti rotor and double sector cells. Sedimentation equilibrium measurements were carried out at 20°C and 15,000 rpm, and sedimentation velocity was monitored at 20°C and 40,000 rpm. The data were recorded at wavelengths of 230, 280, and 405 nm and analyzed using the software provided by Beckman Instruments. Absorption data for kinetics and spectra were recorded with an Ultrospec 4000 spectrophotometer (Amersham Biosciences). When indicated, samples were reduced in the cuvettes with a few grains of sodium dithionite. Peroxidase activity was determined with 10 mM guaiacol and 10 mM H 2 O 2 . One unit corresponds to 1 mol of guaiacol oxidized per min at 22°C in the indicated buffer at 100 mM. The reaction was started by addition of enzyme. Tetraguaiacol formation was followed photometrically at 470 nm ( 470 nm ϭ 26.6 mM Ϫ1 cm Ϫ1 ). For activity stains on native gels, gels were equilibrated with 100 mM sodium citrate (pH 4.0) and 100 M dianisidine, and the peroxidase reaction was started by addition of 100 M H 2 O 2 .

RESULTS
YcdB Is a Tat Substrate, whereas YcdO Is Not-YcdB from E. coli is distantly related to the dye-decolorizing peroxidase (DyP) 2 identified in the fungus G. candidum (16,17). There are bacterial DyP-like proteins in many bacterial species, but none of them has ever been biochemically analyzed. E. coli contains two of these DyP-like proteins, YfeX and YcdB, which share some sequence homology in their C-terminal halves. Of these, YcdB belongs to a subclass of DyP-like proteins with predicted Tat signal sequences, one member of which has been shown to be a Tat substrate in the Gram-positive Bacillus subtilis (18). YcdB was a clear candidate for a novel class of Tat substrates. We thus clarified the Tat dependence of YcdB in E. coli and carried out the first purification and biochemical characterization of a bacterial YcdB homolog.
In Fig. 1A, the signal sequence of YcdB from E. coli is compared with those of YcdB homologs from species of other phyla, including ␣-Proteobacteria, ␤-Proteobacteria, bacilli, and Actinobacteria. As typically found for Tat substrates, YcdB homologs have rather long signal sequences with twin-arginine motifs at the end of a polar n-region of variable length, and the h-regions are rich in alanines and glycines (19). The conservation of these Tat signal sequence characteristics suggested to us that YcdB is indeed very likely to be translocated in a folded state across the cytoplasmic membrane by the Tat system.
When we analyzed the localization of recombinant YcdB in the cell, we detected mature YcdB in the periplasmic fraction (Fig. 1B). The co-detection of the biotin carrier protein in the cytoplasm showed, as an internal control on the same blot, that no cytoplasm was leaking into the periplasmic fraction. Therefore, although the amount of periplasmic YcdB was rather low under these conditions, the presence of YcdB in the periplasm indicated that it could be translocated across the cytoplasmic membrane. As typically observed with recombinant Tat substrates, the translocation was relatively poor with wild-type levels of the Tat translocon. This is because the Tat system is slow and becomes rapidly saturated (20). With YcdB, the high sensitivity of the signal sequence to degradation adds to these effects, as mature-size degradation products are formed in the cytoplasmic and membrane fractions containing the accumulating precursor. Together, the data at the wild-type Tat level showed a limited translocation of recombinant YcdB into the periplasm.
To test whether translocation of YcdB depends on the presence of Tat system components, we tested the translocation in a TatABCDE-defi- 2 The abbreviation used is: DyP, dye-decolorizing peroxidase. Precursor YcdB (pre-YcdB) and mature YcdB (mat-YcdB) were detected in subcellular fractions from MC4100 (wild-type (wt)), from its Tat-deficient derivative DADE (⌬tat), and from DADE with a tatABC complementation system (⌬tat/pRK-tatABC). Ten microliters of periplasmic (P), membrane (M), and cytoplasmic (C) fractions were separated by SDS-PAGE and analyzed by Western blotting using Strep-Tactin-horseradish peroxidase conjugate for development. Expression of ycdB-Strep from pBAD-ycdB-Strep was induced for 2 h with 0.01% arabinose. The positions of marker proteins are indicated on the right. The biotin carrier protein (BCP) was used as an internal cytoplasmic marker and demonstrated the quality of the periplasmic fractions. C, the relative amount of cytoplasmic YcdB is significantly reduced at low expression levels. The YcdB precursor and mature forms were detected in subcellular fractions at varied lower induction levels. As indicated, YcdB production in strain MC4100 with pABS-tatABC (10) and pBAD-ycdB-Strep was induced with 0.00025 or 0.0001% arabinose (ara) for 2 h prior to fractionation. cient derivative strain. Strikingly, there was not any transport of YcdB detectable without the Tat system (Fig. 1B). Thus, YcdB indeed requires the Tat system for translocation into the periplasm. Again, the precursor accumulated in the membrane and cytoplasmic fractions. An interesting aspect on which we did not focus in this study is that the detection of the precursor in the membrane fraction did not depend on the presence of the Tat system.
To complete the evidence for the Tat dependence, we tested the complementation of YcdB translocation by tatABC, which was expressed in trans from its own promoter using the compatible low-copy vector pRK-tatABC (15). We found that the translocation defect in the Tat-deficient strain was fully compensated by the tatABC genes in trans. The complementation system very much improved the translocation compared with the wild-type strain (Fig. 1B). Such a positive effect of the increased Tat system abundance on transport is typically observed with recombinant Tat substrates (20). A significant portion of YcdB accumulated in the cytoplasm, and a part of this was of mature size. Such a "mature" species could point to a natural cytoplasmic subpopulation of YcdB, or it simply could be the result of a translocon limitation: signal sequences of accumulating Tat substrates are often found to be sensitive to proteases, resulting in digestion of the signal sequence while the folded mature part of the protein remains intact. We addressed this aspect by lowering the ycdB induction (Fig. 1C). Strikingly, no precursor of YcdB accumulated at lowered induction, and the cytoplasmic processing decreased markedly relative to the amount of periplasmic YcdB. As cytoplasmic maturation is enhanced by a limited translocation at higher expression levels, it is unlikely to reflect some significant process under natural conditions. Together, our results indicate that YcdB is a novel Tat-dependently translocated periplasmic protein.
We also addressed the possible Tat dependence of the translocation of YcdO. On the basis of signal sequence characteristics, YcdO has been postulated to be a Tat substrate (3). However, the twin-arginine motif in its signal sequence is not conserved in homologs from other phyla. In addition, there is a deviation from the consensus Tat signal pattern that is very unusual. The consensus twin-arginine pattern is (S/T)RRXFLK. In this pattern, the two arginines are almost invariable with the exception of a rarely occurring exchange of the first Arg with Lys (21). Hydrophobic residues are commonly not found at the S/T position preceding the two arginines. YcdO has a hydrophobic Phe residue at that position, which raises additional doubts regarding the prediction that YcdO could be a Tat substrate.
When we examined the subcellular localization of YcdO, we found that Ͼ50% of the detected recombinant YcdO was periplasmic. This rather high translocation efficiency was not Tat-dependent, as YcdO was equally well translocated in the absence of all Tat system components (Fig. 2). In SDS-PAGE analyses, periplasmic YcdO migrated at the same size as cytoplasmic YcdO. Again, as an internal marker on the same blot, the detected biotin carrier protein of the cytoplasmic fraction indicated that no cytoplasm had leaked into the periplasmic fraction. We examined whether the detected periplasmic YcdO was correctly processed. Recombinant YcdO was purified from the periplasmic fraction by affinity chromatography using a Strep-Tactin matrix and was subjected to electrospray mass spectrometry. We determined a molecular mass of 40,458 Da, which indicates a complete and homogeneous cleavage of the signal sequence behind position 26 (supplemental Fig.  S1). The three residues preceding this cleavage site are ANA, which constitute a typical LepB cleavage site. Therefore, all periplasmically detected YcdO corresponded to translocated and correctly processed protein. Analytical ultracentrifugation indicated that YcdO is monomeric in solution (data not shown). Together, the data show that YcdO is a monomeric periplasmic protein that is not Tat-dependently translocated. These data led us to focus on YcdB, as only YcdB was Tat-dependently translocated.
YcdB Contains Fe III -Protoporphyrin IX-Recombinant YcdB with a C-terminal Strep-tag II could be purified from the periplasmic fraction and was analyzed by electrospray mass spectrometry (supplemental Fig.  S2). The mass was found to be 45,127 Da, which exactly matches the mass of the mature protein when cleaved behind the typical LepB signal cleavage site (AHA) at a distance of 35 residues from the N terminus. A second mass of 43,814 Da corresponds to part of the purified mature protein with its C-terminal Strep-tag II degraded. There was no precursor detectable in the periplasmic fraction. When we analyzed the oligomeric state of YcdB by analytical ultracentrifugation, we determined an apparent mass of 89.9 Ϯ 0.8 kDa, which indicates that periplasmic YcdB is a dimer (supplemental Fig. S3).
The periplasmic YcdB preparation was slightly reddish, pointing to the presence of some chromogenic cofactor associated with YcdB. This cofactor could not be covalently bound, as the mentioned mass analysis did not show any alteration of the mass expected for the unmodified mature protein. In addition to the protein absorption at 277 nm, we identified a heme Soret band with a maximum at 406 nm and further weak features between 485 and 660 nm in UV-visible absorption spectra of periplasmic YcdB (Fig. 3A). The purified protein had an A 406 nm / A 277 nm ratio near 1.5. The extinction coefficient of the 406 nm band was calculated to be ϳ65 mM Ϫ1 cm Ϫ1 . Upon reduction with sodium dithionite, the 406 nm band slowly shifted within several minutes to 432 nm, and the weak features at longer wavelengths somewhat condensed and formed maxima at 558 and 622 nm. These data indicate that the heme in YcdB is purified in an oxidized state. Furthermore, the slow reduction of the heme iron by the strongly reducing dithionite suggests that transitions other than the Fe III /Fe II transition might be physiological.
Knowing that YcdB contains a noncovalent heme, we searched for the heme signal by electrospray mass spectrometry. We detected a signal at 616 Da, which was likely to correspond to an iron-containing protoporphyrin IX cofactor. Fragmentation analyses confirmed the identity of the heme cofactor (Fig. 3B). The fragments deduced from the 616 Da mass corresponded to two consecutive losses of 59 Da, which are indicative of cleavages in the heme propionate side chains (22). These data unequivocally demonstrate that YcdB contains an Fe III -protoporphyrin IX complex.
The Heme Iron in YcdB Has a High-spin Electron Configuration-For functional aspects, it is important to determine the spin state of the heme iron in the hemoprotein. Cytochromes with electron transport function are usually low-spin, and the heme iron has six ligands in oxidized and reduced states. High-spin hemes have either five or six ligands in their oxidized state and always only five ligands in their reduced state (23). Therefore, hemoproteins that transiently bind substrates as axial ligands of the heme iron usually have a high-spin electron conformation. Among these high-spin hemoproteins are enzymes such as oxygenases, catalases, peroxidases, and ligand carriers.
A clear classification of the spin state of hemoproteins can be made with spectra of reduced hemes with bound carbon monoxide (CO). The strong ligand field with CO causes a shift in the reduced high-spin heme iron to the low-spin electron configuration upon CO binding. The dithionite-reduced YcdB precursor was incubated with CO, and CO binding resulted in a sharp Soret band at 419 nm and further alterations in the region between 530 and 600 nm (Fig. 4A). The CO redminus-reduced difference spectrum clearly showed the CO red ␣-maximum at 572 nm, the ␤-maximum at ϳ536 nm, and the ␥-maximum at 419 nm (Fig. 4B). The extinction difference between the peak at 419 nm and the trough at 437 nm was ϳ30-fold higher than the corresponding difference for the ␣-band, indicating that the heme of YcdB is high-spin in the reduced state. The Soret maximum of Ͼ418 nm in the CO-bound state is in agreement with the noncovalent binding mode of the heme cofactor (23). Together, the spectroscopic data confirm that YcdB contains a noncovalent high-spin heme. YcdB is therefore likely to function as an enzyme with oxygenase or peroxidase activity.
YcdB Has Peroxidase Activity at Low pH-The presence of a highspin heme and the postulated relation to fungal DyP strengthened the hypothesis that YcdB might be a peroxidase. Peroxidases perform a Fe III /Fe IV transition during their catalytic cycle when they produce the compound I intermediate, in which oxygen forms a double bond with Fe IV (24). To examine the possible peroxidase activity of YcdB, we used the quantitative guaiacol assay. In this assay, the H 2 O 2 -dependent oxidation of the colorless guaiacol results in a guaiacol tetramer, which can be monitored at 470 nm ( 470 nm ϭ 26.6 mM Ϫ1 cm Ϫ1 ). Because YcdB is induced under acidic conditions (7), we measured the activity at different pH values in the acidic to neutral region. We found that YcdB had significant guaiacol peroxidase activity at pH 4.0 (Fig. 5, A and B). The activity was somewhat lower but still significant at pH 3.0. Under less acidic conditions (at pH 5.0 or pH 6.0), the activity was markedly reduced. From the data, we calculated an activity of ϳ200 milliunits/mg purified YcdB with guaiacol as the H 2 O 2 -dependently oxidized substrate. As the natural substrate of YcdB is unknown, the specific peroxidase activity may be manyfold higher in vivo. Having established the peroxidase activity of YcdB, we wondered what influence hydrogen peroxide might have on the spectral characteristics of YcdB. Interestingly, H 2 O 2 addition to oxidized YcdB caused major spectral alterations, indicating that H 2 O 2 could generate a ligand to the ferric iron. The Soret band shifted to 414 nm, and three signals appeared at longer wavelengths of 530, 555, and 603 nm (Fig. 5C). Such spectral changes might reflect the formation of an intermediate similar to compound I in the known peroxidase cycle. Some preparations of YcdB already partially showed the characteristics of this adduct (data not shown), implying that YcdB undergoes this type of reaction also in vivo. Together, the data suggest that YcdB may have a functional role as a periplasmic peroxidase at low pH.
YcdB Can Assemble Heme in the Cytoplasm-It was important to determine whether YcdB can assemble its cofactor in the cytoplasm prior to translocation. We therefore purified YcdB also from the translocation-deficient DADE strain and recorded the UV-visible spectra from these preparations. Any heme in YcdB from this strain would indicate a cytoplasmic assembly. The spectra showed clearly that the heme cofactor was assembled (Fig. 6A). As a significant portion of YcdB in the cytoplasm had proteolytically lost its signal sequence (see Fig. 1B), we wanted to differentiate the cofactor content of the cytoplasmic precursor and mature YcdB. YcdB was purified from the cytoplasmic frac-  tion of MC4100 carrying pBAD-ycdB-Strep, and the heme content of the cytoplasmic YcdB species was assessed by native gel electrophoresis and subsequent activity or Coomassie Blue stains of the gel (Fig. 6B). Three activity bands were separated. A single band corresponding to the mature protein was readily identified by comparison with mature periplasmic YcdB. The two other active bands could be assigned to the YcdB precursor and thus indicated the presence of the cofactor in the precursor. The two bands are likely to reflect two differently interacting populations. Interestingly, not all of the YcdB precursor was active: a significant population of the inactive precursor had not yet acquired the cofactor and migrated as a diffuse and inactive "smear" on native gels. The signal sequence was detected in this fraction by mass spectrometry (trypsin fragment K2DENGVNEPSR2). The diffuse migration was probably due to greater conformational flexibility and aggregation effects of the signal sequence in the apoprecursor. The cytoplasmic heme insertion was independent of the presence of a functional Tat translocon. These data indicate that the precursor of YcdB has the ability to recruit heme in the cytoplasm.

YcdB Is the First Member of a New Class of Tat Substrates-This
study was initiated to reveal the translocation mode and structural characteristics of two postulated protein substrates of the Tat system, YcdB and YcdO. We have demonstrated that, of these two proteins, only YcdB is a Tat substrate in E. coli, and this protein turned out to be the first example of a Tat-dependently translocated hemoprotein.
In E. coli, all other periplasmic hemoproteins analyzed so far are Sec substrates, which fold and assemble their cofactors after translocation.  The biogenesis of periplasmic c-type cytochromes, which are proteins with two cysteine-derived thioether links to the vinyl groups of protoporphyrin IX, has been well studied (25). All c-type cytochromes contain a heme attachment pattern (CXXCH) that forms a disulfide in the oxidative periplasmic environment and therefore has to be reduced by specific reductases to allow heme ligation to the protein. The histidine in this pattern serves as an axial ligand for the heme iron. The ccmAB-CDEFGH genes are responsible for heme delivery and attachment to c-type cytochromes in the periplasm of E. coli. They are also involved in the reduction of the target CXXCH motif prior to heme ligation. However, none of these genes could be demonstrated to carry out heme transport across the membrane (25). The same heme that is used for c-type cytochromes is also used for b-type cytochromes, which do not attach their heme covalently. As b-type cytochromes can assemble their heme in the periplasm without involvement of the ccm genes, heme transport is an unanswered question (26).
With YcdB, the first protoporphyrin IX-containing protein transported by the Tat system has been identified. Tat substrates such as iron-sulfur proteins, molybdoproteins, flavoproteins, and nickel proteins assemble their cofactors in the cytoplasm (19). As heme biogenesis takes place in the cytoplasm and as no periplasmic machinery is known to be required for noncovalent heme assembly, there is no argument against heme cotransport with Tat substrates. YcdB folds in the cytoplasm to an active holoprotein, just as all other Tat substrates do that are known to assemble their cofactors prior to translocation.
It is not surprising that heme can be assembled into Tat substrates prior to translocation, as it has been shown with recombinant Tat signal sequence fusions that, in principle, hemoproteins can be translocated by the Tat system if they carry only their heme before translocation (27). However, with YcdB, we found the first hemoprotein that is naturally translocated by the Tat system.
Why is YcdB not translocated by the Sec system? The Tat dependence indicates a requirement for cytoplasmic folding, but not necessarily for cytoplasmic heme insertion. YcdB is exceptional among the heme-containing proteins in its requirement for cytoplasmic folding. Heme is available on either side of the cytoplasmic membrane; and therefore, the cofactor insertion may not be the reason for the Tat-dependent translocation of YcdB. We clearly detected heme in the cytoplasmic YcdB precursor, suggesting that the holoprotein can be translocated. We also detected an apoprecursor, corresponding to the fraction that had not yet assembled its cofactor. If the apoprecursor can fold without the heme, it is possible that it assembles the heme after translocation.
As heme insertion can also occur in the periplasm, other folding requirements that make the Tat system necessary have to be considered. YcdB does contain four cysteines. One of these is positioned in the hydrophobic region of the signal sequence, and the others are separate cysteines in the mature domain. Folding in the periplasm could result in undesired disulfide formation, making cytoplasmic folding necessary. Another explanation may be that YcdB has to be folded outside even under conditions of acid stress, as the protein is induced under acid conditions and may thus function in response to acid stress (7). Folding in the periplasm could be less effective under such conditions, making translocation as a folded protein necessary. These explanations show that many factors have to be considered as causative for the Tat dependence of YcdB, and further yet unknown circumstances may turn out to be important.
YcdB Is a Structural Homolog of Fungal DyP-Bacterial proteins that share some sequence homology with fungal DyP-type peroxidases have been identified in the past. This resulted in the creation of the term "DyP-type peroxidase family," which has been used in many genome annotations. A Hidden Markow Model-based algorithm was created in 2002 at The Institute of Genomic Research, which recognizes FIGURE 7. Sequence comparison of the mature domains of fungal DyP and E. coli YcdB. The residue numbers refer to the mature protein, starting with position 1 behind the signal sequence cleavage sites. Identities are indicated by asterisks, conservative exchanges by colons, and similar residues by periods. The alignment was generated using ClustalW (30). Note that the C-terminal halves of the sequences contain conserved stretches that may form the heme-binding pocket. The histidine that has been postulated to be the axial ligand of fungal DyP (28) is shown in boldface and underlined.
most of these proteins (accession number TIGR01413, available at www.tigr.org). The Institute of Genomic Research already noted in the description of this postulated family that "a distinct, uncharacterized branch (TIGR01412) of this superfamily has a typical twin-arginine dependent signal sequence characteristic of exported proteins with bound redox cofactors." The assignment of YcdB as a DyP-like protein was indirect, and no bacterial member of this family has ever been characterized. The only characterized peroxidase of the DyP family, an enzyme from the fungus G. candidum, has only 19% sequence identity to YcdB (Fig. 7), which is slightly above the identities of ϳ13-14% often found between structurally unrelated proteins of similar size (e.g. TatC and TatD).
The sequence alignment of YcdB and DyP shows that the postulated ligand of the heme of G. candidum DyP (28) is not at a similar position in YcdB (Fig. 7). His 164 of mature DyP has been tentatively assigned as the axial ligand histidine (29), and an H164A exchange of DyP results in a significant decrease in activity, attributed to loss of the heme (28). If His 164 is indeed the axial ligand in DyP, then there are major differences in ligandation of the hemes in DyP and bacterial DyP-family proteins. This is surprising, as our data strongly suggest that heme binding is similar in DyP and YcdB. The oxidized heme in fungal DyP has a Soret maximum at 406 nm (16), as we found for YcdB (Fig. 3). Another similarity is that YcdB has peroxidase activity at acidic pH with guaiacol as substrate (Fig. 5), which has also been found for DyP (16). It will thus be a very interesting future task to compare the structures of DyP and YcdB. The similarities between DyP and YcdB reside mainly in the C-terminal half of these proteins, and some conserved patterns can be found here, suggesting that this domain comprises the conserved parts of the heme-binding pocket (Fig. 7).
YcdB May Function as a Peroxidase under Acid Stress Conditions-YcdB has been shown to have its heme iron in a high-spin configuration (Fig. 4). This is functionally important, as it implies a five-ligand coordination in the reduced state, whereas the oxidized heme iron may bind either five or six ligands. The high-spin configuration suggests that a substrate ligand can be bound directly to the heme, such as in oxygenases, peroxidases, and ligand-transporting hemoproteins. It was therefore an important observation that YcdB has peroxidase activity, as does fungal DyP (Fig. 5). The natural substrates are not known for DyP or YcdB. However, it is intriguing that peroxidase activity is significantly enhanced at acidic pH, and acidic conditions have been shown previously to induce transcription of the ycdNOB operon, suggesting that YcdB may fulfill some function as a peroxidase under acidic stress conditions (7). We also consider it possible that YcdB could be capable of catalyzing other redox reactions with small ligands under acid stress conditions. However, our results suggest that such redox reactions are unlikely to involve Fe III /Fe II transitions at the heme center. Furthermore, we observed adduct formation with H 2 O 2 , which may well reflect the typical compound I stage of the peroxidase cycle (Fig. 5C). From the physiological point of view, the characterization of YcdB as a peroxidase sheds light on the conserved ycdNOB locus. Although the exact functions of the other encoded components remain to be revealed, the present results on YcdB are surely the first step toward the understanding of this locus on a biochemical level.