Alterations in Epithelial and Mesenchymal Intestinal Gene Expression During Doxorubicin-Induced Mucositis in Mice

In the current study we aimed to gain insight into epithelial-mesenchymal cross-talk and progenitor compartment modulation during doxorubicin (DOX)-induced mucositis in mice. Intestinal segments were collected on various days after DOX treatment. DOX-induced damage at day 1–2 was characterized by increased epithelial proliferation and apoptosis and a decrease in the expression of epithelial differentiation markers. Concurrently, T-cell factor-4 (TCF4) levels increased and the epithelial differentiation enhancing factor, bone morphogenic protein-4 (BMP4), decreased. During severe damage (day 3), BMP4 levels were significantly increased, which inversely correlated with epithelial proliferation. At the same time, the expression of the epithelial differentiation markers was increasing again. At day 7, BMP4 levels were down-regulated, while the levels of the epithelial differentiation markers and TCF4 were normalized again. These data suggest that in response to DOX-induced damage, BMP4 and TCF4 are modulated in such a way that homeostasis of the progenitor compartment is partly preserved.
Introduction
The highly proliferating small intestine is very susceptible to chemotherapy-induced damage [31, 47]. This side effect, often referred to as mucositis, is very painful and can be life threatening due to the enhanced risk of bacterial translocation caused by loss of epithelial barrier function. Each year, around 500,000 patients worldwide suffer from this side effect [7] for which there is still no definitive prophylaxis or treatment.
Drug-induced damage to the highly proliferating stem cells and progenitors located in the crypts of Lieberkühn showed that different classes of cytostatic drugs affect epithelial crypt cells of different topographical and hierarchical status [12, 13, 24]. Methotrexate (MTX), 5-fluorouracil and vincristine for example, induce damage high in the proliferative compartment at crypt position 9–11. Damage induced by MTX is well characterized and shows severe morphological changes characterized by epithelial flattening, villus-atrophy, and specific de-differentiation of enterocytes. The last one is indicated by a decreased expression of the enterocyte-specific enzymes but a maintained expression of goblet and Paneth cell-specific genes [29, 38, 41, 45, 46]. Doxorubicin (DOX) is a cytostatic drug that also frequently causes severe mucositis. However, DOX-induced damage is not well characterized [28]. DOX preferentially attacks cells at crypt position 4–6, which is just above or at the same level as the intestinal stem cells [12, 32]. DOX is therefore thought to induce more severe damage, especially to the proliferating compartment at the bottom of the crypts, as for example MTX. In clinical practice, DOX is often used in treatment of solid tumors, leukemia, and lymphomas in both adult and childhood cancer patients [16, 18, 21, 28]. Recent studies revealed a dynamic cascade of events leading to chemotherapy-induced mucositis [35]. Not only are the specialized epithelial cells affected by cytostatic-drug treatment, but also the underlying submucosal connective tissue. Under normal physiological circumstances, the epithelium maintains a cross-talk with the mesenchyme. Epithelial-mesenchymal interactions are critical for the normal morphogenesis and maintenance of the crypt-villus axis [15, 23].
Bone morphogenic protein (BMP)- and Wnt-signaling pathways are two of the main pathways involved in embryonic and adult intestinal development [5, 33]. BMPs are morphogenes that constitute a large group of structurally and functionally related proteins of the TGF-beta superfamily. BMP signaling pathways have a key role in organogenesis [2, 37], gastrointestinal development and intestinal homeostasis in adults [8–11, 14, 27]. After intestinal development, BMP4 is exclusively expressed in the intravillus and intracrypt mesenchymal cells, including those adjacent to the intestinal stem cells [8, 10]. Paracrine BMP signaling occurs specifically in the villus from the mesenchyme to the adjacent epithelium [8], suggesting involvement in epithelial-mesenchymal interactions as a mesenchymal signaling molecule.
The Wnt-signaling pathway also has been implicated in the regulation of the intestinal epithelial proliferation/differentiation balance in vitro [43]. In the intestine of mice deficient for transcription factor TCF4, the main Wnt pathway transcription factor in the intestinal epithelium, loss of proliferative compartments and epithelial cell differentiation are found [17]. Interestingly, the BMP- and Wnt pathways appear to be linked, as was shown by the fact that BMP signaling suppresses Wnt signaling to ensure a balanced control of stem cell proliferation and subsequent epithelial differentiation [8, 10].
The objective of this study was to develop an experimental mucositis mouse model to characterize DOX-induced intestinal damage and subsequent repair. In addition, we aimed to correlate the alterations in morphology, epithelial homeostasis, and gene expression with changes in BMP4 and TCF4 expression. This, in order to gain insight into possible modulation of the epithelial-mesenchymal cross-talk and progenitor compartment during chemotherapy-induced intestinal damage and regeneration.
Materials and methods
Animals
Animal experiments were performed with permission of the Animal Ethics Committee of the Erasmus MC-Sophia. Upon arrival at our institute, 10-week-old male BALB/c mice (Harlan, Horst, The Netherlands) were housed individually during the whole experiment in micro-isolator cages under specific pathogen-free conditions with free access to a standard palletized diet (Hope farms, Woerden, The Netherlands) and water. After 1 week of adjustment to the new environment, the mice were divided into three groups and injected intravenously with doxorubicin (DOX) (Doxorubicin, Pharma Chemie, Haarlem, The Netherlands) on two subsequent days. At day −1 and 0, the first group of mice was injected with a low dose of DOX of 6 and 4 mg/kg (low dose) respectively, a second group was injected with a medium dose of 8 and 5 mg/kg (medium dose) and a third group was injected with a high dose of 10 and 6 mg/kg (high dose). Controls were given equivalent volumes of 0.9% NaCl. Mice in the low- and high-dose group were sacrificed at day 1, 2, 3 and 7 after the final DOX injection; mice in the medium dose group were only sacrificed at day 3 and 7. One hour before sacrifice, the mice were injected with 120 μl 10 mg/ml 5- Bromo-2’deoxyUridine (BrdU) (Sigma-Aldrich, Zwijndrecht, The Netherlands), an uridine analog, to locate the proliferating cells. Per time point 4–6 DOX-treated animals and 2–4 control animals were sacrificed. Segments of mid-jejunum were collected and either processed immediately for histological analyses or snap-frozen in liquid nitrogen for storage at −80°C and subsequent protein isolation.
Histochemistry
Five-millimeter segments of mid-jejunum were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS), dehydrated and embedded in Paraplast Plus (Sherwood Medical, Den Bosch, The Netherlands) as previously described [45]. Four μm sections were routinely stained with hematoxylin (Vector Laboratories, Burlingame, CA) and eosin (Sigma-Aldrich) to study morphological alterations of the crypts and villi. Immunohistochemistry was performed as described previously [45] with some minor modifications. The sections for BrdU staining required an extra adjustment to this protocol of HCL incubation, washing with borate buffer, and pepsin treatment as described before [34]. In short, the sections were blocked as described and incubated overnight with the following antibodies diluted in PBS: to visualize BrdU incorporation, mouse monoclonal anti-BrdU (1:250, Roche Applied Sciences, Indianapolis, IN) was used, as an enterocyte marker rabbit polyclonal anti-rat Sucrase-Isomaltase (SI) (1:9000 in PBS, kindly provided by Dr. K.Y. Yeh [49]) was used and as a goblet cell-specific marker rabbit polyclonal anti-rat trefoil factor family (TFF3: 1:3000, kindly provided by Prof. Dr. D.K. Podolsky) was used. Furthermore, BMP expression was visualized with anti-BMP4 (1:100, R&D Systems, Abingdon, UK). Immunoreactions were detected using Vectastain ABC Elite Kit (Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich, Zwijndrecht, The Netherlands).
Crypt and -villus length
Longitudinal sections of crypts and their corresponding villi were selected so that the base (marked by Paneth cells), middle and top of the crypt were all in the plane of section and thus well orientated. The depth of ten crypts and the length of ten villi were measured on three slides per animal, four animals per time point, with the use of a Nikon Eclipse E800 microscope and IM 500 software.
Protein dot blotting
The expression of enterocyte markers was detected and quantified as described previously [46]. Briefly, 5-mm segments of the mid-jejunum were homogenized and protein concentrations were measured using the BCA Protein Assay Reagent (Pierce, Rockford, IL) and 50 μg protein of each homogenate was dot-blotted on nitrocellulose (Protran BA83, 0.2 μm; Schleicher & Schuell, Dassel, Germany). Hereafter, blots were blocked for 1 h with blocking buffer containing 50 mM Tris, pH 7.8, 5% (wt/vol) nonfat dry milk powder (Campina Melkunie, Eindhoven, The Netherlands), 2 mM CaCl2, 0.05% (vol/vol) Nonidet P40 (BDH, Brunschwig Chemie, Amsterdam, The Netherlands) and 0.01% Antifoam B (Sigma-Aldrich). Blots were incubated overnight at 4°C with rabbit polyclonal anti-rat SI (1:1000 [49]), and rabbit polyclonal anti-rat trefoil factor family (TFF3: 1:1500) diluted in blocking buffer. After washing with blocking, the buffer blots were incubated with 125I-labeled protein A (specific activity 30 mCi/mg, Amersham Biosciences, Roosendaal, The Netherlands) for 2 h at room temperature. Specific binding of 125I-labeled protein A to the enterocyte marker antibodies was measured using PhosphorImager detection. The elicited signal was quantified by ImageQuant software (Molecular Dynamics, B&L systems, Zoetermeer, The Netherlands) and the expression of TFF3 and SI was expressed per 50 μg protein of tissue. Average expression levels of TFF3 and SI in the mid-jejunum were calculated per mouse, followed by calculation of the mean expression of TFF3 and SI per time point studied. Subsequently, the average expression of TFF3 and SI of control mice was set at 100%. The specificity of the above-described antibodies was previously confirmed by Western-blot analysis [46].
Western-blot analysis
The same protein homogenate was used as described for protein dot blot analysis. Twenty μg of protein was loaded per lane and run on a 12.5% SDS-PAGE. The separated proteins were transferred to nitrocellulose membranes (Protran BA83, 0.2 μm) and blocked for 1 h at room temperature in blocking buffer as described above. The blots were incubated overnight at 4°C with primary antibodies diluted in blocking buffer: mouse monoclonal anti-human PCNA, clone PC10 (1:250, Novo Castra Laboratories, Newcastle upon Tyne, UK) and mouse monoclonal anti-human BMP4, clone 3H2 (1:100), (Novocastra Laboratories, Newcastle upon Tyne, UK), rabbit polyclonal anti-human cleaved Caspase-3 antibody (1:1000, Cell Signaling, Beverly, MA), and mouse monoclonal anti-human TCF-4, clone 6H5-3 (1:250, Upstate, Waltham, MA). After washing with PBS, 0.2% Tween-20 blots bound antibodies were revealed using HRP conjugated goat anti-mouse or rabbit anti-goat secondary antibodies (1:1000) and SuperSignal West Femto Luminol Enhancer kit (Pierce, Rockford, IL). The signal was detected and quantified by the ChemiGenius gel documentation system (Syngene, Cambridge, UK) and the expression of the specific proteins analyzed was expressed per 20 μg protein of tissue. Average expression levels of PCNA, BMP4, TCF4, and caspase-3 in the mid-jejunum were calculated per mouse, followed by calculation of the mean expression of these specific proteins per time point studied. Subsequently, the average expression of PCNA, BMP4, TCF4, and caspase-3 in control mice was set at 100%.
Statistical analysis
Changes in protein expression levels during damage and regeneration were statistically analyzed using the Kruskal-Wallis H-test and the Mann-Whitney U-test. A p < 0.05 was considered statistically significant. Data are presented as the mean ± standard error of the mean (SEM).
Results
Dose-response analysis of DOX-induced mucositis in an experimental mouse model
To optimize the dose of DOX necessary to induce severe intestinal damage (i.e., villus atrophy, crypt loss and flattening of the epithelial cells) a dose-response curve was performed. Thereto, mice were divided in three treatment groups: low dose, medium dose, and high dose (see Materials and methods, section Animals for details). In the high-dose treatment group, two of the six mice died at day 4. Necropsy showed an excess of fluid in the abdominal cavity of unknown source. Because of the elapsed time after death, morphological evaluation could not be performed.
Morphological analysis of the low-dose treatment group at days 1 and 2 (data not shown), revealed only a slight increase in crypt length, which became more pronounced at day 3 (Fig. 1B). An increase in crypt length was also seen in the medium dose group at day 3. (Note the medium-dose treatment group was not studied at days 1 and 2). No other morphological changes were seen in the low dose- (days 1–3) and medium-dose group (day 3). In contrast, in the high-dose treatment group we did see severe morphological damage (Fig. 1D–F). Specifically, severe villus atrophy, crypt lengthening, crypt loss, perturbation of crypt arrangement, flattening of crypt and villus epithelium and inflammation were observed at day 3 (Fig. 1F). Moreover, the latter morphological damage was already seen, although in a milder degree, at days 1 and 2 after the high-dose DOX treatment (Fig. 1D, day 1 and Fig. 1E, day 2). At day 7, the intestinal morphology was completely restored in the low-dose and medium-dose group (data not shown), but was still affected in the high-dose group. As in the latter group, the crypt epithelium still showed signs of regeneration, like new crypt formation and less epithelial flattening (Fig. 1G).
Quantitative analysis of crypt and villus length
To quantify the amount of morphological damage induced by the different doses of DOX, the length of the crypts and villi were measured (Fig. 1H–I). In the high-dose group, but not in the low-dose or medium-dose group, a significant increase in crypt length was already observed at day 2 (data not shown). At day 3 the crypt length was significantly increased in each treatment group compared to the control group (Fig. 1H). By day 7, the length of the crypts had returned to control levels in the low-dose and medium-dose group, but were still increased in the high-dose group. This increase in crypt length, however, showed a trend towards normal levels because the crypt length at day 3 was 41% above control level but at day 7 this was still only 13% above control level. The length of the villi did not change in the low-dose or the medium-dose treatment group on each day investigated. However, in the high-dose treatment group the length of the villi were significantly decreased on days 1–3 and 7 (Fig. 1H, day 3 and 1I, day 7). Because severe intestinal mucositis (day 3) and morphological regeneration (day 7) were seen only in the high-dose treatment group, we continued our studies with the high-dose DOX treatment model.
Effects of high-dose DOX treatment on enterocyte-specific gene expression
To gain insight into the functional capacity of the intestinal epithelium after DOX treatment, a sucrase-isomaltase (SI) immunohistochemical staining was performed. SI is an enterocyte-specific disaccharidase responsible for sucrose degradation. SI is expressed in the brush border of differentiated villus enterocytes directly after weaning [42] and is considered an intestinal epithelial differentiation marker. At day 1 and 2 after DOX treatment SI was expressed in the brush border of enterocytes along the entire villi, comparable to the control epithelium (Fig. 2A, control; B, day 1; C, day 2). At day 3, SI staining showed a patchy pattern with weak staining on less affected parts of the epithelium and even absence of staining on severely damaged epithelial parts of the sections (Fig. 2D). At day 7, during the regenerative phase, the SI staining pattern was comparable to control again (Fig. 2E).
To quantify SI protein expression, protein dot-blot analyses were performed using SI-specific antibodies (Fig. 2F). The SI protein expression level at day 1 was similar to the control situation. At day 2, SI expression levels were significantly decreased and remained significantly decreased at day 3. At day 7, the SI expression levels regained to control levels.
Effects of DOX treatment on goblet cell-specific gene expression
Goblet cell-specific gene expression after high-dose DOX treatment was analyzed by the expression of trefoil factor family 3 (TFF3). TFF3 is a bioactive peptide, produced and secreted by goblet cells, that is involved in epithelial protection and repair [6, 22]. Immunohistochemical staining of TFF3 showed TFF3 expression by goblet cells in crypts and villi of the jejunum (Fig. 3). At day 1, the TFF3 staining was not visibly altered compared to the control situation (Fig. 3B). At day 2, the immunohistochemical detection of TFF3 decreased visibly, especially in the goblet cells localized in the crypts and lower part of the villi where TFF3 protein could hardly be detected/was absent (Fig. 3C). At day 3, during most severe morphological damage, TFF3 staining by goblet cells in the crypt was reconstituted (Fig. 3D). Day 7 showed complete regeneration of TFF3 (Fig. 3E).
Subsequently, a protein dot-blot analysis was performed (Fig. 3F) using TFF3-specific antibodies to quantify TFF3 levels. At day 1, the TFF3 expression was already significantly decreased to 50% of control level and maintained at this low level at day 2. At day 3, concomitant with the most severe morphological damage, TFF3 expression returned to control level. TFF3 protein expression was again comparable to control situation at day 7.
Effect of high-dose DOX treatment on epithelial proliferation
Localization of proliferation was studied by immunohistochemical detection of incorporated BrdU (Fig. 4). In controls (Fig. 4A) BrdU was mainly localized in cells from the bottom of the crypt up to three quarter of the crypt length. At day 1 and 2 the proliferative zone broadened and progressively moved upwards to the crypt-villus junction. At day 3, during severe morphological damage, BrdU-positive cells formed a scattered pattern throughout the remaining crypt structures of the small intestine. In the regenerative phase, at day 7 BrdU-positive cells were located in the lower part of the crypts again.Fig. 1Morphology of the murine small intestine after low, medium- or high-dose DOX treatment. Morphology of the jejunum of a control mouse (A), at day 3 after low-dose DOX (B) and at day 3 after medium-dose DOX (C), both were mildly affected by DOX treatment. Morphology of the jejunum at day 1 (D) and day 2 (E) after high-dose DOX treatment, which appeared progressively affected and severely deteriorated at day 3 (E). At day 7, the regenerative phase, the intestinal morphology of the mice in the low- and medium-dose group was completely restored to control situation (data not shown). The morphology at day 7 of the mice treated in the high-dose group was regenerating, showing new crypt formation but still villus atrophy. These photographs are representative examples of a group of four animals per time-point. To quantify the effect of these different doses of DOX treatment, crypt and villus lengths were measured. Crypt and villus lengths of control mice were set at 100%. At day 3 (A), the length of the crypts in all three DOX treatment groups was significantly increased in comparison to the control group. Villus length in the low- and medium-dose group remained at control level. The length of the villi of the high-dose group decreased significantly, indicating significant villus atrophy after high-dose DOX treatment. At day 7 (B) crypt and villus lengths of the low- and medium-dose DOX treatment groups showed complete restoration. The crypts of the high-dose treatment group were still significantly longer, but this increase in crypt length was declining compared to day 3 (day 3 41%, day 7 13%). The length of the villi was still significantly decreased at day 7, showing no signs of regeneration. *P < 0.05 versus control. The bars are expressed in mean + SEMFig. 2Effect of high-dose DOX treatment on the enterocyte-specific SI expression in the small intestinal epithelium. SI expression of the jejunum of a control mouse (A), at day 1 (B) and at day 2 (C). The brush border staining at day 1 and 2 is comparable to control situation. At day 3 (D), SI staining showed a patchy pattern; some parts of the brush border showed SI expression, on some other parts of the brush border SI expression was absent (→). SI expression at day 7 (E) showed complete regeneration, and was equal to the control situation. SI protein expression levels were analyzed by dot-blot technique and the SI expression of control mice was set at 100% (F). The expression of SI remained stable at day 1, but decreased significantly at day 2 and 3. During the regenerative phase, at day 7, the SI expression reached control level again. *P < 0.05 day 2 and 3 versus control. The bars are expressed in mean + SEMFig. 3Effect of high-dose DOX treatment on the goblet-specific TFF3 expression in the small intestine. Immunohistochemical staining of the goblet cell-specific protein TFF3 (A–E). TFF3 expression of the jejunum of a control mouse (A), at day 1 (B), at day 2 (C), at day 3 (D) and at day 7 (E). In the control situation, TFF3 was expressed in all goblet cells in the crypts and villi, at day 1 (B), the amount of goblet cells expressing TFF3 maintained at control level. At day 2 (C), TFF3 expression decreased visibly, the goblet cells localized in the crypts showed no staining. At day 3 (D), during severe morphological damage, TFF3 expression of goblet cells in the crypt was reconstituted. TFF3 expression at day 7 (E) was regenerated and comparable to control situation. TFF3 protein expression levels were analyzed by dot-blot technique, the TFF3 expression of control mice was set at 100% (F). TFF3 expression decreased significantly at day 1 and day 2, the expression was decreased to 50% of control level. During severe morphological damage at day 3, the TFF3 expression increased almost to control level. Day 7 showed TFF3 expression even above control level. *P < 0.05 day 1 and 2 versus control. The bars are expressed in mean + SEMFig. 4Effect of high-dose DOX treatment on epithelial proliferation. The localization of epithelial proliferation was visualized by detection of BrdU incorporation. BrdU incorporation of the jejunum of a control mouse (A) and mice at day 1 (B), at day 2 (C), at day 3 (D) and at day 7 (E). At day 1, the BrdU-positive cells were slightly migrated upwards in the crypt, at day 2 the BrdU-positive cells were seen along the entire length of the crypts. At day 3 the BrdU expression decreased and showed a scattered pattern on some crypts and villi. The location of BrdU-positive cells at day 7 was confined again to the lower part of the crypts. PCNA, Caspase-3, TCF4 and BMP4 protein expression levels were analyzed by Western-blot technique (F), the PCNA expression of control mice was set at 100% (G). The expression of PCNA protein showed a non-significant trend of increased expression at day 1, and a significant increase at day 2. PCNA expression at day 3 and 7 showed a decreasing trend to around 20% of control level. *P < 0.05 day 1 and 2 versus control. The bars are expressed in mean + SEM
The changes in proliferation induced by DOX were quantified by Western-blot analysis of PCNA protein expression [4, 40] using a specific monoclonal PCNA antibody. PCNA protein expression after MTX treatment (Fig. 4F, G) showed a trend of increased expression at days 1. At day 2, this increased expression was significant compared to control levels, indicating that high DOX treatment did not induce an arrest in proliferation at day 1 and 2. At day 3, PCNA expression was decreased to around 20% of the normal level and was still 50% of control level at day 7. The latter data indicate that the inhibition of epithelial proliferation sustained during the regenerative phase. Overall, the PCNA levels correlated with the immunohistochemical data (Figs. 4A–E vs. 4G)
Effect of high-dose DOX treatment on enterocyte apoptosis
Besides proliferation, the influence of DOX treatment on apoptosis was quantified with a polyclonal antibody against cleaved Caspase-3 (Fig. 4F, 5A). Caspase-3 is one of the key executioners of apoptosis [26]. The expression of cleaved Caspase-3 protein was significantly increased at day 1 and 2, at the beginning of DOX-induced morphological damage. At day 3, during severe morphological damage, and day 7, during regeneration, expression of cleaved caspase-3 was comparable to control level. Increased apoptosis at day 1 and 2 was primarily located along the crypt axis (Fig. 5B showing day 1 after MTX treatment))Fig. 5Effect of high-dose DOX treatment on epithelial apoptosis. Caspase-3 protein expression of the control mice, analyzed by Western-blot technique (Fig  4F), was set at 100% (A). At day 1 and 2, the Caspase-3 levels increased significantly in comparison to the control levels. At day 3, during severe morphological damage the caspase-3 expression maintained at control level, the same was seen at day 7, during the regenerative phase. The increase in apoptosis at day 1 is primarily located in the crypt region (B). *P < 0.05 day 1 and 2 versus control. The bars are expressed in mean + SEM. → indicates apoptotic cells
Effect of high-dose DOX treatment on TCF-4 protein expression
To gain insight into the effects of DOX on the epithelial stem cell compartment, the expression of TCF-4 was quantified. TCF-4 is a transcription factor of the Wnt-signaling pathway and is expressed in the gut epithelium in a gradient fashion that is highest at the base of the crypts [1, 19]. TCF-4 −/− mice lose the intestinal epithelial progenitor and stem cell population before crypt formation can be established [17]. Therefore, TCF-4 has a role in intestinal epithelial stem cell maintenance. The TCF-4 expression (Figs. 4F and 6) remained very stable despite DOX treatment. Only at day 1 the TCF-4 expression increased significantly, although little in absolute sense. But at day 2, day 3 and day 7, the TCF4 protein expression remained at control level.Fig. 6Effect of high-dose DOX treatment on TCF-4 protein expression. TCF-4 protein expression of control mice, analyzed by Western-blot technique (Fig. 4F), was set at 100%. At day 1, TCF-4 protein expression increased significantly. At day 2, day 3, and day 7, the TCF-4 protein expression remained stable at control level. *P < 0.05 day 1 versus control. The bars are expressed in mean + SEM
Effect of high-dose DOX treatment on expression of Bone morphogene 4 (BMP4)
To examine the role of BMP4 signaling in intestinal homeostasis the expression of BMP4 was quantified. BMP4 is crucial for epithelial-mesenchymal cross-talk and intestinal homeostasis. BMP4 is expressed in the intravillus and intracrypt mesenchymal cells [8, 10], inhibition of BMP signaling causes intestinal architectural abnormalities [8] and decreased differentiation as seen in tumorigenesis [27].
BMP4 protein expression showed a mild decrease in expression level at day 1 after DOX treatment (Figs. 4F, 7A). At day 2, this decrease in BMP4 expression level progressed and was almost significant (P=0.05) in comparison to the control level. Thereafter, BMP4 day 2 but not to control. Day 7 showed a non-significant decrease in expression compared to control mice. BMP4 was predominantly expressed in the intravillus mesenchym as shown by immunohistochemistry (Fig. 7B, C), both in control as in MTX-treated mice.Fig. 7Effect of high-dose DOX treatment on expression of the morphogen BMP4. The BMP4 protein expression of the control mice, analyzed by Western-blot technique (Fig. 4F), was set at 100% (A). Expression of the morphogen BMP4 protein showed a decreasing trend at day 1, which was at the brink of significance (P=0.05) at day 2 in comparison to control expression. At day 3, the BMP4 expression showed an increasing trend. This increase in expression was significant in comparison to day 2. Day 7 showed a decrease in expression again beneath the level of control expression. The localization of BMP4 expression did not change at day three (C) in comparison to control situation (B). *P < 0.05 day 2 versus control, **P < 0.05 day 2 versus day 3. The bars are expressed in mean + SEM
Discussion
This study revealed that DOX, in a dose of 10 and 6 mg/kg induced severe morphological damage to the small intestine of mice within 3 days, which was almost completely regenerated by day 7. Moreover, it revealed that the intestine was virtually not or much less affected by lower doses of DOX. Mucositis induced by the chosen dose of DOX was characterized by an increasing degree of intestinal morphological damage at day 1 and 2, which correlated with a significant increase in both apoptosis and proliferation. During this phase of epithelial hyper-proliferation, the epithelial cells lost their highly differentiated status as measured by a significant down-regulation of epithelial-specific SI at days 2–3. The decreased expression of TFF3 at days 1–2 could be caused by a decrease in goblet cell differentiation, but on the other hand, could also be the result of increased TFF3 secretion. At day 3, the time-point when intestinal damage was most severe, the morphology was characterized by severe villus atrophy, a significant rise in crypt length, epithelial flattening, crypt loss, inhibition of proliferation and impaired epithelial differentiation. During morphological regeneration, at day 7, proliferation started to return to control level, and SI and TFF3 expression levels were normalized again.
In order to be able to prevent or treat chemotherapy-induced mucositis, it is essential to know if different cytostatic drugs induce the same or different kinds of intestinal damage. Potten et al. demonstrated that there is a general tendency for antibiotics, like DOX, and radiation to damage the lower cell positions in the crypt near or at the position of the stem cells (position 4–6) [12]. Alkylating agents on the other hand mainly damage cells at position 6–8. Anti-metabolites like MTX and a microtubule dissociating agents act on higher cell positions [9–11]. It is, however, unclear if cytostatic drugs attacking at the lowest positions in the crypts cause a different kind of damage than drugs damaging cells at higher positions. If we compare the DOX-induced mucositis as studied presently with the well-characterized MTX-induced mucositis [34, 38, 44–46, 48], then it is clear that there are many similarities and few discrepancies between the two. Although the two drugs affected cells of different hierarchical height [12], they both caused apoptosis, villus atrophy, epithelial flattening, crypt loss and a temporary loss of SI expression and TFF3 expression [45, 46, 48]. Since SI is involved in carbohydrate metabolism and TFF3 is involved in mucosal repair [6, 22, 30, 42], these data suggest impaired absorption and mucosal repair after DOX as well as after MTX treatment. In contrast, both MTX [45] and DOX hardly affect the expression of lysozyme by Paneth cells in the crypts (data not shown).
Decreased levels of TFF3 after both DOX and MTX was at the same time as epithelial hyper-proliferation [45, 48], but changes in proliferation, induced by MTX treatment followed a different time-line compared to DOX treatment. MTX causes proliferation inhibition within 1 day, followed by a period of hyper-proliferation during severe intestinal damage [34, 45]. DOX treatment leads to immediate hyper-proliferation (day 1 and 2) with subsequent inhibition of proliferation during severe morphological damage (day 3). Moreover, the cell-fate specific affect of MTX on goblet cells causing goblet cells to accumulate in the crypt and at the top of the villus [45, 46] was not seen after DOX treatment. The reason for these discrepancies remains to be further investigated, but might be directly related to the difference in topographical height (and thus status) of the cells vulnerable to the two different drugs. Overall, however, the similarities in intestinal responses after DOX or MTX treatment are striking. This suggests that there may be common pathways involved in intestinal damage and repair.
Historically, chemotherapy- or radiation-induced mucositis was believed to be solely due to damage to dividing epithelial cells at the bottom of the crypts [20]. However, recently it has become clear that other parts of the intestinal mucosa and submucosa might also be involved [35, 36]. Here we provide evidence for a mesenchymal contribution to the damage by showing that BMP4, a very important lamina propria derived-morphogen in the small intestine [8, 10], is affected by DOX treatment. BMP4 was modulated by DOX during the onset of damage at days 1 and 2. BMP4 expression decreased almost significant at day 2, which correlated well with an increasing degree of morphological damage, increased proliferation, and loss of epithelial differentiation as measured by the decreased SI and likely TFF3 expression. Very recently, a link between the BMP and the Wnt pathways has been demonstrated. It was shown that BMP signaling suppresses Wnt signaling to ensure a balanced control of stem cell proliferation and subsequent epithelial differentiation [8, 10]. Here we show that there indeed might be a close relationship between BMP and Wnt pathways, because at day 1 when BMP4 expression is decreased, expression of TCF4, a Wnt effector, increased significantly, which correlated well with increased proliferation and inhibited epithelial differentiation. At day 2, BMP4 remained low, whereas TCF4 returned to a normal level and remained at control level during the following days. At day 3, when damage was most severe, BMP4 was increased, which inversely correlated with proliferation, and correlated with epithelial differentiation, as suggested by the recovery of TFF3 expression level.
BMP is involved in epithelial-mesenchymal signaling [8] and therefore we conclude that the data presented in this study indicate that epithelial-mesenchymal cross-talk is modulated during onset of DOX-induced damage in order to stimulate proliferation instead of differentiation and during severe intestinal damage to induce differentiation and inhibit proliferation. During the regenerative phase, at day 7, BMP4 expression level was down-regulated again, which could be a response to the shortage in number of crypts, since a blockage of BMP4 has been shown to cause stem cells to divide, leading to newly formed crypts [8, 10]. Therefore, our findings are in line with the roles of the BMP and Wnt/TCF pathway in epithelial homeostasis/morphogenesis. Furthermore, the decrease in BMP4 might also indirectly cause the observed decrease in SI expression, because inhibition of BMP4 stimulates Wnt signaling, and Wnt signaling itself induces SOX9, a negative regulator of Cdx2, which is a SI transcriptional activator [3, 10, 39]. In addition, BMP4 has been shown to be directly involved in HNF-1α expression, also a well-known activator of SI transcription [25]. Thus, the decrease in BMP4 might result in a decrease in Cdx2 and HNF-1α expression, two of the most important activators of SI transcription [39]. Currently, it is not known whether the Wnt or BMP4 signaling pathways regulate TFF3 gene expression.
In conclusion, high-dose DOX induces severe damage to the epithelium, which closely resembles damage induced by MTX, indicating that general mechanisms of damage and repair are involved. We show that signaling pathways involving BMP4 and TCF4 and thus epithelial-mesenchymal cross-talk are modulated by DOX-induced damage in such a way that homeostasis of the progenitor compartment is restored by initially inducing cell proliferation and inhibiting differentiation and subsequently inducing differentiation, inhibiting proliferation and promoting crypt fission. Understanding these mechanisms is essential to develop clinical strategies to prevent chemotherapy-induced mucositis.

tcf4
morphogens
epithelial homeostasis
intestinal differentiation
bone morphogenetic proteins


Spectroscopic evidence for an all-ferrous [4Fe–4S]0 cluster in the superreduced activator of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans

The key enzyme of the fermentation of glutamate by Acidaminococcus fermentans, 2-hydroxyglutarylcoenzyme A dehydratase, catalyzes the reversible syn-elimination of water from (R)-2-hydroxyglutaryl-coenzyme A, resulting in (E)-glutaconylcoenzyme A. The dehydratase system consists of two oxygen-sensitive protein components, the activator (HgdC) and the actual dehydratase (HgdAB). Previous biochemical and spectroscopic studies revealed that the reduced [4Fe–4S]+ cluster containing activator transfers one electron to the dehydratase driven by ATP hydrolysis, which activates the enzyme. With a tenfold excess of titanium(III) citrate at pH 8.0 the activator can be further reduced, yielding about 50% of a superreduced [4Fe–4S]0 cluster in the all-ferrous state. This is inferred from the appearance of a new Mössbauer spectrum with parameters δ = 0.65 mm/s and ΔEQ = 1.51–2.19 mm/s at 140 K, which are typical of Fe(II)S4 sites. Parallel-mode electron paramagnetic resonance (EPR) spectroscopy performed at temperatures between 3 and 20 K showed two sharp signals at g = 16 and 12, indicating an integer-spin system. The X-band EPR spectra and magnetic Mössbauer spectra could be consistently simulated by adopting a total spin St = 4 for the all-ferrous cluster with weak zero-field splitting parameters D = −0.66 cm−1 and E/D = 0.17. The superreduced cluster has apparent spectroscopic similarities with the corresponding [4Fe–4S]0 cluster described for the nitrogenase Fe-protein, but in detail their properties differ. While the all-ferrous Fe-protein is capable of transferring electrons to the MoFe-protein for dinitrogen reduction, a similar physiological role is elusive for the superreduced activator. This finding supports our model that only one-electron transfer steps are involved in dehydratase catalysis. Nevertheless we discuss a common basic mechanism of the two diverse systems, which are so far the only described examples of the all-ferrous [4Fe–4S]0 cluster found in biology.
Introduction
The anaerobic bacterium Acidaminococcus fermentans ferments glutamate via the 2-hydroxyglutarate pathway to ammonium, carbon dioxide, acetate, butyrate, and molecular hydrogen [1]. A key enzyme in this fermentation is 2-hydroxyglutaryl-CoA dehydratase (CoA is coenzyme A), which catalyzes the reversible syn-elimination of water from (R)-2-hydroxyglutaryl-CoA, resulting in (E)-glutaconyl-CoA. This reaction is of considerable interest since a nonactivated β-C–H bond has to be cleaved (pK ≈ 40); for reviews see [2–4]. The enzyme system from A. fermentans is composed of two protein components, the homodimeric activator or archerase (HgdC) and the heterodimeric dehydratase (HgdAB), whose genes have been cloned and sequenced. After overexpression of hgdC in Escherichia coli, the activator could be purified to homogeneity aided by a C-terminal Strep-tag. The protein [γ2, 2 × (27 ± 1) kDa] contains 4.0 ± 0.1 iron and 3.8 ± 0.1 acid-labile sulfur, which form one [4Fe–4S]+/2+ cluster between the two subunits [5]. The activator can be readily reduced with dithionite or titanium(III) citrate. Most likely in vivo a reduced clostridial [4Fe–4S]-type ferredoxin [6] or the hydroquinone form of a flavodoxin [7], both isolated from A. fermentans, can act as reducing agents for the activator. The activator is extremely oxygen-sensitive; its half life under air is only about 10 s. The oxygen sensitivity results from destruction of the [4Fe–4S] cluster, which is exposed to the solvent [8]. Furthermore, the activator also precipitates in the presence of the dyes that are commonly used for the determination of redox potentials; herefore, the redox potential of the [4Fe–4S]+/2+ cluster (E0′ ≈ −320 mV) can only be roughly estimated from its almost complete reduction by ferredoxin or flavodoxin (E0′ = −420 mV) [7]. The native 2-hydroxyglutaryl-CoA dehydratase HgdAB (αβ, 54 + 42 kDa) contains 4.5 ± 0.3 iron, 3.5 ± 0.4 acid-labile sulfur and 1.0 FMNH2 [5]. In order to obtain catalysis, incubation of the dehydratase with reduced activator in the [4Fe–4S]+ state, ATP, and MgCl2 is necessary. Spectroscopic studies revealed that thereby one electron is transferred from the activator to the [4Fe–4S] cluster of the dehydratase [5, 7, 9], which cannot be reduced by any other means, except by deazaflavin and light in the presence of EDTA as shown for the related 2-hydroxyisocarproyl-CoA dehydratase (J. Kim and W. Buckel, unpublished results).
A mechanism has been proposed in which the dehydration is initiated by injection of this electron into the thiol ester carbonyl. The resulting substrate-derived ketyl radical eliminates the adjacent hydroxyl group. The enoxy radical formed can now be deprotonated to the product-related allylic ketyl radical, because radical formation has lowered the pK of the β-hydrogen by about 26 U [10]. In the last step the electron is returned from the allylic ketyl radical to the [4Fe–4S] cluster of the dehydratase and a new cycle can start. With the related 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile this radical has been identified by electron paramagnetic resonance (EPR) spectroscopy [11] and it has been shown that such a cycle can endure for about 10,000 turnovers until a new ‘shot’ by the activator is required [12].
Homologous activators with around 60% sequence identity have been characterized from 2-hydroxyglutaryl-CoA dehydratase in Fusobacterium nucleatum [13], lactyl-CoA dehydratase in C. propionicum (T. Selmer and W. Buckel, unpublished results), phenyllactyl-CoA dehydratase in C. sporogens [14, 15], and 2-hydroxyisocaproyl-CoA dehydratase in C. difficile [12]. Furthermore, each genome of almost all anaerobically thriving bacteria and archaea, even that of E. coli (yjiL gene), encodes up to four different homologues of the A. fermentans activator with 30–40% sequence identity. Only the function of the two homologues in Thauera aromatica and other anaerobic benzoate-degrading bacteria could be assessed as the α- and δ-subunits of the four subunits comprising benzoyl-CoA reductase. The catalysis of this enzyme, which in addition requires ATP and reduced ferredoxin as substrates, has been proposed to proceed also via a ketyl radical [16].
Hence such activators, which probably all contain a single [4Fe–4S] cluster between two subunits with a helix–cluster–helix architecture [8], are widespread in the anaerobic world. Proteins with almost the same architecture but with unrelated sequences are the nitrogenase reductases, which appear to have the same function as the activators [17]. The proteins of both families increase the reductive power of an electron by ATP hydrolysis. Nitrogenase reductase from Azotobacter vinelandii, also called Fe-protein or Av2, has been shown to become superreduced to the all-ferrous state by titanium(III) citrate [18–26]. The energy efficiency of nitrogenase would be twofold enhanced if the catalysis proceeded via superreduced Av2, owing to the transfer of two electrons from the corresponding [4Fe–4S]0 cluster per hydrolysis of two ATP molecules [27, 28]. However, the low redox potential Em = −790 mV reported for the (+1/0) couple of the iron–sulfur cluster virtually excludes any physiological relevance [measured with Cr(II)EDTA as a reductant] [29]. Interestingly, a much higher value, Em = −460 mV, was initially obtained by using methyl viologen [26]. Moreover, a recent report indicates that nitrogenase catalysis actually can be supported by superreduced Av2 and may proceed via the (+2/0) couple of the iron–sulfur cluster, if the in vivo reductant flavodoxin hydroquinone (FldHQ) is used [30]. In this new state of the Fe protein, the all-ferrous [4Fe–4S]0 cluster is difficult to discriminate from the +1 and +2 redox states using the absorption spectra, and it appears to be diamagnetic. In contrast, the Av2 [4Fe–4S]0 cluster made with titanium(III) citrate has a distinct absorption spectrum [18], and an unusually high spin state S = 4, as was inferred from elaborate integer-spin EPR and magnetic Mössbauer studies [18, 19, 31, 32] and theoretically explored by density functional theory studies [33, 34]. Here we report an all-ferrous state for the [4Fe–4S] cluster in the activator from A. fermentans after treatment with a tenfold excess of titanium(III) citrate. To our knowledge this is the second biological system which stabilizes a [4Fe–4S]0 cluster. Remarkable spectroscopic and biochemical similarities between activator and the A. vinelandii Fe-protein give reason to favor a common functional principle of these exciting proteins.
Materials and methods
Metallic 57Fe was purchased from Chemotrade (Düsseldorf, Germany). The metal (20 mg) was dissolved in 0.5 mL 8 M HCl at 80 °C for 12 h. This stock solution was added directly to the medium. Standard I nutrient broth was from Merck (Darmstadt, Germany). E. coli strain XL1-blue was purchased from Stratagene (Heidelberg, Germany). Streptavidin–Sepharose and the plasmid pASK-IBA3 were obtained from IBA (Göttingen, Germany). All other chemicals and substances were from Sigma or Fluka (Deisenhofen, Germany).
Anaerobic manipulations were performed in an anaerobic chamber (Coy Laboratories, Ann Arbor, MI, USA) with an atmosphere of 5% H2 in N2. X-band EPR measurements were performed using a Bruker E500 ELEXSYS continuous-wave spectrometer with the Bruker dual-mode cavity (ER4116DM) or a standard cavity (ER4102ST) and an Oxford Instruments helium-flow cryostat (ESR 910). Microwave frequencies were measured with a Hewlett–Packard frequency counter (HP5352B), and the field control was calibrated with a Bruker NMR field probe (ER035M). The spectra were simulated on the basis of a spin-Hamiltonian description of the electronic ground state: where St is the spin of the coupled system and D and E/D are the axial and rhombic zero-field parameters. The simulations were performed with a program which was developed from the S = 5/2 routines of Gaffney and Silverstone [35] and which specifically makes use of the Newton–Raphson iterative search method for transition fields as described there. Frequency-dependent Gaussian line shapes were used with full-width-at-half-maximum σν given in millitesla at g = 1. Alternatively also the Bruker XSOPHE suite [36] was used for the calculation of energy-level schemes and transition roadmaps. Mössbauer spectra were recorded using two spectrometers with alternating constant acceleration. The minimum experimental line width was Γfwhm = 0.24 mm/s (full width at half maximum). The sample temperature was maintained constant either in an Oxford Variox or an Oxford Mössbauer-Spectromag cryostat. The latter is a split-pair superconducting magnet system for fields up to 8 T in which the sample temperature can be kept constant in the range 1.5–250 K. The field is perpendicular to the γ-beam. The 57Co/Rh source was positioned at room temperature inside the gap of the magnet in a reentrant bore tube at a distance of about 85 mm from the sample. The field is zero at this position. All isomer shifts are quoted relative to iron metal at 300 K. Magnetic Mössbauer spectra were simulated with a computer program based on the same spin-Hamiltonian for St = 4 as for the EPR simulations (Eq. 1). The hyperfine interactions for 57Fe were calculated by using the usual nuclear Hamiltonian [37].
Purification of recombinant activator from A. fermentans
Standard I medium (8–10 L, Merck, Germany) was inoculated with 25 mL overnight culture of recombinant E. coli (XL1-blue/hgdC in pASK-IBA3) and grown in tightly closed 2-L bottles at 30 °C. When an optical density at 600 nm of about 0.3 was reached, cells were induced with anhydrotetracycline (30 μg/L). After anaerobic growth for a further 3 h, cells were harvested by centrifugation and suspended in 20 mM 3-(N-morpholino)propanesulfonic acid, pH 7.2, 100 mM NaCl, 10 mM MgCl2, 1 mM ATP, 2 mM dithiothreitol or 4 mM dithionite (buffer A). The bacteria were lysed anaerobically using a French press system. Cell debris and membranes were removed by ultracentrifugation for 60 min at 100,000g. The filtered supernatant was loaded with gravity flow onto an 8-mL streptavidin–Sepharose column, which was equilibrated with buffer A. After complete binding, the column was washed with at least ten column volumes of buffer A. The activator was eluted with 2.5 mM d-desthiobiotin in buffer A, then concentrated with an Amicon stirred cell, and immediately used for further experiments.
Results
Preparation and characterization of superreduced activator
The activator as isolated was concentrated to 60–80 mg protein/mL (1.2–1.6 mM) in 50 mM tris(hydroxymethyl)aminomethane hydrochloride pH 8.0. A tenfold excess of titanium(III) citrate was added, which yielded a color change from brown to red. Correspondingly, a new shoulder appeared in the UV–vis spectrum at about 500 nm (Fig. 1). The orange-red color apparently is a typical feature of superreduced [4Fe–4S]0 clusters, and does not originate from titanium(III) citrate directly, because this was removed by gel filtration on a Sephadex G-25 desalting column. It is noteworthy that the same color change was also observed upon full reduction of the nitrogenase Fe-protein from A. vinelandii [18].
Fig. 1UV–vis absorption spectrum of activator reduced with dithionite, and superreduced with titanium(III) citrate
As described previously, the activator had to be used right after purification for biochemical studies because of its tendency to precipitate within 4 days [5]. To our surprise, the superreduced activator was nearly as stable as the singly reduced or oxidized enzyme; the activity decayed only with a half life of 4–5 days or more. Remarkably, dehydratase activity could not be observed with 2-hydroxyglutaryl-CoA when titanium(III) citrate reduced activator was used in the activity assay [38].
EPR spectroscopy
In a previous EPR and Mössbauer study we demonstrated that dithionite-reduced activator1 possesses a [4Fe–4S]+ cluster with spin S = 3/2 [5]. The unusual magnetic ground state was inferred from X-band EPR spectra as shown in the inset in Fig. 2. The EPR resonances extend over a wide field range of at least 800 mT with a dominating strong absorption-like peak at g ≈ 4.5 and a broad trough at g ≈ 1.3–2. The large anisotropy and particularly the presence of high effective g values indicate the high spin multiplicity with large zero-field splitting (D > gμBB). Although the broad derivative spectrum does not have the classical appearance known for a quartet species from a transition metal complex [39], the pattern could be nicely simulated by using the usual spin-Hamiltonian for S = 3/2 with reasonable zero-field splitting parameters D = 1.3 cm−1 and E/D = 0.213. Just a simple Gaussian distribution of the rhombicity parameter with half width σ(E/D) = 0.11 was sufficient to account for the observed line broadening caused by g strain [5]. Moreover, a minor fraction of the [4Fe–4S]+ clusters (approximately 3%) was found to have the more common spin state S = 1/2, which gives rise to the narrower resonances around g = 2. The corresponding simulations for both spin states of [4Fe–4S]+, S = 1/2 and 3/2, are shown as gray and blue lines in the inset in Fig. 2.
Fig. 2Normal-mode (B1⊥B0) X-band electron paramagnetic resonance (EPR) spectrum of titanium(III) citrate reduced activator (low-field part, microwave frequency 9.6337 GHz; power 1.6 mW; modulation 2 mT at 100 kHz). The simulation for St = 4 (green line) is obtained with D = −0.66 cm−1, E/D = 0.165, g = (2.1, 1.99, 2.03) (Footnote 2) and a frequency-dependent Gaussian line width of σν = 70 mT. The inset shows the full range X-band spectrum of dithionite-reduced activator at 10 K with simulations for the [4Fe–4S]+ clusters as published previously [5]. The arrows in the main panel and in the inset indicate the field position of the strongest integer-spin signal from [4Fe–4S]0 clusters with St = 4
The strong absorption-like band at g ≈ 4.5 from S = 3/2 persists also in the EPR spectrum of titanium(III) citrate treated activator (Fig. 2, bold trace). We assign it to [4Fe–4S]+ clusters remaining from incomplete conversion of the reduced activator. The relative abundance of this contribution is estimated by corresponding the Mössbauer spectra to account for about 50% of the total iron (see below). Formation of superreduced clusters, however, is also discernible in the EPR spectrum from the appearance of two new lines in the low-field range at g ≈ 11 and g ≈ 16. Narrow absorption-like signals at such high g values without related strong derivative lines at higher field are completely unexpected for half-integer-spin systems, but rather suggest integer-spin transitions. The interpretation is corroborated by parallel-mode measurements (B1||B0) for which the new low-field resonances are the strongest signals, whereas the spectrum from the reduced activator, particularly the absorption-like band at g ≈ 4.5, is completely attenuated, as shown in Fig. 3. The striking difference of the two detection modes results from the different selection rules Δm = ±1 for B1⊥B0 and Δm = 0 for B1||B0, which essentially discriminate half-integer-spin and integer-spin transitions [40, 41]. Therefore, the low-field transitions at g ≈ 11 and g ≈ 16 must be assigned to an all-ferrous [4Fe–4S]0, cluster for which coupling of the four Fe(II) sites with local spin SFe = 2 can yield only an integer total spin. This clearly indicates superreduction of the activator by titanium(III) citrate. The ground state for such a tetrameric all-ferrous cluster can have, in principle, any total spin ranging from St = 0 to St = 8, depending on the six spin coupling constants between the four ferrous ions. But sophisticated quantum chemical studies for the titanium(III) citrate reduced [4Fe–4S]0 cluster from the Fe protein suggest only St = 0, 4, or 8 as possible solutions for that particular system [33, 34]. Below we show that the all-ferrous [4Fe–4S]0 cluster in superreduced activator actually has total spin St = 4, with properties that closely resemble those of the superreduced state of the Fe protein from A. vinelandii [18, 31].
Fig. 3Parallel-mode (B1||B0) X-band EPR spectra of titanium(III) citrate reduced activator recorded at 3, 10, and 20 K (microwave frequency 9.3404 GHz; power 1.6 mW; modulation 0.57 mT at 100 kHz). The green lines represent simulations for St = 4 with D = −0.66 cm−1, E/D = 0.165, g = (2.1, 2.0, 2.1) (Footnote 2), and a frequency-dependent Gaussian line width of σν = 30 mT
The integer-spin signals are remarkably sharp in the parallel-mode EPR spectra with full width at half maximum of only Γfwhm ≈ 4–5 mT for the main derivative peaks, but with very broad and asymmetric underlying shoulders. Such a superposition of broad and narrow powder signals may be caused by complex distributions of spin-Hamiltonian parameters, which owe their origin to microheterogeneity in the surrounding of the [4Fe–4S] clusters. In normal mode, however, the corresponding lines appear to be almost Gaussians of moderate 9–12 mT line width (Fig. 2). The difference may be related to the different orientational selections that contribute to the respective powder spectra [41]. Other ms components of the wave functions determine the transition probabilities between the zero-field split sublevels of the integer-spin system for parallel-mode detection, and thus may respond differently on the structural distortions. In the simulations, however, we could not reproduce the complex experimental line shape of the parallel-mode spectra of the superreduced activator by a simple distribution model for any of the spin-Hamiltonian parameters. Since we wanted to refrain from further sophistications other than Gaussian distributions, we just focused on the main features of the EPR spectra, i.e., the well-resolved sharp lines in the parallel-mode spectra and the Gaussian lines of the normal-mode spectra recorded at base temperature.
The EPR spectra of superreduced activator recorded in normal and parallel mode can be consistently simulated with total spin St = 4, as shown in Figs. 2 and 3. The zero-field parameters obtained from global optimization are D = −0.66 (±0.1) cm−1 and E/D = 0.165 (±0.01), with g = (2.2, 1.99, 2.03).2 Particularly the unusual temperature dependence of the two narrow parallel-mode signals in Fig. 3 is basically well reproduced with those values, i.e., at 3 K the simulated line at g ≈ 11 is stronger than the line at g ≈ 16, whereas the intensity ratio is inverted at 20 K. According to the energy splitting scheme shown on the left in Fig. 4, the derivative signals arise from transitions within the low-lying “ms = ±4” and the excited “ms = ±3” non-Kramers doublets, respectively. At the actual conditions of D and E/D, both of these ms doublets are virtually unsplit at zero magnetic field. For fields applied in the molecular z-direction, their X-band resonances are expected at apparent g values of 16 and 12, respectively. However, the transition probability is zero for the “ms = ±4” transition at exact B0||z and is very low for the “ms = ±3” transition, but the Zeeman effect is very anisotropic and causes mixing of the ms wave functions for both doublets, so EPR transitions become allowed for B0 oriented between the principal axes. The major intensity in a parallel-mode powder spectrum actually arises from orientations close to the z-axis. The variation of the resonance field as a function of the field orientation is visualized by the transition roadmaps shown on the right in Fig. 4. Powder derivative signals are expected from these diagrams only at the turning points of the respective traces. The corresponding g values for the “ms = ±3” and “ms = ±4” derivative spectra are indicated by the vertical dotted lines and g labels in diagram B in Fig. 4. Interestingly, there is a third resonance traced in the diagrams, coming from the highest pair of ms levels for the field close to the y-axis (marked with an asterisk). That also has a turning point at g ≈ 16, so the contribution in the EPR spectrum entirely overlaps the “ms = ±4” transitions.
Fig. 4A Energy level scheme for St = 4 with D = −0.66 cm−1, E/D = 0.165, g = 2.03, and B0 applied in the z-direction. The ms values given in quotation marks are labels for the magnetic sublevels but do not reflect the true composition of the wave functions. The vertical bold bars indicate parallel-mode transitions for ν = 9.6 GHz. B–D Transition roadmap diagrams for the same system, which are plots of the resonance fields for hypothetical single-crystal rotation experiments. The resonances of the “ms = ±4” and “ms = ±3” non-Kramers doublets are shown in red and blue, whereas transitions within the highest two ms levels are marked with an asterisk: B rotation of B0 in the z → x-plane; C rotation in the y → x-plane; and D rotation in the y → z-plane
The roadmap diagram also explains why the g ≈ 11 and the g ≈ 16 derivative signals have different intensities in the parallel-mode spectra at 20 K, although the Boltzmann law arrogates almost equal population of the corresponding non-Kramers doublets at this condition of temperature and field strength. Apparently, the ground-state “ms = ±4” doublet has a higher magnetic anisotropy, so the resonances become spread over a wider field range than for the “ms = ±3” transitions and the signal amplitudes are attenuated. Additionally, also lower intrinsic transition probabilities can be found at g ≈ 16, because higher ms functions become less mixed as a result of the competition between Zeeman- and zero-field interaction. At 3 K, in contrast, the g ≈ 11 signal fades out because the excited states become mostly depopulated. The effect yields substantial information about the magnitude of D and E/D, although the constraint on the parameters obtained from the intensity variations in the parallel-mode spectra in Fig. 3 may be somewhat weakened by the odd overall experimental line shapes of the two signals. However, the actual D and E/D values are nicely supported by the successful simulation of the resonance lines in the normal-mode spectrum in Fig. 2.
The parallel-mode spectra of the superreduced activator (Fig. 3) resemble remarkably well the EPR features of the all-ferrous cluster in the Fe-protein [21, 24] with a ground-state spin St = 4 [18, 31]. This cluster also shows a very sharp line in the parallel-mode X-band spectra around g ≈ 16 (actually at g = 16.4), but at g ≈ 11 only a weak, broad feature is observed at elevated temperature, whereas for the activator the line at g ≈ 11 is sharp and appears already at 3 K as a strong sharp signal. The inherent similarity of the spectra illustrates the same spin state for the all-ferrous clusters of the activator and the Fe protein and reveals similar physical properties. The differences in the EPR spectra arise from only small differences in the zero-field interaction, which slightly modulate the properties of the resonating non-Kramers doublets as shown in Fig. 4 (D = −0.75 cm−1, E/D = 0.30 for the Fe protein [31] compared with −0.66 cm−1 and 0.165 for the activator). In view of the fact that the zero-field splitting of the cluster spin manifold is inherited from the single-ion properties of four ferrous sites [42], and that there are 15 different ways to obtain St = 4 from such a spin tetrade [31], the close spectral similarity suggests very similar electronic properties of the all-ferrous clusters from activator and the Fe protein.
Mössbauer spectroscopy
We used 57Fe Mössbauer spectroscopy to quantify superreduction of the activator samples and to further substantiate the comparison with the Fe protein. Figure 5 shows the zero-field Mössbauer spectrum of titanium(III) citrate reduced activator recorded at 140 K. The asymmetric pattern could be fitted with three quadrupole doublets (I, IIa, and IIb), of which the prominent subspectrum I can be clearly assigned to [4Fe–4S]+ clusters from remaining starting material, because its isomer shift, δ = 0.51 mm/s, and quadrupole splitting, ΔEQ = 0.94 mm/s, are virtually identical to those observed for reduced activator at 80 K (δ = 0.53 mm/s, ΔEQ = 0.95 mm/s) [5]. The relative intensity of subspectrum I accounts for 54% of the total iron.
Fig. 5Zero-field Mössbauer spectrum of titanium(III) citrate reduced activator at 140 K. Subspectrum I and the superposition of subspectra IIa and IIb represent the contributions from reduced [4Fe–4S]+ and superreduced [4Fe–4S]0 clusters, respectively. δ(I) = 0.51 mm/s, ΔEQ(I) = 0.94 mm/s, 54% relative intensity; δ(IIa, IIb) = 0.65 mm/s, ΔEQ(IIa) = 1.51 mm/s, ΔEQ(IIb) = 2.59 mm/s; intensity ratio of IIa and IIb fixed at 3:1
Superreduction of the activator and formation of all-ferrous [4Fe–4S]0 clusters is revealed by the presence of the wide split component with higher isomer shift shown as the shaded area in Fig. 5. The corresponding all-ferrous [4Fe–4S]0 clusters from Av2 showed a similar pattern, but one that could be fitted with four subspectra according to four different iron sites. However, three subspectra were strongly overlapping and had similar quadrupole splittings in the range ΔEQ = 1.2–1.7 mm/s, but one deviated, showing ΔEQ = 3.08 mm/s [18, 31]. Such distinct differences cannot be resolved from the spectra of the all-ferrous cluster of the activator because of broad lines and limited experimental resolution, but the basic distribution pattern of three similar sites and one different site seems to persist also for the activator. We could fit our data with two Lorentzian doublets, IIa and IIb, having identical isomer shifts and a fixed intensity ratio of 3:1, so subspectrum IIa accounts for three similar iron sites of the all-ferrous cluster with unresolved signals, and subspectrum IIb represents a fourth, discernibly different site. Details on the significance of the fit model are given in the electronic supplementary material.3 The Mössbauer parameters obtained are δ = 0.65 mm/s for IIa and IIb and ΔEQ = 1.51 mm/s for IIa and ΔEQ = 2.59 mm/s for IIb, respectively. The high isomer shift and large quadrupole splittings are typical for tetrahedral Fe(II)S4 sites with localized valences. Particularly the isomer shift, which is the key parameter for the assignment of valence states, compares very well with that of the monomeric Fe(II) centers in reduced rubredoxin (δ = 0.70 mm/s at 4.2 K) [32, 43] or with that of the diferrous [2Fe–2S] cluster in a plant-type ferredoxin (δ = 0.71 mm/s at 4.2 K) [44]. Finally it matches exactly the value found for the all-ferrous cubane clusters in the superreduced Fe-protein (δ = 0.65 mm/s at 130 K). We assume that the rather strong line broadening found for the activator, Γfwhm(IIa) = 0.7 mm/s, Γfwhm(IIb) = 0.47 mm/s, is caused by further heterogeneity of the cluster sites in the frozen protein sample. We can exclude broadening by paramagnetic relaxation at 140 K since basically the same pattern was observed at 80 K.
It was not possible to achieve higher yields of superreduced [4Fe–4S]0 clusters than the approximately 50% mixture with remaining [4Fe–4S]+ clusters observed in the Mössbauer spectra shown here. Prolonged incubation with titanium(III) citrate or a higher excess of reducing agent did not improve the result. All attempts failed to fully convert a sample by the use of any chemical means, whereas photolytic reduction of the activator with deazaflavin led to cluster deterioration and precipitation of the protein. Because of this restriction, the midpoint of the [4Fe–4S]+ → [4Fe–4S]0 transition could not be exactly determined, but we estimate that the redox potential is lower than that of titanium(III) citrate, for which the value E0′1/2 = −720 mV against the standard hydrogen electrode is reported [45] (E0′ = −420 mV; or E0 = 0 mV). This estimation is based on the fact that most likely two protons per electron are released during the oxidation of TiIII to TiIV, which forms a titanyl ion: Hence at pH 8.0, at which the all-ferrous cluster was prepared, the redox potential of titanium(III) citrate should be—according to the Nernst equation—2 × 60 mV more negative, i.e., E0′ = −840 mV.
Magnetic Mössbauer measurements of titanium(III)-treated activator were performed at 4.2 K with applied fields of 0.2, 4, and 7 T. They yielded a complex magnetic hyperfine pattern with a large overall splitting of about 10 mm/s as shown in Fig. 6. On the basis of the model applied for the fit of the zero-field data, the spectra could be decomposed into three components. The inner part in the range from −2.5 to 3 mm/s is mostly determined by the contribution from the reduced activator having [4Fe–4S]+ clusters with spin S = 3/2 [5]. The component could be reasonably well simulated (subspectrum I, blue lines) by using isomer shift, quadrupole splitting, zero-field splitting, and magnetic hyperfine parameters as published previously for the S = 3/2 species4 [5]. The relative intensity of subspectrum I was fixed to 54% as taken from the zero-field spectrum.
Fig. 6Applied-field Mössbauer spectra of titanium(III) citrate reduced activator recorded at 4.2 K with B = 0.2, 4 and 7 T. The blue lines are spin-Hamiltonian simulations for subspectrum I from reduced [4Fe–4S]+ clusters with spin S = 3/2 [5] (Footnote 3). The thick green lines and the shaded areas mark the superposition of the two subspectra IIa and IIb from superreduced [4Fe–4S]0 clusters with the parameters as given in Table 1. The individual contribution of subspectrum IIb from the unique Fe(II) site with antiparallel aligned local spin is additionally shown as a thin green line above the other spectra
The outer hyperfine lines in the magnetic Mössbauer spectra found at about −4 and +5.5 mm/s are the best indicators for the paramagnetic contribution from the all-ferrous [4Fe–4S]0 clusters, which must have large internal fields at the iron nuclei because of the large spin. The magnetic splitting of the corresponding spectra is clearly outside the typical range of hyperfine splitting known for [4Fe–4S]+ clusters with spin S = 3/2 or 1/2. We could readily simulate that contribution from the superreduced activator (Fig. 6, green lines with shaded areas) by using the spin-Hamiltonian for spin St = 4 (Eq. 1), amended with the 57Fe hyperfine interaction with four sites grouped in the two subspectra IIa and IIb introduced above. Particularly the field dependence of the Mössbauer spectra, which depends critically on the zero-field splitting, is well reproduced with the corresponding zero-field splitting parameters D = −0.66 cm−1 and E/D = 0.165 obtained from the EPR analysis. The complete set of Mössbauer parameters used in the simulation of the magnetic spectra are summarized in Table 1. We have to mention, however, that particularly the results obtained for subspectrum IIb are not unique, because of the relatively poor signal-to-noise ratio of the broad spectra and the strong overlap with the remaining starting material, which perturb particularly the analysis of the otherwise informative weak-field spectra. Therefore, the solution given for the anisotropy of the A tensors and the components of the electric field gradient tensors (η and the Euler angles α, β, and γ) have to be regarded as generic. The main objective of presenting the analysis of the magnetic Mössbauer spectra is to show that the data are nicely consistent with the EPR analysis.
Table 1Mössbauer parameters for the all-ferrous [4Fe–4S]0 cluster of superreduced activator with spin St = 4 at 4.2 KSubspectrum IIa (three sites, 75% intensity)Subspectrum IIb (one site, 25% intensity)δ (mm/s)0.71 (3)0.71 (6)ΔEQ (mm/s)+1.80 (5)+2.6 (1)η0.8 (4)0.7 (4)α (°)90a90aβ (°)54 (10)125 (40)Ax/gNβN (T)−10.2 (1)+5.0 (5)Ay/gNβN (T)−6.4 (1)+8.0 (5)Az/gNβN (T)−7.23 (2)+7.8 (1)A0/gNβN (T)b−7.95 (7)+6.6 (4)The zero-field parameters used for the simulation were D = −0.66 cm−1 and E/D = 0.165. The Euler angles α, β, and γ  rotate the electric field gradient tensor with respect to the principal axes system of the zero-field splitting. The numbers given in parentheses are rough estimates of the error margins obtained by single-parameter variation and visual inspectionaThe angles α and γ were fixed at 90° and 0° to reduce the number of parametersbIsotropic value is 1/3 tr(A)
The three iron sites of subspectrum IIa that are indistinguishable at zero field also could not be further discriminated with applied fields, neither by sign or size of the quadrupole splitting nor by the magnetic hyperfine interaction. The isomer shift and quadrupole splitting appear to be uniform, with values close to those at 140 K with appropriate corrections for the lower temperature, and the hyperfine splitting can be described by only a single A tensor. Any possible heterogeneity in subspectrum IIa is masked by the anisotropy of A and the rather poor experimental resolution. The isotropic part A0 of the hyperfine tensor is negative for subspectrum IIa, which is best seen from the behavior of the resolved outer magnetic lines. They both have a strong component that is moving “inward” with increasing strength of the applied field from B = 0.2–7 T, owing to opposing internal and applied fields. Since the corresponding negative sign of A0 is like the sign intrinsically expected for iron(II) with respect to the local spin SFe = 2, the three iron sites forming subspectrum IIa must be basically coupled “parallel” to the total spin St = 4 of the [4Fe–4S]0 cluster.
The isotropic value A0 for subspectrum IIb, in contrast, is taken to be positive. This causes the increasing magnetic splitting of a part of the outer hyperfine lines moving outward with increasing applied field. For demonstration of this effect the corresponding subspectrum IIb is also shown separately as a thin green line above the superposition of subspectra IIa and IIb and the experimental traces in Fig. 6. Thus, the local spin of the corresponding unique Fe(II) site is predominantly opposite to the total spin of the [4Fe–4S]0 cluster. The specific scheme of negative and positive A0 values was adopted from the study of the [4Fe–4S]0 cluster in superreduced Fe-protein [31]. There it was also demonstrated that the occurrence of one positive and three negative A values is a necessary common property for any ground multiplet with total spin St = 4 that one can obtain from a tetrade of four spins SFe,i = 2 with isotropic exchange interaction [31].
The effective A-tensor components found for the [4Fe–4S]0 cluster of the superreduced activator are relatively weak compared with the intrinsic a0 value for high-spin Fe(II) sites like that of rubredoxins (isotropic part A0/gNβN = −7.94 and 6.6 T for the activator cluster compared with a0/gNβN = −16.2 T for rubredoxin) [32, 43], but they are in the range of those of the [4Fe–4S]0 centers from the Fe-protein [A0 = −11.2 (±0.2) and 5 MHz [31], which corresponds to A0/gNβN = −8.1 (±0.14) and +3.6 T, respectively]. The apparent reduction of the effective hyperfine coupling in the cubane clusters is caused by competing antiferromagnetic spin coupling. In the corresponding coupling scheme invoked for the all-ferrous cluster of the Fe-protein [31], the spin-projection factors Ki that describe the effective hyperfine interaction with respect to the total spin of the individual sites, A0(i) = Kiao(i), have been obtained as K1 = K2 = K3 = 7/15 (subsumed in subspectrum IIa here) and K4 = −2/5 (subspectrum IIb). With the a0 value for rubredoxin, a0/gNβN = −16.2 T, one obtains A0(IIa)/gNβN = −7.6 T and A0(IIb)/gNβN = 6.5 T. Both values are in reasonable agreement with the experimental result found for the superreduced activator, similar to what was found for the Fe-protein. In summary the spectroscopic properties found for the all-ferrous [4Fe–4S]0 cluster from the superreduced activator resemble quite closely those of the superreduced Fe-protein from A. vinelandii nitrogenase.
Discussion
In the past, cubane [4Fe–4S]n+ clusters had only been known to undergo one-electron redox reactions using either the [4Fe–4S]+/2+ couple or the [4Fe–4S]2+/3+ couple, but not both [46]. According to which couple the clusters are supporting, the iron–sulfur proteins were graded as ferredoxin-type proteins or high-potential iron proteins (HIPIPs) [47]. The latter are most abundant in phototrophic organisms. Except for the case of the [4Fe–4S]n+ cluster in the HIPIP from Chromatium vinosum, which can be artificially cycled from the +3 to the +1 state [48], the classification appeared to be unique and generally valid, until Watt and Reddy [26] reported that the [4Fe–4S]+ cluster of the nitrogenase Fe-protein from A. vinelandii could be reversibly reduced by methyl viologen to an all-ferrous [4Fe–4S]0 state where all irons have oxidation state +2. The midpoint potential for this reaction was initially given as −0.46 V versus the standard hydrogen electrode (SHE), but later studies with other reductants reported −0.79 V versus SHE [29], in contrast to −0.31 V for the first reduction (+2 ↔ +1). Titanium(III) citrate reduced nitrogenase Fe-protein was the subject of elaborate EPR and Mössbauer spectroscopic investigations, which yielded detailed insight in the electronic structure of the all-ferrous cluster and revealed spin S = 4 for the ground state [18, 31]. More recently, the formation of another superreduced state of Av2 with a diamagnetic [4Fe–4S]0 cluster and a different electronic absorption spectrum was observed, if the in vivo reductant FldHQ was used [30]. Notably, that system has a sufficiently high redox potential to support nitrogenase catalysis with two-electron transfer by using the (+2/0) couple of the iron–sulfur cluster.
Hitherto, nitrogenase Fe-protein was the only known enzyme which supported the all-ferrous [4Fe–4S]0 cluster. Also the first example of a synthetic all-ferrous [4Fe–4S]0 complex with ligands other than phosphane was reported only recently [49, 50]. It represents a very valuable analogue of the biological all-ferrous cluster, since it has been structurally and spectroscopically characterized. The Mössbauer spectra of the compound closely match those of titanium(III) citrate reduced Av2. In this paper we have demonstrated that superreduction in biological systems is not a unique phenomenon of the Fe-protein, but also the activator from 2-hydroxyglutaryl-CoA-dehydratase is able to support the [4Fe–4S]2+/+ as well as the [4Fe–4S]+/0 redox cycles of a single cubane cluster.
The electronic structures of the superreduced [4Fe–4S]0 cluster from the Fe-protein and from the activator appear to have a series of remarkable spectroscopic similarities. First, the UV–vis spectra of both systems exhibit a shoulder-type signal around 500–530 nm which causes distinct color changes from brown to orange-red upon reduction. We note that reduced iron(II) rubredoxin does not exhibit a similar absorption in the visible region [51]. Moreover, both [4Fe–4S]0 clusters exhibit the same total spin St = 4 in their ground state, and even the zero-field-splitting parameters compare remarkably well (activator, D = −0.66 cm−1, E/D = 0.165; Fe-protein, D = −0.75 cm−1, E/D = 0.33 [31]). The moderate difference of the rhombicity parameters for the superreduced activator, deviating from the almost fully rhombic limit encountered with superreduced Fe-protein, gives rise to the two distinct lines in the parallel-mode EPR spectra of the activator instead of the strong single line found for the Fe protein.
The Mössbauer isomer shifts for both centers are very similar and uniform for the respective cluster sites, owing to the same ferrous character of the metal ions. Our data are consistent with the specific distribution pattern of three iron(II) sites with similar quadrupole splitting and similar magnetic hyperfine coupling, and one different iron(II) site with larger quadrupole splitting and positive A tensor found for the Fe protein. In both enzymes the clusters are suspended at the interface of a protein dimer, but the spectra lack any indication of twofold symmetry, as one might expect from the respective protein structures [8, 52]. Since the electric field gradient, which gives rise to the quadrupole splitting, senses only local charge asymmetries, the more uniform appearance of the activator might reflect a smaller asymmetry of the actual cluster coordination in the protein and solvent moiety. Apart from that, the Mössbauer lines of the superreduced activator are unusually broad. Therefore we suppose that the molecular structure is probably not very well defined in the frozen solutions and exhibits microheterogeneity at the cluster site which leads to a distribution of quadrupole splittings for each of the iron(II) sites.
Microheterogeneity seems also to be the reason for the unusual shape of the EPR signals measured from the superreduced activator. The resonances show a superposition of sharp lines and very broad shoulders centered at similar resonance fields. The corresponding distribution of spin-Hamiltonian parameters, like the distribution of electric field gradients, may be related to the location of the cluster at a sensitive position in the protein structure between two protein monomers [8], where variations in the conformational substates of the protein may have a particularly strong impact on the geometry and electronic properties of the cluster. Such a distribution of spin-Hamiltonian parameters has been observed previously also for the dithionite-reduced activator with [4Fe–4S]+ clusters having the unusual spin state S = 3/2 [5] (the unusually broad EPR spectrum is redepicted in the inset in Fig. 2). Here, it is interesting to mention that the [4Fe–4S]+ cluster of the Fe-protein has spin S = 1/2 under physiologically reduction conditions, but it also changes to S = 3/2 if it is reduced in the presence of urea [53].
In contrast to the situation for the Fe protein from nitrogenase, it was never possible to obtain a complete superreduction of the activator; at best a ratio close to 1:1 could be achieved for [4Fe–4S]+ and [4Fe–4S]0 clusters. One may speculate whether this is related to the conformational distributions invoked above for interpretation of the spectroscopic heterogeneities. Probably, some fraction within the ensemble of variations may impose such unfavorable conditions on the molecular structure of the cluster and the resulting electronic properties such that further reduction is practically impossible. This interpretation seems to be supported by the observation that hard photoreduction by using deazaflavin as a potent electron source leads to inactivation and precipitation of the protein. However, for the time being also the trivial explanation cannot be excluded, namely, the midpoint redox potential for the [4Fe–4S]+/0 couple could be lower than that of titanium(III) citrate at pH 8.0 (see above), and thus even with a tenfold excess of this agent the cluster only becomes half reduced. We also cannot readily explain why titanium(III) citrate reduced activator is found to be enzymatically inactive, whereas the EPR and Mössbauer spectra reveal the presence of about 50% [4Fe–4S]+ clusters remaining, which should be catalytically competent. We presume that in the enzymatic assay the activator is fully reduced to the [4Fe–4S]0 state because of the very low protein concentration in micromolar range, in contrast to the situation in the highly concentrated spectroscopic samples where the protein is in millimolar abundance.
Since the ability of a [4Fe–4S] cluster to undergo two one-electron redox reactions using both the [4Fe–4S]+/2+ and the [4Fe–4S]+/0 couples should be basically related to its geometry, i.e., the metric details of molecular structure, as well as the (dielectric) properties of the protein site harboring the metal center. In this sense it may not be accidental that both proteins, the nitrogenase Fe-protein and the activator of 2-hydroxyglutaryl-CoA dehydratase, have very similar quaternary structures: homodimeric proteins in which a [4Fe–4S] cluster bridges the subunits. In both proteins an α-helix from each subunit points with its N-terminus towards the cluster and thus may stabilize with its dipole a negative charge, which should increase the redox potential. In the Fe-protein of nitrogenase this helix–cluster–helix architecture forms an angle of 150°, which upon binding of ATP and interaction with the iron–molybdenum protein opens to 180° [17]. Whether this conformational change further influences the redox potential of the cluster remains to be established. The helix–cluster–helix architecture of the activator of 2-hydroxyglutaryl-CoA dehydratase forms an angle of 105°, which is 45° closer than that of the Fe-protein of nitrogenase [8]. Upon ATP binding, the activator angle is thought to open in a similar manner [54], but a crystal structure of the complex between the activator and dehydratase stabilized by AlF4−-ADP (J. Kim and W. Buckel, unpublished results) is not available yet. Most likely the common electronic properties of the [4Fe–4S] clusters as well as the common structural features favor a related mechanism of both enzyme systems. Redox potentials are lowered by hydrolysis of ATP in order to achieve chemically difficult reductions of the substrates molecular nitrogen and 2-hydroxyglutaryl-CoA, respectively. However, owing to the observed inability of the superreduced activator to catalyze the enzyme reaction, the [4Fe–4S]2+ → [4Fe–4S]+ cluster transition and the subsequent electron transfer to the dehydratase component (HgdAB) appears to be the only biologically relevant charge transfer. The situation seems to be more complex for the nitrogenase mechanism. It was postulated that the all-ferrous nitrogenase Fe-protein indeed is an active redox state for nitrogen reduction. First, Watt and Reddy [26] reported that the ATP consumption for nitrogen fixation using all-ferrous Fe-protein is only half of that reported for the [4Fe–4S]+ cluster containing Fe-protein (one ATP per two transferred electrons compared with one ATP per one electron) [26]. In the meantime, the principal physiological relevance of the [4Fe–4S]0 cluster appears to have been corroborated for the nitrogenase system [30]. Maybe the enzyme can switch the redox pathway to choose between different electron donors for nitrogen reduction available in the cell. A similar role for the all-ferrous cluster of the 2-hydroxyglutaryl-CoA dehydratase system is still elusive.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Comparison of the fit model for the zero-field Mössbauer spectra of Ti(III) citrate-treated activator
with one and two Lorentzian doublets for the all-ferrous cluster. (PDF 374 kb)