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Originally published In Press as doi:10.1074/jbc.M411664200 on December 21, 2004

J. Biol. Chem., Vol. 280, Issue 9, 7671-7676, March 4, 2005
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Two Heme Binding Sites Are Involved in the Regulated Degradation of the Bacterial Iron Response Regulator (Irr) Protein*

Jianhua Yang{ddagger}, Koichiro Ishimori§, and Mark R. O'Brian{ddagger}

From the {ddagger}Department of Biochemistry, State University of New York, Buffalo, New York 14214 and §Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 615-8510, Japan

Received for publication, October 13, 2004 , and in revised form, December 7, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The iron response regulator (Irr) protein from Bradyrhizobium japonicum is a conditionally stable protein that degrades in response to cellular iron availability. This turnover is heme-dependent, and rapid degradation involves heme binding to a heme regulatory motif (HRM) of Irr. Here, we show that Irr confers iron-dependent instability on glutathione S-transferase (GST) when fused to it. Analysis of Irr-GST derivatives with C-terminal truncations of Irr implicated a second region necessary for degradation, other than the HRM, and showed that the HRM was not sufficient to confer instability on GST. The HRM-defective mutant IrrC29A degraded in the presence of iron but much more slowly than the wild-type protein. This slow turnover was heme-dependent, as discerned by the stability of Irr in a heme-defective mutant strain. Whereas the HRM of purified recombinant Irr binds ferric (oxidized) heme, a second site that binds ferrous (reduced) heme was identified based on spectral analysis of truncation and substitution mutants. A mutant in which histidines 117–119 were changed to alanines severely diminished ferrous, but not ferric, heme binding. Introduction of these substitutions in an Irr-GST fusion stabilized the protein in vivo in the presence of iron. We conclude that normal iron-dependent Irr degradation involves two heme binding sites and that both redox states of heme are required for rapid turnover.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Heme is the prosthetic group or active moiety of proteins involved in a wide range of cellular functions that include catalysis, electron transport, and oxygen transport. In addition, heme proteins can be sensors that detect and respond to O2 (1, 2), CO (3, 4), NO (5), and the cellular redox state (6). Moreover, the heme moiety itself can have regulatory functions that control gene expression at the levels of transcription (7, 8), translation (9), protein localization (10), protein stability (11–13), and cell differentiation (14). Heme (protoheme) biosynthesis involves a multistep pathway culminating with the insertion of ferrous iron into protoporphyrin IX by the enzyme ferrochelatase (15). Iron can be a limiting nutrient for both prokaryotes and eukaryotes. The iron response regulator (Irr)1 protein from the bacterium Bradyrhizobium japonicum coordinates the heme biosynthetic pathway to prevent the accumulation of toxic porphyrin precursors under iron limitation (16). Loss of function of the irr gene is sufficient to uncouple the pathway from iron-dependent control as discerned by the accumulation of protoporphyrin under iron limitation. Irr belongs to the Fur family of transcriptional regulators involved in metal-dependent control of gene expression. The Irr protein accumulates in cells under iron limitation, where it negatively regulates the pathway at hemB, the gene encoding the heme biosynthetic enzyme {delta}-aminolevulinic acid dehydratase (16).

Irr is a conditionally stable protein that degrades rapidly when cells are exposed to iron, allowing derepression of heme synthesis (13). This iron-dependent degradation is mediated by heme (13). Accordingly, Irr persists in heme synthesis mutant strains in the presence of iron, and a mutation in an Irr heme binding site stabilizes the protein in the presence of the metal (13). Thus, heme is an effector molecule in Irr degradation that reflects the availability of iron for heme synthesis. Irr interacts directly with the heme biosynthesis enzyme ferrochelatase (17). Moreover, a regulatory activity for ferrochelatase was identified, distinct from its catalytic function, which allows Irr function to be controlled by the substrates of ferrochelatase. In the presence of iron, ferrochelatase inactivates Irr followed by heme-dependent degradation. Under iron limitation, protoporphyrin relieves the inhibition of Irr by ferrochelatase, probably by promoting protein dissociation, allowing genetic repression. Thus, Irr responds to iron via the status of protoporphyrin and heme locally at the site of heme synthesis. This allows heme to regulate a cellular process in the absence of a free heme pool (17).

Heme binds directly to Irr near the N terminus at a region termed the heme regulatory motif (HRM), which is necessary for rapid degradation (13). HRMs are found in functionally diverse proteins, and Cys-Pro is the only absolutely conserved sequence within the short motif. Numerous heme-mediated functions of proteins require an HRM, including mitochondrial targeting of mammalian {delta}-aminolevulinic acid synthase precursor (10), transcriptional activation of yeast HAP1 (18), repressor activity and nuclear export of human Bach1 (8, 19, 20), and the activities of cytochrome c heme lyase (21) and heme oxygenase-2 (22). It is not known how heme exerts its effect through the HRM.

In the present study, we further addressed the mechanism of iron-dependent degradation of Irr. We provide evidence for a second heme binding site that is necessary for degradation. The HRM and the new heme site bind the ferric and ferrous forms of heme, respectively, showing that normal Irr degradation involves both redox states of heme.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Media—B. japonicum I110 and LO were parent strains used in the present work. Strain MLG1 is a hemA disruption strain (23). Strain IrrC29A contains an irr allele that encodes an Irr protein with a Cys29->Ala mutation (13). Strains I110(pGST), I110(pIrr-GST), I110(pC29A-GST), and I110(pH117/118/119A-GST) contain plasmids that express GST fusion proteins of wild-type or mutant Irr proteins. Strains I110(pIrr1–150-GST), I110(pIrr1–135-GST), I110(pIrr1–116-GST), and I110(pIrr1–36-GST) contain plasmids that express GST fusion proteins of Irr C-terminal truncation. B. japonicum strains were routinely grown at 29 °C in GSY medium (35) with appropriate antibiotics. Strain MLG1 was grown with 50 µg/ml kanamycin. Strain IrrC29A was grown with 50 µg/ml tetracycline and 50 µg/ml streptomycin. Strains that contain plasmids were grown with 50 µg/ml tetracycline. For low iron medium, modified GSY medium was used, which contains 0.5g/liter yeast extract instead of 1g/liter and no exogenous iron source. The actual iron concentration of the medium was 0.3 µM, as determined with a PerkinElmer Model 1100B atomic absorption spectrometer. The high iron medium was supplemented with 6 µM FeCl3 and 15 µM hemin for heme-containing medium. Escherichia coli strain DH5{alpha} was used for propagation of plasmids in LB medium containing 100 µg/ml ampicillin.

Mutagenesis of the irr Gene—The irr gene was amplified by PCR from pUC{Delta}SES7, which contains the irr gene within pUC19, with HindIII and EcoRV sites at 5' and 3' ends, respectively. The product was ligated into HindIII/EcoRV sites of pSK to construct pSK/irr. Mutant irr genes with His->Ala mutations were amplified from pSK/irr using QuikChange (Stratagene, La Jolla, CA), using two complementary primers containing the nucleotide changes. Nucleotide sequences of mutant irr genes were confirmed by sequencing. The mutations changed the histidine codon (CAA) to an alanine codon (GCC) used highly in B. japonicum. Thus, single mutants (H117A, H118A, and H119A), double mutants (H117A/H118A, H117A/H119A, and H118A/H119A), and the triple mutant (H117A/H118A/H119A) were constructed in pSK with HindIII and EcoRV sites at 5' and 3' ends, respectively.

Construction of GST Fusion Proteins—The irr-GST gene fusion contains a GST coding region at the C terminus and irr gene, including a 211-bp promoter region and a 492-bp coding region at the N terminus. To do this, the coding region of GST was amplified by PCR from pGEX-6P-2 (Amersham Biosciences) with EcoRV and PstI sites at 5' and 3' ends, respectively. The product was ligated into EcoRV/PstI sites of pSK to construct pSK/GST. The nucleotide sequence of GST was confirmed by sequencing. To construct GST gene fusions, the EcoRV/PstI fragment from pSK/GST was ligated into the EcoRV/PstI sites of pSK constructs of irr genes, yielding pSK/Irr-GST, pSK/C29A-GST, and pSK/H117A-H118A-H119A-GST.

GST fusions of C-terminal truncations of the irr gene were amplified from pSK/Irr-GST by QuikChange using two complementary primers that encompass the deleted region, yielding pSK/Irr1–36-GST, pSK/Irr1–116-GST, pSK/Irr1–135-GST, pSK/Irr1–150-GST, and the control pSK/Pirr-GST. The primers were complementary to both sides of the deleted region. Nucleotide sequences of C-terminal truncations and the GST control were confirmed by sequencing. Truncations encode proteins that retain the N-terminal 36, 116, 135, and 150 residues of Irr, respectively.

Plasmids pGST, pIrr-GST, pC29A-GST, pH117/118/119A-GST, pIrr1–36-GST, pIrr1–116-GST, pIrr1–135-GST, and pIrr1–150-GST were constructed by ligating HindIII/PstI fragments from their pSK constructs into the broad host range vector pLAFR3. Plasmids were then introduced into B. japonicum I110 by conjugation, as described previously (24), yielding strains I110(pGST), I110(pIrr-GST), I110(pC29A-GST), I110(pH117/118/119A-GST), I110(pIrr1–36-GST), I110(pIrr1–116-GST), I110(pIrr1–135-GST), and I110(pIrr1–150-GST), respectively.

Analysis of Irr-GST Fusion Proteins—GST fusion protein levels were measured in cells grown under varying conditions of iron or heme. Aliquots of cells grown to mid-log phase were analyzed for Irr-GST protein on 12% SDS-PAGE by immunoblots using antibodies against GST (Zymed Laboratories, San Francisco, CA) or Irr (16). Detection of GroEL was carried out as a control using anti-GroEL antibodies (StressGen, Vancouver, Canada).

The turnover of GST fusion proteins in cells was examined by pulse-chase followed by GST pull-down with glutathione-Sepharose 4B beads. The cells were grown to A540 of 0.5 in low iron medium to allow the Irr-GST derivatives to accumulate in the cells. The cultures (50 ml) then were centrifuged, washed, and resuspended in 50 ml of defined medium lacking cysteine and methionine and were incubated at 29 °C for 1 h. The defined medium was as follows (per liter of media): 670 mg of yeast nitrogen base, 243 mg of amino acid dropout powder, 0.2 mg of riboflavin, 0.08 mg of p-aminobenzoic acid, 0.5 mg of nicotinic acid, 0.12 mg of biotin, 0.8 mg of thiamin·HCl, 0.8 mg of calcium pantothenate, 0.48 mg of inositol, 300 mg of KH2PO4, 300 mg of Na2HPO4, 100 mg of MgSO4·7H2O, 50 mg of CaCl2·2H2O, 10 mg of H3BO3, 1 mg of ZnSO4·2H2O, 0.5 mg of CuSO4·5H2O, 0.5 mg of MnCl2·4H2O, 0.5 mg of Na2MoO4·2H2O, and 4 g of glycerol (pH6.8). The cells were then radiolabeled by adding 20 µCi/ml trans 35S-labeled L-[35S]methionine and L-[35S]cysteine (MP Biomedicals) and were incubated at 29 °C for 2.5 h. The cells were then centrifuged, washed, and resuspended in 40 ml of modified GSY. At time 0 of the chase, 12 µM FeCl3 was added to the cultures, and the cells were then incubated at 29 °C. 20-ml aliquots were removed at 0 and 4 h and washed with phosphate-buffered saline. The cell pellets were resuspended in 10 cell volumes of lysis buffer (50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8, 4 mg/ml lysozyme, and 1 mM phenylmethylsulfonyl fluoride) followed by incubation at room temperature for 10 min. The lysed cells were mixed with 1 ml of radioimmune precipitation assay buffer (50 mM Tris, pH 8, 150 mM NaCl, 1 mg/ml SDS, 5 mg/ml deoxycholate, 1% (v/v) Nonidet P-40) containing 1 mM phenylmethylsulfonyl fluoride and were incubated on ice for 30 min with occasional mixing. The suspension was centrifuged, and the supernatant was used for GST pull-down. The lysate (1 ml) was incubated with 10 µl of glutathione-Sepharose 4B beads at 4 °C for 30 min. The beads were washed four times with radioimmune precipitation assay buffer and then resuspended in 25 µl of SDS sample buffer. 15-µl samples were analyzed by SDS-PAGE followed by autoradiography using x-ray film or a Bio-Rad phosphorimaging device.

Analysis of Irr in Vivo—Irr levels were measured in cells grown under varying conditions of iron or heme. Aliquots of I110, IrrC29A, or MLG1 cells grown to mid-log phase were analyzed on 12% SDS-PAGE by immunoblots using antibodies against Irr (16).

Overexpression and Purification of Recombinant Irr Proteins—The IMPACT I system (New England Biolabs) was used for synthesis of recombinant Irr proteins. The coding regions of wild-type and mutant Irr proteins were amplified by PCR from their pSK constructs with NdeI and SapI sites at 5' and 3' ends, respectively, and ligated into NdeI/SapI sites of pCYB1. C-terminal truncations were amplified by PCR from pSK/irr using the 5' end of the coding region as the forward primer and the corresponding C-terminal region as the reverse primer with NdeI and SapI sites at 5' and 3' ends, respectively, and ligated into NdeI/SapI sites of pCYB1. The constructs in pCYB1 were confirmed by nucleotide sequencing. Protein overexpression and purification were carried as described previously (13).

Heme Binding Experiments—The effects of wild-type, mutant, and truncated Irr proteins on the absorption spectrum properties of ferric heme were determined by recording the absorption spectra of 4 µM hemin in the presence or absence of 8 µM protein. To determine ferrous heme binding properties, 30 mM dithionite was added to reduce ferric heme (hemin) to ferrous heme. The spectra were recorded between 320 and 450 nm on a DW-2000 UV-VIS spectrophotometer (SLM Instruments, Inc.).

Interaction between Ferrochelatase and Irr—GST and GST-FC were overexpressed and purified using pGEX-6P-2 vector, as described previously (17). Glutathione-Sepharose 4B beads containing 2.5 µg of GST or 5 µg of GST-FC were incubated with 10 µg of purified recombinant Irr proteins for 1 h at 4 °C. The beads were then washed four times with phosphate-buffered saline containing 1% Triton X-100 and boiled in SDS-PAGE sample buffer. Irr proteins were detected by immunoblots of 12% SDS-PAGE gel using antibodies against Irr. Equal amounts of GST or GST-FC were confirmed by Ponceau S staining of the immunoblots.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a Region of Irr That Confers Regulated Instability on the Protein—GST expressed in B. japonicum from a plasmid is unaffected by the iron status of the cells as determined by immunoblots using anti-GST antibodies (Fig. 1A). However, the protein level of an Irr-GST fusion expressed from the same promoter as GST was iron-dependent, with very little detected in cells grown in iron-containing medium. This is similar to what was observed for authentic Irr (13) (see "An HRM Mutant of Irr Degrades Slowly in a Heme-dependent Manner"). Anti-GST antibodies were used to follow the fusion proteins in the experiments shown, but similar results were obtained using antibodies directed against Irr (data not shown). GroEL is not regulated by iron and was used as a control (Fig. 1A).



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  		FIG. 1.
Regulated degradation of Irr-GST fusion proteins. A, effects of iron and heme on Irr-GST fusion proteins. Fusion proteins were expressed under the control of native irr promoter on plasmid pLAFR3 in parent strain I110. Cells were grown in medium containing either low iron (–), high iron (Fe) or heme (H), and Irr-GST levels were detected by immunoblot analysis using anti-GST antibodies. GroEL was used as a control for an unregulated protein and was detected with anti-GroEL antibodies. B, pulse-chase pull-down of Irr-GST. Cells were labeled with [35S]methionine and [35S]cysteine in low iron media. At t = 0 during the chase, 12 µM ferric chloride was added. Samples were taken at 0 and 4 h, and GST fusion proteins were pulled down by glutathione-Sepharose beads. Aliquots were analyzed by SDS-PAGE followed by autoradiography.

 
To assess whether the dependence of Irr-GST on iron was due to protein turnover, cells were pulse-labeled with [35S]methionine and [35S]cysteine in low iron medium, and the fate of Irr-GST was followed during the chase by precipitation with glutathione-Sepharose beads at 0 and 4 h after the addition of 12 µM FeCl3 (Fig. 1B). Although GST was detected on autoradiograms after 4 h of exposure to iron, the Irr-GST fusion turned over in an iron-responsive manner. These results are qualitatively similar to what is observed for authentic Irr (13). These findings show that the conditional instability of Irr can be conferred on a stable protein when fused to it. Furthermore, these fusions can be used to identify regions of Irr necessary for degradation.

Irr(1–36)-GST contains the N-terminal amino acids of Irr, including the HRM, fused to GST. Unlike the full-length fusion, Irr(1–36)-GST was stable in the presence of iron (Fig. 1), suggesting that the HRM is not sufficient to confer iron-dependent instability on GST. Additional fusions containing truncations from the C-terminal end of Irr were constructed (Fig. 1). Irr(1–150)-GST and Irr(1–135)-GST accumulated to very low levels when grown in iron-containing media and turned over in response to iron in pulse-chase experiments (Fig. 1). However, Irr(1–116)-GST was unresponsive to iron in the growth and turnover experiments, indicating that a region of Irr between residues 116 and 135 is critical for iron responsiveness. Endogenous Irr disappeared in response to iron and heme in all cases (data not shown).

Irr interacts with ferrochelatase, the enzyme that catalyzes the final step of heme biosynthesis, and ferrochelatase is required for normal iron-dependent turnover of Irr (17). This requirement can be overridden by supplementation of the growth medium with heme. Irr-GST and the truncations that were iron-responsive also degraded in response to heme (Fig. 1A). However, Irr-(1–116) was stable in the presence of heme as well as iron. These observations indicate that the stability of Irr-(1–116) was not because of an inability to interact with ferrochelatase, but rather the truncation is missing a heme-dependent instability element. We attempted to make N-terminal truncation fusions, but none of these constructs were expressed in B. japonicum, orin E. coli. We have no explanation for those observations.

An HRM Mutant of Irr Degrades Slowly in a Heme-dependent Manner—Iron-dependent degradation of Irr is mediated by heme, which binds directly to the HRM (13). Irr accumulates under low iron conditions but disappears within 60–90 min upon exposure of cells to iron (Fig. 2A) (13). By contrast, IrrC29A, which contains a Cys29->Ala substitution within the HRM, does not degrade within this time frame in response to iron (Fig. 2A) (13).



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  		FIG. 2.
Effects of iron and heme on Irr accumulation in wild-type, HRM mutant strain C29A, and heme biosynthesis mutant strain hemA. A, iron-dependent Irr turnover in vivo. Cells were grown to the mid-log phase under low iron concentration. At t = 0, 6 µM FeCl3 was added, and aliquots were taken at various times afterward up to 90 min and analyzed for Irr by immunoblot analysis using antibodies raised against Irr. B, cells of strains I110 (wt), C29A, MLG1 (hemA) were grown in modified GSY medium containing either low iron (–), high iron (Fe) or high heme (H) concentrations. Irr levels in cells were analyzed by immunoblot analysis using anti-Irr antibodies. Thirty micrograms of protein were loaded/lane. C, the protocol is the same as for A, except Irr was followed over 4 h.

 
Surprisingly, we observed that when strain IrrC29A cells were grown from an inoculum in medium containing FeCl3, the mutant Irr protein was expressed at very low levels, as discerned in immunoblots, similar to that observed in the wild type (Fig. 2B). Thus, it appeared that iron negatively affected Irr levels even in the HRM mutant. Because the cells were exposed to iron for a longer period of time in the growth experiments compared with the initial time course experiments, we repeated the latter over a longer time period (Fig. 2C). One hour after exposure of the cells to iron, wild-type Irr was almost undetectable in immunoblots, whereas IrrC29A was easily discerned. However, IrrC29A levels were diminished substantially at 3 h and almost undetectable by 4 h. Thus, Irr responds to iron independently of the HRM, although more slowly. This observation agrees with the conclusion that Irr degradation involves elements in addition to the HRM.

To determine whether the slow disappearance is heme-dependent, we monitored the responsiveness of Irr to iron in a heme-deficient background. The hemA mutant strain MLG1 is deficient in {delta}-aminolevulinic acid synthase, the enzyme that catalyzes the first step in heme biosynthesis. Irr was completely stable in the hemA strain throughout the 4-h time course (Fig. 2C) and in the cultures of the mutant grown in the presence of iron (Fig. 2B), suggesting that heme is needed for iron-responsiveness of both the wild-type and mutant Irr proteins. In addition, Irr was nearly undetectable in cultures of the wild-type, IrrC29A, or the hemA strain when media were supplemented with heme (Fig. 2B). The data indicate that normal, rapid iron-dependent degradation of Irr requires the HRM but that degradation can occur slowly without the HRM in a heme-dependent manner.

Irr Binds Ferrous Heme Independent of the HRM—Heme-dependent degradation of Irr in the absence of a functional HRM raises the possibility that Irr has another heme binding site involved in turnover. Heme can exist in the ferrous (heme iron is Fe2+) or ferric (Fe3+) forms. We showed previously that purified recombinant Irr has two ferric heme binding sites with different affinities (13). One of them is the HRM, which is responsible for a 377-nm peak in the absorption spectrum of heme in the presence of Irr that is absent in the IrrC29A mutant protein (13) (Fig. 3A). A second ferric heme binding site was discerned from binding studies (17) and from an absorption shoulder at 419 nm in wild-type Irr and a peak at 413 nm in IrrC29A (Fig. 3A) (13). This second ferric heme site remains uncharacterized and, as described below, is distinct from the newly described heme binding site.



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  		FIG. 3.
Effects of Irr or C29A on the absorption spectra of ferric (A) or ferrous (B) heme. A, absorption spectra of ferric heme were taken between 320 and 450 nm for 8 µM protein alone, 4 µM heme alone, Irr plus heme, and C29A plus heme. B, absorption spectra of ferrous heme were taken between 380 and 480 nm for 8 µM protein alone, 4 µM heme alone, Irr plus heme, and C29A plus heme, with 30 mM dithionite as the reductant in all reactions.

 
Here, the effects of Irr on the absorption spectrum of reduced (ferrous) heme were examined. A peak at 423 nm was observed in the presence of Irr, whereas free heme had only a very slight, broad feature in that region (Fig. 3B). Unlike ferric heme, the spectrum of ferrous heme bound to IrrC29A was identical to the wild-type protein, showing that Cys29 within the HRM was not a ligand for reduced heme. Thus, Irr binds ferrous heme independently of the HRM.

Effects of Irr Truncations on Ferrous and Ferric Heme Binding—It seemed plausible that a heme binding site on Irr other than the HRM participates in turnover and that one or more residues necessary for heme binding was absent in the truncations. To address this, we analyzed the effects of truncations of purified recombinant Irr derivatives on the spectral properties of heme (Fig. 4). The 419-nm shoulder of ferric heme bound to Irr was lost in Irr-(1–80), and the 377-nm peak was shifted slightly to 380 nm, indicating that this truncation retained the HRM but lost the other ferric heme binding site (Fig. 4A). The ferric heme spectra of Irr-(1–116) was similar to the wild type, with spectral features at 377 and 419 nm. However, the 423-nm peak of the ferrous heme spectrum of wild-type Irr was lost in Irr-(1–116), as well as in Irr-(1–80) (Fig. 4B), showing that this truncated protein retained the ability to bind ferric heme but not ferrous heme. These data show that one or more ligands binding ferrous heme must be different from those binding either of the ferric heme binding sites. In addition, the loss of ferrous heme binding in Irr-(1–116) in vitro correlates with the loss of iron- and heme-dependent degradation of the Irr-(1–116) fusion protein in vivo.



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  		FIG. 4.
Effects of C-terminal truncations on the absorption spectra of ferric (A) or ferrous (B) heme. A, absorption spectra of ferric heme were taken between 320 and 450 nm. B, absorption spectra of ferrous heme were taken between 380 and 480 nm, with 30 mM dithionite as the reductant in all reactions. The spectra are as follows: 8 µM protein alone, 4 µM heme alone, Irr plus heme, Irr(1–116) plus heme, and Irr(1–80) plus heme.

 
Ferrous Heme Binds an Irr Histidine and Is Required for Degradation—Histidine residues are often involved in heme binding (25). As an initial search for candidate histidine residues in Irr, we aligned numerous Irr homologs from different organisms using ClustalW (26). Histidines are completely conserved at positions 63, 117, and 119 (B. japonicum numbering) and His118 is conserved in many, but not all, of the Irr homologs. We mutagenized each of the three conserved histidines, as well as His118, to alanine and analyzed the spectra of ferrous heme bound to each of the four mutants. None of the single mutations abrogated the 423-nm peak (data not shown). It is possible that mutation of a heme binding ligand can be compensated by another residue (e.g. Ref. 27), which seems particularly plausible with the three adjacent histidines. Therefore, we studied that region further by constructing and analyzing the three double mutations and the triple mutation H117A/H118A/H119A. All of the double mutations retained the ferrous heme peak, but it was slightly diminished (data not shown). However the 423-nm peak was severely diminished in the triple mutant (Fig. 5A), indicating that this region is involved in ferrous heme binding. Unlike ferrous heme, ferric heme binding was retained in the triple mutant as discerned by the spectral features that were very similar to the wild type (Fig. 5B). These data reinforce the conclusion that ferrous heme binds to different residues on Irr than does ferric heme.



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  		FIG. 5.
Effects of His->Ala substitutions at various positions on the absorption spectra of ferrous (A) or ferric (B) heme. A, absorption spectra of ferrous heme were taken between 380 and 480 nm, with 30 mM dithionite as the reductant in all reactions. B, absorption spectra of ferric heme were taken between 320 and 450 nm. The spectra are as follows: 8 µM protein alone, 4 µM heme alone, H117A/H118A/H119A plus heme, C29A plus heme, and Irr plus heme.

 
We addressed the effects of the H117A/H118A/H119A mutant on iron responsiveness in vivo using fusion proteins (Fig. 6). Unlike the wild-type or IrrC29A fusions, the triple mutant was stable in cells grown in high iron medium (Fig. 6A). Furthermore, the triple mutant did not turnover in response to iron in pulse-chase experiments (Fig. 6B). Two lines of evidence suggest that the stability of the H117A/H118A/H119A fusion is not caused by the lost ability to interact with ferrochelatase. First, supplementation of the media with heme overrides the need for ferrochelatase for turnover, and the triple mutant was stable in heme-containing cultures (Fig. 6A). In addition, we addressed the interaction between Irr and ferrochelatase by examining the ability of a GST-ferrochelatase fusion to pull down purified recombinant Irr proteins (Fig. 7). Wild-type Irr and both the C29A and H117A/H118A/H119A mutants were precipitated by GST-ferrochelatase, thus ruling out a disruption of the mutant Irr with ferrochelatase as an explanation for its stability. The data show that ferrous heme binds Irr in the histidine 117–119 region, and this region is necessary for iron-dependent degradation.



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  		FIG. 6.
Effects of mutations in the HRM or the ferrous heme binding site on iron-dependent degradation of Irr-GST fusion proteins. A, effects of iron and heme on Irr-GST fusion proteins. Fusion proteins were expressed under the control of native irr promoter on plasmid pLAFR3 in parent strain I110. Cells were grown in medium containing either low iron (–), high iron (Fe) or heme (H), and Irr-GST levels were detected by immunoblot analysis using anti-GST antibodies. B, pulse-chase pull-down of Irr-GST fusions. The protocol was the same as described in the legend to Fig. 1.

 



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  		FIG. 7.
Interaction of Irr and mutant derivatives with GST-Ferrochelatase. The GST-FC fusion protein was used to precipitate purified recombinant Irr and mutant derivatives in solution. GST was used as a negative control. 2.5 µg of GST, 5 µg of GST-FC, and 10 µgof Irr, C29A, and H117A/H118A/H119A were used for the corresponding individual experiment. Irr proteins were detected by immunoblots using antibodies against Irr.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We showed previously that heme binds to a HRM of Irr, which is necessary for normal degradation. In the present study, we found that a mutant Irr protein defective in the HRM degrades in response to iron, although much more slowly than the wild-type protein, and that this turnover is heme-dependent. Moreover, Irr binds ferrous heme at a site different from the HRM or from the other uncharacterized ferric heme binding site. Irr mutants with deletions or amino acid substitutions that mitigate ferrous heme binding are stable in vivo independent of iron. These findings indicate that both ferrous and ferric heme are necessary for Irr degradation and that different ligands bind the two forms of heme.

Irr decay follows first order kinetics (13), indicating a single mechanism for degradation. This suggests that the two hemes participate in a single degradation process rather than independent processes that occur at different rates. This is supported by the observation that mutation of the histidine(s) involved in ferrous heme binding abrogates degradation completely, even with an intact HRM (Fig. 6). The need for heme in both the oxidized and reduced forms leads us to speculate that the redox activity of heme may be important for Irr turnover. One possibility is that heme catalyzes the oxidation of Irr, promoting its degradation by a protease. Heme can react with oxygen to form reactive oxygen species that oxidatively damage proteins (28) Thus, Irr may be similarly damaged. The mammalian iron regulatory protein 2 (IRP2) is oxidized by iron and subsequently degraded (29), and evidence implicates a role for heme (30–32). Heme-bound IRP2 is ubiquitinated in vitro in an O2-dependent manner, but it has not been established that heme catalyzes oxidation of the protein (32).

Another possibility is that the redox activity of Irr induces a conformational change that makes Irr accessible to a protease or another factor necessary for degradation. This idea is based on the observation that the reduced and oxidized forms of heme bind different Irr residues, and redox-dependent ligand switching is accompanied by conformational changes in the transcription factor CooA from Rhodospirillum rubrum (3) and the redox sensor EcDos from E. coli (33). Additional studies are required to further characterize the basis of heme-dependent degradation of Irr.

Although Irr has been studied extensively only in B. japonicum, an irr mutant in Rhizobium leguminosarum has the same fluorescent colony phenotype as the B. japonicum mutant (34), and the genomes around the irr homologs show a high degree of synteny. Thus, Irr likely has a similar function in other organisms. However, examination of the Irr homologs reveals that only some of them have the Cys-Pro sequence and an HRM-like domain, whereas His117 and His119 of B. japonicum Irr are completely conserved in all of the homologs. The current work suggests that the proteins lacking an HRM may degrade nevertheless. It is possible that the second, uncharacterized ferric heme binding site partially compensates for the defective HRM in IrrC29A, allowing slow degradation. By analogy, Irr homologs lacking an HRM may have a compensatory mechanism that allows turnover. Characterization of these other homologs should shed additional light on bacterial Irr function.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant GM-067966 (to M. R. O.) and Grant-in-aid 14658217 from the Ministry of Education, Culture, Sports, Science, and Technology in Japan (to K. I.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Biochemistry, 140 Farber Hall, State University of New York at Buffalo, Buffalo, NY 14214. Tel.: 716-829-3200; Fax: 716–829-2725; E-mail: mrobrian{at}buffalo.edu.

1 The abbreviations used are: Irr, iron response regulator; GST, glutathione S-transferase; HRM, heme regulatory motif; FC, ferrochelatase. Back



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