Bärbel Schröfelbauer

Johanna Raffetseder

Maria Hauner

Andrea Wolkerstorfer

Wolfgang Ernst

Oliver H J Szolar
email: oliver.szolar@onepharm.com

Glycyrrhizin, the Main Active Compound in Licorice, Attenuates Pro-Inflammatory Responses by Interfering with Membrane-Dependent Receptor Signaling

Keywords: Glycyrrhizin, Toll-like receptor signaling, inflammation, cell membrane Glycyrrhizin, TLR, Toll-like receptor, TCR, T-cell receptor, BCR, B-cell receptor, BH, Benjamini-Hochberg

The triterpene glycoside glycyrrhizin (GL) is the main active compound in licorice. It is used as a herbal medicine owing to its anti-cancer, anti-viral, and anti-inflammatory properties. Its mode of action however remains widely unknown. In this study, we aimed to elucidate the molecular mechanism of GL in attenuating inflammatory responses in macrophages. Using microarray analysis, we found that GL caused a broad block in the induction of pro-inflammatory mediators induced by the toll-like receptor 9 (TLR9) agonist CpG-DNA in RAW 264.7 cells. Furthermore, we found that GL also strongly attenuated inflammatory responses induced by TLR3 and TLR4 ligands. The inhibition was accompanied by reduced activation not only of the NF-B pathway but also of the parallel MAPKs signaling cascade upon stimulation with TLR9 and TLR4 agonists. Further analysis of upstream events revealed that GL treatment reduced cellular attachment and/or uptake of CpG-DNA and strongly impaired TLR4 internalization. Moreover, we found that the anti-inflammatory effects were specific for membrane-dependent receptormediated stimuli, as GL was ineffective in blocking Tnfa induction upon stimulation with phorbol myristate acetate (PMA), a receptor and membrane-independent stimulus. These observations suggest that the broad anti-inflammatory activity of GL is mediated by the interaction with the lipid bilayer thereby attenuating receptor-mediated signaling.

Introduction

The innate immune system responds to invading microbes by detecting highly conserved structures of pathogens through interaction with toll-like receptors (TLRs). In humans, ten TLRs (TLR1-10) have been identified to date, detecting various pathogen specific molecules [1;2]. TLR9 is activated by unmethylated CpG motifs within single stranded DNA which are present at high amounts in bacteria. These motives can be mimicked by synthetic DNA oligonucleotides (ODNs) [START_REF] Krieg | Toll-like receptors 7, 8, and 9: Linking innate immunity to autoimmunity[END_REF][START_REF] Vollmer | Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8[END_REF][START_REF] Agrawal | Medicinal chemistry and therapeutic potential of CpG DNA[END_REF]. In unstimulated cells, TLR9 is localized in the endoplasmatic reticulum . Upon activation, it translocates to the lysosomal compartment were signaling is initiated [3;6;7]. TLR3 also localizes in endosomal compartments [8;9]. It can be activated by double stranded RNA (dsRNA) derived from the viral RNA genome or produced during viral replication . To mimic viral infection, polyriboinosinic:polyribocytidylic acid (poly(I:C)), a stable synthetic dsRNA analogue can be used [START_REF] Okahira | Interferon-ß induction through toll-like receptor 3 depends on double-stranded RNA structure[END_REF]. TLR3 activation results in induction of several pro-inflammatory cytokines and the expression of type I interferons (IFNs), which in turn activate numerous antiviral proteins including Mx GTPase, protein kinase R, and 2'-5'-oligoadenylate synthetases [START_REF] Kenny | Signalling adaptors used by Toll-like receptors: An update[END_REF]. TLR4 which is expressed on the cell surface can be activated by several bacterial surface molecules including lipopeptides and lipopolysaccharide (LPS) from gram-negative bacteria. Activation of TLR4 triggers strong induction of pro-inflammatory responses [START_REF] Popova | Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus[END_REF][START_REF] Rassa | Murine retroviruses activate B cells via interaction with toll-like receptor 4[END_REF][START_REF] Beutler | Innate immune sensing and its roots: The story of endotoxin[END_REF][START_REF] Lu | LPS/TLR4 signal transduction pathway[END_REF].

TLR4 activation depends on intact plasma membrane microdomains [START_REF] Schmitz | CD14 signalling in lipid rafts: New ligands and co-receptors[END_REF]. They aid TLR4 signaling initiation by facilitating the formation of the active receptor complex consisting of TLR4-MD2 and CD14 [START_REF] Schmitz | CD14 signalling in lipid rafts: New ligands and co-receptors[END_REF]. The cellular uptake of CpG-DNA mediated by scavenger receptors is similarly membrane-dependent [START_REF] Steward | Co-administration of polyanions with a phosphorothioate oligodeoxynucleotide (CGP 69846A): a role for the scavenger receptor in its in vivo disposition[END_REF][START_REF] Takagi | Involvement of Specific Mechanism in Plasmid DNA Uptake by Mouse Peritoneal Macrophages[END_REF][START_REF] Jozefowski | Role of scavenger receptor MARCO in macrophage responses to CpG oligodeoxynucleotides[END_REF][START_REF] Kim | Lipid raft microdomains mediate class A scavenger receptor-dependent infection of Brucella abortus[END_REF]. In addition, membrane microdomains have been demonstrated to play a critical role in the initiation of a broad variety of signaling events including activation of T-cell receptor (TCR) and B-cell receptor (BCR) signaling. They act through facilitating the pre-assembly and stabilization of receptor complexes, which permits quick, efficient, and sustained connection to the signaling cascade upon receptor engagement [20;21]. Accordingly, disruption of plasma membrane microdomains results in strong attenuation of receptor-mediated signaling [15;20;21]. Activation of TLRs triggers the formation of distinct signaling complexes. Stimulus-specific responses are achieved by the recruitment of different adopter proteins. The activation of these pathways leads to the expression of various pro-infammatory cytokines and chemokines such as TNF , IL1-and RANTES, the secretion of IFNs and to apoptotic responses. The induction of these responses are mainly triggered by the activation of the nuclear factor-kappa B (NF-B) and mitogen-activated protein kinase (MAPKs) pathways, a consequence common to the stimulation of all TLRs [2;3;14].

Licorice root has been used in traditional Chinese, Indian, and Greek medicine since the middle ages [22;23]. It contains several compounds including triterpene saponins, flavonoids, and polysaccharids [START_REF] Asl | Review of pharmacological effects of Glycyrrhiza sp. and its bioactive compounds[END_REF]. Out of these, the triterpene glycoside glycyrrhizin (GL) has been reported to be beneficial against viral infections. In fact, the GL formulation Stronger-Neo Minophagen C (SNMC) has been used to treat viral hepatitis C in Japan [25;26]. Anti-viral activity has also been reported against Influenza, SARS and HIV-1 [START_REF] Hattori | Preliminary evidence for inhibitory effect of glycyrrhizin on HIV replication in patients with AIDS[END_REF][START_REF] Ito | Mechanism of inhibitory effect of glycyrrhizin on replication of human immunodeficiency virus (HIV)[END_REF][START_REF] Harada | The broad anti-viral agent glycyrrhizin directly modulates the fluidity of plasma membrane and HIV-1 envelope[END_REF][START_REF] Cinatl | Glycyrrhizin, an active component of liquorice roots, and replication of SARS-associated coronavirus[END_REF]. Apart from its anti-viral activity, GL has been shown to influence inflammatory responses by up-regulating IL-10, IL-2, IL-12, and IFN-, while down-regulating pro-inflammatory cytokines including IL-8, IL-1 , TNF , eotaxin-1 as well as cyclooxygenase activity in different experimental systems [START_REF] Takei | Glycyrrhizin inhibits the manifestations of anti-inflammatory responses that appear in association with systemic inflammatory response syndrome (SIRS)-like reactions[END_REF][START_REF] Takei | Glycyrrhizin Inhibits Interleukin-8 Production and Nuclear Factor-kappaB Activity in Lung Epithelial Cells, but Not Through Glucocorticoid Receptors[END_REF][START_REF] Yuan | Antiinflammatory effect of Diammonium Glycyrrhizinate in a rat model of ulcerative colitis[END_REF][START_REF] Yoshida | Inhibitory effect of glycyrrhizin on lipopolysaccharide and d-galactosamine-induced mouse liver injury[END_REF][START_REF] Matsui | Glycyrrhizin and related compounds down-regulate production of inflammatory chemokines IL-8 and eotaxin 1 in a human lung fibroblast cell line[END_REF][START_REF] Matsui | Glycyrrhizin derivative inhibits eotaxin 1 production via STAT6 in human lung fibroblasts[END_REF][START_REF] Raphael | Effect of naturally occurring triterpenoids ursolic acid and glycyrrhizic acid on the cell-mediated immune responses of metastatic tumor-bearing animals[END_REF].

It is speculated that due to its structure, GL might act as glucocorticoid receptor agonist [START_REF] Yoh | Effects of glycyrrhizin on glucocorticoid signaling pathway in hepatocytes[END_REF] while another study demonstrated a glucocorticoid receptor-independent block of NF-B transcriptional activation [START_REF] Takei | Glycyrrhizin Inhibits Interleukin-8 Production and Nuclear Factor-kappaB Activity in Lung Epithelial Cells, but Not Through Glucocorticoid Receptors[END_REF]. In fact, inhibition of the NF-B activity by GL and other triterpenoids has been suggested for quite some time [32;39-42]. Interestingly, a study by Harada reported the anti-viral activity to be a result of changes in membrane fluidity possibly caused by incorporation of GL into the cellular membrane [START_REF] Harada | The broad anti-viral agent glycyrrhizin directly modulates the fluidity of plasma membrane and HIV-1 envelope[END_REF]. However, the exact mode of action remains unclear.

In this study, we aimed at elucidating the mode of action of GL as inhibitor of inflammatory responses mediated by TLRs. Initially the effect of GL on the expression profile of CpG-DNA stimulated macrophages was characterized followed by an in depth evaluation of the most significantly affected proteins. We further extended these experiments by analyzing the effects of GL on inflammatory responses caused by TLR3-and TLR4-agonists. Furthermore, its inhibitory effect on NF-B and MAPK activation was investigated. Eventually, the effects of GL on membrane-dependent ligand binding/uptake and signaling were evaluated. labeled cRNA targets for hybridization with mouse (4 x 44k) oligonucleotide arrays (G4122F), the Agilent Low RNA Input Fluorescent Linear Amplification Kit was used. 500 ng of total RNA were used for reverse transcription, the subsequent T7 polymerase cRNA target synthesis and for the labeling reaction with either Cyanine 3-CTP or Cyanine 5-CTP nucleotides. Each of the biological duplicates was processed in duplicate reactions. RNeasy mini spin columns (Qiagen) were used for purifying labeled cRNA targets. The cRNA was digested and hybridizations were performed in a rotisserie at 65°C for 17 h using Agilent's hybridization kit.

Microarray data analysis. Slides were scanned with the dual laser Agilent microarray scanner at 5 micron pixel resolution and 10 % and 100 % PMT (photo multiplier tube) power setting to allow extended dynamic range (XDR) data calculation. For raw data generation and normalization, feature extraction software (version 9.1) was used. The Linear&Lowess algorithm was used with default settings for signal characteristics and normalization spots. To correct for intensity variations at different regions on the array spatial and multiplicative detrending (removing the surface trend) was selected (default). Feature extraction output files were imported into Genespring GX v 9.0.2 (Agilent) for visualization and analysis of expression data. Data filtering according to a fold-change criterion and a certain confidence level (p-value) was completed to identify targets that are statistically significant. Where indicated, Benjamini-Hochberg-correction (BH-correction) was performed. Gene Ontology-(GO) analyses was made with Genespring software.

Quantification of gene expression. mRNA expression levels of stimulated and unstimulated cells with or without GL treatment were quantified using QuantiGene technology (Panomics). Gene-specific probe-sets were obtained from Panomics and luminescence was detected using a Synergy HT microplate reader (Biotek). Analysis was performed according to manufactures' instructions. Briefly, cells were lyzed in provided lysis buffer (Panomics) supplemented with proteinase K, by boiling at 60°C for 30 min. Supernatants were stored at -80°C until further analysis. Results were normalized to housekeeping genes (Gapdh or Actin), which were analyzed in parallel. Fold change was calculated in relation to unstimulated cells, which were set to 1.

ELISA. Amount of cytokine release was determined in culture supernatants of cells stimulated with the corresponding ligand over night by ELISA according to manufactures' instructions. TNF , IL-1 , IL-1 , and IL-10 ELISA kits were obtained from Biolegend, RANTES and MIP-1 from R&D, IFN-ELISA from PBL and IL-6 ELISA from Bender. Absorbance was detected using a Synergy HT microplate reader (Biotek). In parallel, cell viability was determined using CellTiter-Glo Luminescent Cell Viability Assay kit (Promega).

Immunoprecipitation and Western blotting. Cells were stimulated for indicated times followed by lysis in M-PER buffer (Pierce) or RIPA buffer containing protease inhibitor cocktail (Pierce) and phosphatase inhibitor cocktail (Sigma). Protein concentration was determined by Bradford (Bio-Rad). For IL-1 detection, cells treated with indicated amounts of GL were stimulated with 10 nM CpG-DNA over night. Cells were lyzed in RIPA-buffer and equal amounts of cell lysates (1 mg protein) were incubated with goat anti-mouse IL-1 antibody (R&D) followed by precipitation with protein-G-sepharose beads (Amersham). Beads were washed 3x in lysis buffer, followed by elution with SDS-sample buffer by boiling at 90°C for 5 min. For Western blot analysis, equal amounts of cell lysate (10-30 g) or IP sample were loaded onto 4-12 % or 12 % Bis-Tris Gel (Invitrogen) and blotted onto PVDF membranes (Invitrogen). Antibodies against A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. mouse p-Erk1/2 (T202/Y204), Erk1/2, p-p38 (T180/Y182), p38, p-SAPK/JNK (T183/Y185), SAPK/JNK and p-I B and I B were obtained from Cell Signaling Technologies, anti-mouse IL-1 from R&D, antibodies against mouse FAS, Cox-2 and Tubulin were obtained from Abcam. HRP conjugated secondary antibodies were purchased from GE Healthcare. Blots were developed using ECL substrate (GE healthcare) on a BioSpectrum Imaging System using BioChemi HR camera (UVP).

CpG-uptake. RAW 264.7 cells were incubated for 60 min with 40 nM FITC-labeled CpG-ODN (Invivogen) with or without GL, washed twice in PBS, fixed with 4 % paraformaldehyde for 10 min and analyzed by flow cytometry using a FACScalibur (Becton Dickinson). A minimum of 20.000 cells per sample were acquired. TLR4 internalization. RAW 264.7 cells were preincubated for 2 h with 2.5 mM GL prior to stimulation with 2 g/ml LPS. Upon stimulation for 60 min, cells were stained with rat anti-TLR4 antibody (Sa15-21 [START_REF] Akashi | Lipopolysaccharide interaction with cell surface toll-like receptor 4-MD-2: Higher affinity than that with MD-2 or CD14[END_REF]) and FITC conjugated anti-rat antibody and directly analyzed by flow cytometry.

Results

GL causes changes in gene expression upon CpG-DNA stimulation. GL is well known for its anti-inflammatory actions in vivo and in vitro. Its effect on inflammatory responses induced by the activation of specific TLRs however has not been studied in detail so far. Thus, we aimed to analyze the effects of GL on gene expression patterns in response to CpG-DNA by microarray analysis. RAW 264.7 cells were stimulated with 10 nM CpG-DNA in the presence or absence of GL. 4 h post stimulation, RNA was isolated, labeled and hybridized to a mouse oligonucleotide array. Resulting data was normalized as described in experimentals and analyzed using Genespring software. To compensate for multiple comparisons, BH procedure was used for pvalue adjustment. Twenty genes were identified that were down-regulated more than 2-fold with a corrected p-value of <0.01. A list of these genes is shown in Table 1. The genes that were down-regulated strongest by GL were the pro-inflammatory cytokines Il1a (17-fold) and Il1b (13-fold). Other strongly regulated genes included the chemokines Ccl5/Rantes, Ccl4/Mip1b, Cxcl2/Mip2a and Ccl3/Mip1a. In addition, some genes that are involved in the regulation of signaling were found to be down-regulated significantly (Cish and Nfkbiz). Using the same criteria (BH-adjusted p-value <0.01), 8 genes were found to be up-regulated by GL by more than 1.5-fold. However, these effects were very mild with the highest induction being 3-fold (see supplemental table ST1). GL treatment alone did not cause significant changes in gene regulation in unstimulated cells (data not shown).

Strong down-regulation of pro-inflammatory responses by GL.

To further analyze the effect of GL on the gene expression pattern of CpG-DNA activated cells, we performed GO-analysis. For this analysis, we did not adjust for multiple comparisons in order to obtain a more extensive gene list. The input list consisted of 102 genes that were down-regulated and 14 genes that were up-regulated more than 2-fold (see supplemental table ST2 and ST3) with a p-value <0.01. As shown in Table 2, highly significant gene enrichment could be observed for the categories 'immune response', 'immune system process', 'defense response' and 'cytokine activity'. 24 genes fell into the category of 'immune response' and 'immune system process'. In addition, GOterms including 'inflammatory response', 'receptor binding', 'response to wounding', 'taxis', and 'response to stimulus' were found to be enriched with high statistical significance. Taken together, these data indicate that GL broadly alters the pro-inflammatory response induced by CpG-DNA.

Confirmation of the anti-inflammatory activity on RNA level. To verify the results obtained by microarray analysis, expression of several inflammation-associated genes that showed strong down-regulation upon treatment with GL were individually analyzed by QuantiGene. Therefore, cells were stimulated with CpG-DNA and expression of selected genes involved in inflammation (Il1b, Il1a, Ccl4/Mip1b, Tnfa, Ptgs2/Cox2, Ccl5/Rantes) was analyzed at different time points. The most striking result was obtained for Il1b, which was induced about 12-fold after 4 h and 48fold 8 h post stimulation, while barely any induction was observed in cells treated with GL (Fig. 1). Ccl5/Rantes was induced about 20-fold 4 h, and as much of 40-fold at 8 h post stimulation. In GL treated cells, Ccl5/Rantes expression remained low with a maximum of only 4-fold increase as compared to unstimulated cells. CpG-DNA caused a 6-fold induction of Ptgs2/Cox2 at 4 h post stimulation which increased to 12-fold at 8 h post stimulation. By contrast, in cells treated with GL, the induction of Ptgs2/Cox2 was significantly attenuated, with only a 2-fold increase 8 h after addition of CpG-DNA. The effect of CpG-DNA on the induction of Tnfa, Il1a, Fas, and Ccl4/Mip1b showed similar effects. Tnfa and Il1a expression was induced 5-to 6-fold at 2 h post stimulation with CpG-DNA and stayed high until 8 h post stimulation, while no Tnfa and Il1a mRNA was detected in cells treated with GL. Mild Fas-induction was observed upon stimulation with CpG-DNA, which was not detected in cells treated with GL. Ccl4/Mip1b expression was only weakly induced 2 and 4 h post stimulation, but expression increased to 7-fold after 8 h. In the presence of GL, Ccl4/Mip1b mRNA levels remained low. These findings are consistent with the microarray data and confirm that numerous inflammation-associated genes are downregulated by GL.

GL inhibits the production of pro-inflammatory proteins.

To further confirm the antiinflammatory properties of GL, its effect on the production and release of cytokines and chemokines that were shown to be attenuated by GL on mRNA level, was analyzed. MIP-1 TNF , and RANTES were induced by CpG-DNA and GL reduced their release in a dosedependent manner (Fig. 2A-C). Despite considerable efforts, we were unable to detect IL-1 in the supernatant of cell cultures. However, CpG-DNA induced significant intracellular expression of IL-1 which was reduced in cells treated with increasing amounts of GL (Fig. 2D, top panel). In addition, Western blot analysis showed that CpG-DNA induced intracellular expression of Cox-2 and FAS which was again inhibited by GL in a dose-dependent manner (Fig. 2D, bottom panel). These data further demonstrate that GL down-regulates expression of pro-inflammatory proteins upon stimulation with CpG-DNA.

GL inhibits pro-inflammatory responses induced by TLR4 ligands.

To evaluate if GL specifically blocks CpG-DNA-induced inflammatory responses, we investigated mRNA and protein secretion of several pro-inflammatory mediators upon stimulation with LPS, a TLR4 agonist that in contrast to TLR9 signals both, in a MyD88-dependent and independent manner. LPS triggered strong induction of Ccl5/Rantes, Il1b, Ptgs2/Cox2, Ccl4/Mip1b and weaker induction of Tnfa mRNA levels (Supplemental Figure S1). In GL treated cells, induction of all these pro-inflammatory genes was almost completely suppressed. Similar results were obtained on protein level (Fig. 3A). GL reduced LPS-induced secretion of TNF RANTES, and IFN-by 70-80%. As observed for CpG-DNA, the reduction was dose-dependent (data not shown). Thus, GL was able to inhibit pro-inflammatory responses induced by LPS. Unlike RANTES and IFN-the induction of TNF is dependent on MyD88, suggesting that GL blocks inflammatory responses in a pathway-independent manner.

GL inhibits responses induced by TLR3.

To further confirm the broad and pathway independent anti-inflammatory properties of GL, we tested its ability to block responses induced by a TLR3 agonist, which in contrast to TLR4 and TLR9 signals solely in a MyD88-independent manner. Poly(I:C)-induced IL-6 and IFN-secretion was strongly reduced by GL (Fig. 3B). Again, the reduction was dose-dependent (data not shown). Taken together, these data demonstrate that GL indeed has a broad inhibitory effect on the cellular responses to several distinct TLR agonists, implying that it interferes with a target common in all TLR pathways.

GL inhibits NF-B activation induced by CpG-DNA and LPS.

To deepen our understanding of the molecular mechanism by which GL exerts its anti-inflammatory effects, we analyzed the signaling pathways activated by TLR9 and TLR4. Since the effect of GL could readily be explained by a block in the NF-B pathway, which is activated by all TLRs, we first analyzed the effect of GL on the activation of NF-B by measuring the phosphorylation (hence activation) and expression levels of I B . As shown in Fig. 4A, left panel, low levels of I B phosphorylation could be detected at 30 and 60 min post CpG-DNA stimulation which peaked at 120 min. In the presence of GL, I B phosphorylation was appreciably reduced as compared to untreated cells. Total I B expression levels correlated with these data. Compared to unstimulated cells, protein levels were weakly reduced at 30 min post stimulation, and I B further declined up to 60 min post stimulation in untreated cells, indicating NF-B activation. At 120 min and 240 min post stimulation, I B expression increased again. In cells treated with GL, I B expression was considerably higher during the whole course of the experiment with the most pronounced difference detected at 60 and 120 min post stimulation. The effect of GL on the activation of NF-B appears to be less pronounced compared to gene expression data; however, it should be mentioned that the amount of CpG-DNA used in experiments described earlier (10 nM) was not sufficient to induce detectable levels of activation. Thus, these experiments were conducted using 100 nM CpG-DNA, a concentration at which GL overall shows considerably weaker antiinflammatory activities (data not shown).

Similar results, yet more pronounced were obtained upon stimulation with LPS (Fig. 4A, right panel). At 5 min post LPS stimulation, phosphorylation of I B could be observed in untreated cells, while in GL treated cells barely any activation of I B could be detected up to 60 min. In agreement with this, I B remained stable until 30 min post stimulation in GL treated cells, while it was almost completely degraded at 30 min post stimulation in untreated cells. These data indicated that GL reduces phosphorylation and subsequent degradation of I B thereby reducing downstream activation of NF-B, both upon stimulation with CpG-DNA and LPS. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

GL reduces

4B, left panel) at 60 min and stronger at 120 min post stimulation which was mildly reduced in GL treated cells while total JNK expression was unaffected. Compared to unstimulated cells, enhanced phosphorylation of p38 was detected at 30 min and 60 min post CpG-DNA stimulation while the signal declined to background levels after 120 min. In the presence of GL, p38 phosphorylation was significantly weaker at both, 30 and 60 min post stimulation, indicating that GL treatment reduced p38 activation. The expression of total p38 was comparable for all time points and independent of the treatment. Similar results were obtained for ERK1/2. Weak activation was detected 30 min post stimulation, which strongly increased at 60 min post stimulation in untreated cells, while only low levels of activation were detectable in cells treated with 1 mM GL at 30 and 60 min post CpG-DNA stimulation. The overall ERK1/2 protein levels remained unchanged.

Comparable results were obtained upon stimulation with LPS (Fig. 4B, right panel). JNK activation was detectable at 30 and 60 min post stimulation in untreated cells, while barely any JNK phosphorylation could be observed until 30 min post stimulation and only weak activation at 60 min post stimulation in GL treated cells. Total JNK expression remained unaffected throughout the course of the experiment. Strong activation of p38 was detectable at 30 and 60 min post stimulation, which was dramatically reduced in cells treated with GL while treatment had no effect on total p38 levels. Similar results were obtained for ERK1/2 phosphorylation which was strongest at 30 and 60 min post LPS stimulation. In GL treated cells activation was decreased throughout the course of the experiment. Total ERK1/2 expression was comparable in treated and untreated cells. Collectively, this analysis indicates that GL does not specifically block the NF-B pathway but similarly reduces the activation of MAPKs, strongly suggesting that GL interferes with TLR signaling upstream of MAPK and NF-B activation. Moreover, given that GL is active against various TLRs that signal through distinct pathways it is likely to target an upstream event common to all TLR pathways.

GL reduces cellular uptake/attachment of CpG-DNA. GL appears to act early upon TLR induction and seems to cause a rather broad block to TLR-mediated responses; therefore, we asked whether GL affects the overall stimulation by reducing cellular uptake or attachment of TLR ligands. RAW 264.7 cells were incubated with FITC-labeled CpG-DNA for 60 minutes and after extensive washing, fluorescence was measured by FACS analysis. As shown in Figure 5A, incubation of cells with CpG-DNA induced a significant fluorescence shift. In cells treated with GL, the shift was reduced dose-dependently. The effect with 1 mM GL was considerably weak which is most likely due to the increased amount of CpG-DNA (40 nM) used in this experiment. Consistently, under these conditions GL only shows reduced anti-inflammatory effects, while it completely blocked pro-inflammatory responses at a concentration of 2.5 mM GL (data not shown).

TLR4 internalization is attenuated in GL treated cells. Stimulation of cells with LPS is known to trigger TLR4 internalization [START_REF] Kagan | TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon[END_REF]. To further confirm that GL does indeed block initial responses to various stimuli we tested its effect on the internalization of TLR4 upon stimulation with LPS. Therefore RAW 264.7 cells were preincubated with GL followed by stimulation with LPS. TLR4 internalization was measured upon staining with specific antibodies by FACS. As shown in Figure 5B surface TLR4 staining was readily detected in unstimulated cells. LPS stimulation for 60 minutes resulted in a strong reduction of the signal, indicating TLR4 internalization. In contrast, in cells treated with GL, LPS stimulation had no effect on TLR4 surface expression. These data clearly demonstrate that GL interferes with responses to LPS by inhibiting the initiation of TLR4 signaling.

GL does not affect PMA induced Tnfa induction. Uptake of CpG-DNA is known to be mediated by scavenger receptors [START_REF] Steward | Co-administration of polyanions with a phosphorothioate oligodeoxynucleotide (CGP 69846A): a role for the scavenger receptor in its in vivo disposition[END_REF][START_REF] Takagi | Involvement of Specific Mechanism in Plasmid DNA Uptake by Mouse Peritoneal Macrophages[END_REF][START_REF] Jozefowski | Role of scavenger receptor MARCO in macrophage responses to CpG oligodeoxynucleotides[END_REF]. Efficient uptake of pathogens by scavenger receptors requires intact membrane microdomains [START_REF] Kim | Lipid raft microdomains mediate class A scavenger receptor-dependent infection of Brucella abortus[END_REF]. Similarly, for efficient TLR signaling lipid raft formation is critical. Interestingly, it has previously been reported that GL causes altered membrane fluidity [START_REF] Harada | The broad anti-viral agent glycyrrhizin directly modulates the fluidity of plasma membrane and HIV-1 envelope[END_REF]. Thus, we hypothesized that GL might possess the ability to incorporate into membranes thereby disrupting the membrane architecture. To test this, we analyzed the effect of GL on stimulation with PMA, a receptor-and thus membrane-independent stimulus [START_REF] Doan | Differential regulation of TNF-R1 signaling: Lipid raft dependency of p42mapk/erk2 activation, but not NF-B activation[END_REF]. RAW 264.7 cells were stimulated with different amounts of PMA with or without GL treatment and mRNA levels of Tnfa were measured 4 h post stimulation. PMA dose-dependently induced Tnfa mRNA (Figure 5C). Strikingly, GL treatment had no effect on Tnfa expression, and remained unaffected in all experimental conditions. These results strongly suggest that GL does indeed cause its broad anti-inflammatory actions by interfering with membrane integrity.

Discussion

Although the anti-inflammatory properties of GL are widely described, a detailed analysis of its effects on the molecular level has not been conducted thus far. By using microarray analysis, we found that GL blocked inflammatory responses induced by CpG-DNA, a TLR9 ligand. The expression of a wide range of cytokines and chemokines was also strongly reduced by GL upon stimulation of TLR4. Moreover, treatment with GL resulted in reduced secretion of IL-6 and IFN-induced by the TLR3 agonist poly(I:C). These effects were accompanied by a broad block in TLR signaling. GL not only inhibited the activation of NF-B but also the activation of the MAPKs p38, ERK1/2, and JNK induced by both, TLR4 and TLR9 ligands. Further analysis revealed that cellular attachment and/or uptake of the TLR9 ligand CpG-DNA was strongly reduced and TLR4 internalization was blocked in the presence of GL. The anti-inflammatory effects of GL were specific for receptor-mediated stimuli as GL did not show any effect on Tnfa induction triggered by PMA, which signals in a receptor-independent manner. These findings indicate that the anti-inflammatory actions of GL are mediated by an interaction with the cell surface rather than by a specific block in the downstream signaling cascades.

Comparison of gene expression patterns of untreated and GL-treated cells demonstrated a strong down-regulation of numerous pro-inflammatory genes at 4 h post stimulation with the TLR9 agonist CpG-DNA. Strikingly, analysis of microarray data by GO analysis revealed that a subset of 24 GL-regulated genes were over-represented in the categories 'immune response' and 'immune system process'. Other categories that were found to be overrepresented with high statistical significance included 'cytokine activity' and 'inflammatory response'. These data imply that GL causes a rather broad block to CpG-DNA induced responses.

Notably, GL efficiently blocked the expression of a wide range of pro-inflammatory mediators at very early times post stimulation. These data strongly indicate that GL acts early upon stimulation, by blocking TLR signaling directly rather than by down-regulating one or a few specific genes, resulting in reduced autocrine/paracrine stimulation. We also detected a reduction in the expression of Ifnb and of numerous IFN regulated genes. Induction of IFN inducible genes is not caused by CpG-DNA directly but is most likely a consequence of an autocrine/paracrine stimulation mediated by IFN-. Thus, the reduced expression of IFN-regulated genes in the presence of GL is likely a consequence of the initial reduced induction of IFN-.

The anti-inflammatory actions of GL in response to various TLRs could easily be explained by a block of NF-B activation. In agreement with this, several reports describing anti-inflammatory actions of GL suggest a specific and direct block of the NF-B pathway based on data demonstrating reduced I B degradation or reduced NF-B transcriptional activation analyzed by EMSA or nuclear localization studies of p65/RelA [32;33;40;41]. However, these studies failed to analyze possible effects on parallel pathways such as MAPK activation and events further up-stream in the signaling cascade to proof specificity for a direct block of NF-B activity. Our data in contrast clearly demonstrate that GL does not block a specific target in the signaling cascade but instead causes a broad inhibition not only of NF-B but also of MAPK activation upon stimulation of TLR4 and TLR9. Thus, these data are suggestive for the hypothesis that GL blocks inflammatory responses either by rather unspecifically targeting several distinct molecules within the different TLR pathways, or more likely, by directly interacting with TLRs or, even more globally, by interfering with cell membrane-associated mechanisms.

The hypothesis that GL interferes with membrane-dependent processes is supported by our data that demonstrate that GL interferes with cellular attachment and/or uptake of CpG-DNA providing an explanation for the broad, early inhibitory effects of GL. Treatment with GL also inhibited TLR4 internalization, a process for which lipid raft formation is critical. These observations lead to the hypothesis that GL might be incorporated into lipid bilayers thereby altering the integrity of the plasma membrane. Indeed, Harada demonstrated reduced membrane fluidity in the presence of GL causing the suppression of infectivity of HIV-1, influenza virus, and vesicular stomatitis virus [29;46]. As a consequence to changes in membrane integrity, it could be hypothesized that GL impedes receptor dimerization, complex formation, or ligandreceptor binding resulting in reduced signal transduction. CpG-DNA uptake depends on scavenger receptors which have been described as being membrane-dependent [START_REF] Steward | Co-administration of polyanions with a phosphorothioate oligodeoxynucleotide (CGP 69846A): a role for the scavenger receptor in its in vivo disposition[END_REF][START_REF] Takagi | Involvement of Specific Mechanism in Plasmid DNA Uptake by Mouse Peritoneal Macrophages[END_REF][START_REF] Jozefowski | Role of scavenger receptor MARCO in macrophage responses to CpG oligodeoxynucleotides[END_REF][START_REF] Kim | Lipid raft microdomains mediate class A scavenger receptor-dependent infection of Brucella abortus[END_REF]. Reduced membrane fluidity or disruption of membrane microdomains could therefore interfere with scavenger receptor formation and might thus account for the observed reduction of CpG-DNA surface binding in the presence of GL. Interestingly, intact membrane microdomains are also critical for the initiation of TLR4 signaling. Its disruption has been shown to interfere with the formation of the functional complex of TLR4-MD2 with CD14 resulting in a block in LPS signaling [START_REF] Schmitz | CD14 signalling in lipid rafts: New ligands and co-receptors[END_REF]. Altered membrane integrity triggered by GL could therefore provide an explanation not only for the inhibition of TLR9 signaling but also for its anti-inflammatory actions upon TLR4 activation. The fact that GL has no effect on Tnfa expression induced by PMA, a stimulus that is known to induce signaling in a membrane-independent manner [START_REF] Doan | Differential regulation of TNF-R1 signaling: Lipid raft dependency of p42mapk/erk2 activation, but not NF-B activation[END_REF] even further supports this hypothesis.

In addition to its block of anti-inflammatory responses caused by TLR agonists, GL similarly blocks TNF release induced by various additional stimuli, including the pro-inflammatory cytokine HMGB1 and other RAGE ligands such as S100, S100b, and AGE-BSA as well as antiviral responses induced by type I interferons (data not shown). Strikingly, signaling induced by these stimuli has also been demonstrated to depend on intact membrane microdomains [47;48]. Since all these stimuli act through distinct signaling cascades, yet are dependent on an intact membrane integrity, it is likely that the mode of action is indeed mediated by the ability of GL to attach onto or incorporate into the plasma membrane thereby attenuating various membranedependent processes including initiation of receptor signaling. Interestingly, GL does not show any toxicity even if cells are treated with very high concentrations (up to 5 mM). In case of purely unspecific 'coating' of the cell membrane, one would expect cellular maintenance and growth as well as intercellular communication being severely impaired, which would be accompanied by a significant reduction in cell viability. However, cell integrity was fully maintained throughout all studies suggesting that GL targets specific microdomains of the plasma membrane rather than interfering with the entire lipid bilayer.

It should be noted that rather high concentrations of GL were required in our experiments to exert anti-inflammatory effects; one reason might be the rather hydrophilic character of GL limiting its interaction with the cellular system. So at present, it is not clear whether such doses can be reached in vivo especially in the case of systemic administration. Sufficient doses will most likely require a local (e.g. topical) application of a suitable glycyrrhizin formulation. On the other hand, substitution of polar residues with hydrophobic groups in the molecule could be a promising approach, which is, however, beyond the scope of this study.

In conclusion, this study clearly indicates a broad inhibitory effect of GL on pro-inflammatory responses induced by various TLRs in RAW 264.7 macrophages that appears to be mediated by interactions with the cell membrane (such as alteration of membrane microdomains) rather than by a block of a specific intra-cellular target as has been suggested in the literature thus far. These data further highlight the importance of membrane integrity for the initiation of efficient innate immune responses. Interestingly, altered plasma membrane architecture has also been reported to be associated with several diseases including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), by inducing hyperactivation of cell signaling [START_REF] Kabouridis | GO:0002376 immune system process (24) Isgf3g, Il10, Cxcl10, Ddx58, Fas, Csf3, Tnf, Mx[END_REF]. As GL appears to alter the membrane integrity, thereby attenuating signaling it might be a useful therapeutic not only for the treatment of a broad variety of chronic inflammatory diseases but also for the treatment of autoimmune disorders. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 14 May 2009 as manuscript BJ20082416 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082416

  CpG-DNA and LPS induced activation of MAPKs. Beside the activation of NF-B, TLR signaling also involves the activation of MAPK pathways. To evaluate the specificity of GL for the NF-B pathway, we next examined its role in MAPK activation by analyzing the phosphorylation of JNK, p38, and ERK1/2. As observed for I B activation, no phosphorylation of either MAPK could be detected by stimulation with 10 nM CpG-DNA, thus again, subsequent experiments were performed with 100 nM CpG-DNA, explaining the mild effects observed with GL. Weak activation of JNK could be detected upon stimulation with 100 nM CpG-DNA (Fig. Biochemical Journal Immediate Publication. Published on 14 May 2009 as manuscript BJ20082416 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082416 A c c e p t e d M a n u s c r i p t

Figure 1 .

 1 Figure 1. Effect of GL on the expression of pro-inflammatory genes upon stimulation with CpG-DNA. RAW 264.7 cells were stimulated with 10 nM CpG-DNA in the presence or absence of 1 mM GL. Cells were harvested at indicated times and mRNA levels of indicated genes were analyzed by QuantiGene using the corresponding probe sets. Data were normalized to the housekeeping gene Gapdh and fold change calculated relative to unstimulated cells. Data represent means of triplicates +/-standard deviation. Data was reproduced in at least two independent experiments.

Figure 2 .

 2 Figure 2. Dose-dependent reduction of expression of pro-inflammatory proteins by GL. Release of MIP-1 (A), TNF (B) and RANTES (C) was determined by ELISA from supernatants of cells that were stimulated with 10 nM CpG-DNA in the presence or absence of indicated amounts of GL over night. Data represent means +/-standard deviation. (D) Expression of IL-1 (upper panel) was analyzed by immunoprecipitation and Western blotting of IL-1 from cells stimulated with 10 nM CpG-DNA in the presence of indicated amounts of GL. Expression of Cox-2 and FAS (lower panel) was analyzed in cells stimulated with 10 nM CpG-DNA treated with indicated amounts of GL by Western blotting. Blots were probed for Tubulin to verify equal protein loading. Data was reproduced in at least two independent experiments.

Figure 3 .

 3 Figure 3. GL blocks inflammatory responses induced by TLR3 and TLR4 ligands. (A)RAW 264.7 cells were stimulated with 1 ng/ml LPS with or without 500 M GL treatment. Release of TNF , RANTES, and IFN-was analyzed by ELISA from supernatants of cells stimulated with LPS over night. The release of the corresponding proteins in untreated cells was set to 100% (not shown) and the release in treated cells was calculated relative to the untreated samples. (B) Release of IL-6 and IFN-was analyzed from cells stimulated with 1 g/ml poly (I:C) over night in the presence or absence of 500 M GL. The release of the corresponding proteins in untreated cells was set to 100% and the release in treated cells was calculated relative to the untreated samples. Data represent means of duplicates and was reproduced in at least two independent experiments.

Figure 4 .

 4 Figure 4. GL blocks NF-B and MAPKs signaling upon stimulation with CpG-DNA and LPS. (A) Phosphorylated and total I B was detected by Western blotting of RAW 264.7 cells upon stimulation with 100 nM CpG-DNA (left panel) or 10 ng/ml LPS for indicated times with or without GL treatment (1 mM). Equal protein loading was verified by probing with anti-Tubulin antibody. (B) For analysis of MAPKs activation, cells were stimulated with CpG-DNA (left panel) or LPS (right panel) as described above. Antibodies specific for the indicated phosphorylated and total MAPKs were used. In addition, blots were probed for Tubulin to verify equal protein loading.

Figure 5 .

 5 Figure 5. Reduced cellular attachment/uptake of CpG-DNA and TLR4 internalization in the presence of GL. (A) RAW 264.7 cells were incubated with 40 nM FITC-labeled CpG-DNA for 60 min in the presence or absence of indicated amounts of GL. After the incubation, cells were washed extensively and fixed with 4 % paraformaldeyde. Fluorescence was measured by FACS. (B) RAW 264.7 cells were stimulated with 2 g/ml LPS for 1h in the presence or absence of 2.5 mM GL. Surface TLR4 expression was measured by FACS. (C) RAW 264.7 cells were stimulated with indicated amounts of PMA with or without GL (1 mM) treatment and Tfna

  Fig.1

  FIG 3

Table Legends Table 1 .

 Legends1 Effect of GL on gene expression 4 h post CpG-DNA stimulation. Genes that were down-regulated more than 2-fold with a BH-adjusted p-value of <0.01 are shown.

Table 2 .

 2 Function of the genes regulated by GL upon CpG-DNA stimulation using GO enrichment analysis. The input list consisted of a total of 116 genes that were up-and downregulated by GL >2 fold with a p-value of <0.01. Journal Immediate Publication. Published on 14 May 2009 as manuscript BJ20082416 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082416 A c c e p t e d M a n u s c r i p t

	Biochemical

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Table 1 .

 1 Biochemical Journal Immediate Publication. Published on 14 May 2009 as manuscript BJ20082416 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082416 A c c e p t e d M a n u s c r i p t

	gene	fold reduction
	Il1a	17.2
	Il1b	13.1
	Cish	9.9
	Ch25h	9.9
	Ccl4	6.5
	Nfkbiz	5.9
	Ccl5	5.6
	Mx2	5.5
	Cxcl2	5.3
	Ccl3	3.9
	Ifi204	3.8
	Gstt4	3.1
	Pdcd1	2.9
	Ifit3	2.5
	Cd83	2.5
	Fndc7	2.3
	Ifi205	2.2
	Ifi44	2.1
	Ier3	2.1
	Tnfaip3	2.0

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Table 2 .

 2

	Go Accession	GO term	genes	p -val

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgment

We thank Prof. K. Miyake (Institute for Medical Sciences, University of Tokyo) for providing the Sa15-21 antibody.

Funding

This work was in part supported by the 'Austrian Research Promotion Agency (FFG)' and 'The ZIT Center for Innovation and Technology'.

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Leonor Morgado

Ana P Fernandes

Yuri Y Londer

P Raj Pokkuluri

Marianne Schiffer

Carlos A Salgueiro
email: csalgueiro@dq.fct.unl.pt

Thermodynamic characterization of the redox centers in a representative domain of a novel c-type multiheme cytochrome

Keywords: Multiheme, cytochrome c, Geobacter, axial coordination Gs, Geobacter sulfurreducens, PpcA, Geobacter sulfurreducens triheme cytochrome (GSU0612), PpcB Geobacter sulfurreducens triheme cytochrome

Multiheme cytochromes that could form protein "nanowires" were identified in Geobacter sulfurreducens genome and represent a new type of multiheme cytochromes. The sequences of these proteins, two with 12-hemes (GSU1996, GSU0592) and one with 27-hemes (GSU2210) suggest that they are formed by domains homologous to triheme cytochromes c 7 . While all three hemes have bis-His coordination in cytochromes c 7 , in each domain of the above polymers the heme equivalent to heme IV has His-Met coordination. We previously determined the structure and measured the macroscopic redox potential of one representative domain (domain C) of a dodecaheme cytochrome (GSU1996). In the present study, the microscopic redox properties of the individual heme groups of domain C were determined using NMR and UV-visible spectroscopies. The reduction potentials of the hemes for the fully reduced and protonated protein are different from each other (heme I, -106 mV; heme III, -136 mV; heme IV, -125 mV) and are strongly modulated by redox-interactions. This result is rather surprising since the His-Met coordinated heme IV does not have the highest potential as was expected. The polypeptide environment of each heme group and the strong heme pairwise redox interactions must play a dominant role in controlling the individual heme potentials. The strong redox interactions between the hemes extend the range of their operating potentials at physiological pH (heme I, -71 mV, heme III, -146 mV and heme IV -110 mV). Such a modulation in heme potentials is likely to have a functional significance in the metabolism of Geobacter sulfurreducens.

INTRODUCTION

Microrganisms of the Geobacteraceae family have a major role in the dissimilatory reduction of metals and radionuclides coupled to the oxidation of organic matter in soils and sediments [START_REF] Lloyd | Microbial detoxification of metals and radionuclides[END_REF]. Geobacter species are metabolically diverse; in particular, they can use both soluble and insoluble electron acceptors. Especially interesting is the biological reduction of insoluble electron acceptors [START_REF] Shi | Respiration of metal (hydr)oxides by Shewanella and Geobacter: a key role for multihaem c-type cytochromes[END_REF]; since the reduction occurs outside the cell rather than in the periplasm, it requires electron transfer through the outer membrane to the outside of the cell [START_REF] Mehta | Outer membrane ctype cytochromes required for Fe(III) and Mn(IV) oxide reduction in Geobacter sulfurreducens[END_REF][START_REF] Reguera | Extracellular electron transfer via microbial nanowires[END_REF]. This electron transfer capability relies on a group of electron transfer proteins, mainly cytochromes c, which are abundant in Geobacteraceae. Indeed, more than a hundred genes encoding for c-type cytochromes were identified in Geobacter sulfurreducens (Gs) genome [START_REF] Methé | Genome of Geobacter sulfurreducens: metal reduction in subsurface environments[END_REF] and most of them contain multiple hemes. The best studied cytochromes in Gs are the periplasmic triheme cytochromes c 7 , PpcA and PpcB [START_REF] Lloyd | Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens[END_REF][START_REF] Pessanha | Thermodynamic characterization of triheme cytochrome PpcA from Geobacter sulfurreducens: evidence for a role played in e -/H + energy transduction[END_REF][START_REF] Morgado | Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes[END_REF][START_REF] Pokkuluri | Family of cytochrome c 7 -type proteins from Geobacter sulfurreducens: structure of one cytochrome c 7 at 1.45 Å resolution[END_REF]. The family of cytochromes c 7 is structurally homologous to the tetraheme cytochromes c 3 with heme II deleted [START_REF] Pokkuluri | Structure of a novel c 7 -type three-heme cytochrome domain from a multidomain cytochrome c polymer[END_REF]. To maintain consistency with the literature, the heme groups in the triheme cytochromes are numbered I, III and IV. In cytochromes c 7 and cytochromes c 3 all the heme groups have bis-His axial coordination.

Gs genome encodes two proteins with 12 heme binding motifs (GSU0592 and GSU1996) and one with 27 heme binding motifs (GSU2210). Homologous dodecaheme cytochromes were identified in the genomes of Geobacter metallireducens and Geobacter uraniireducens. These proteins represent a new class of multiheme cytochromes. Their sequences suggest a multidomain organization of highly homologous cytochrome c 7 -type domains with a mixed type of heme coordination (His-His and His-Met) within each domain [START_REF] Pokkuluri | Family of cytochrome c 7 -type proteins from Geobacter sulfurreducens: structure of one cytochrome c 7 at 1.45 Å resolution[END_REF][START_REF] Pokkuluri | Structure of a novel c 7 -type three-heme cytochrome domain from a multidomain cytochrome c polymer[END_REF][11]. The physiological function of these polymers is not known though one of their putative functions is to contribute to the enhancement of the cellular electron-storage capacity. Indeed, by having a large number of redox centers, these molecules, in the absence of external electron acceptors are suggested to allow the continuous flow of electrons from the inner membrane to maintain cellular energy demands until new acceptors can be reached in the environment [START_REF] Esteve-Núñez | Fluorescent properties of c-type cytochromes reveal their potential role as an extracytoplasmic electron sink in Geobacter sulfurreducens[END_REF]. The putative structure of the 12-heme cytochrome GSU1996 is a one-dimensional array composed of four triheme cytochrome domains (termed A, B, C and D) covalently bound by short linkers. Each domain of the mature protein GSU1996 was expressed individually in E. coli [START_REF] Pokkuluri | Structure of a novel c 7 -type three-heme cytochrome domain from a multidomain cytochrome c polymer[END_REF]. The crystal structure of the single domain C was determined at 1.7Å resolution together with its macroscopic midpoint potential [START_REF] Pokkuluri | Structure of a novel c 7 -type three-heme cytochrome domain from a multidomain cytochrome c polymer[END_REF]. Both the structural data and the sequence homology among each of the four domains revealed that each domain has structural features that are unique for multiheme c-type cytochromes: two of the hemes (I and III) have bis-His axial coordination while the third one (heme IV) has His-Met axial coordination.

To shed light on the redox properties of the hemes in this new class of multiheme cytochromes isolated from Gs we fully characterized the redox properties of the heme groups in the domain C of the dodecaheme cytochrome GSU1996 using NMR and UV-visible spectroscopic techniques.

Our study shows that the reduction potentials of the heme groups are different from each other and are strongly modulated by the structure of the protein and by redox interactions that expand the reduction potential working range of the cytochrome. The heme with His-Met axial coordination does not have the highest reduction potential contrary to what is expected.

4

EXPERIMENTAL Domain C sample preparations

Gs domain C was expressed in E. coli and purified as described previously [START_REF] Pokkuluri | Structure of a novel c 7 -type three-heme cytochrome domain from a multidomain cytochrome c polymer[END_REF]. The buffer used in the purification was exchanged to 20mM NaCl by ultrafiltration methods for all samples. For NMR studies the protein was lyophilized twice with 2 H 2 O (99.9% atom) and then re-suspended in 600µl of 80mM phosphate buffer with NaCl (final ionic strength of 250mM) solution prepared in 99.96% 2 H 2 O. Protein concentration (~70µM for redox titrations and ~140µM for structural studies) was determined using the specific absorption coefficient of the α-band of the reduced form for PpcA [13]. The pH was adjusted by the addition of small amounts of NaO 2 H or 2 HCl. The reduced sample was obtained by adding gaseous hydrogen in the presence of catalytic amounts of Fe-hydrogenase isolated from Desulfovibrio vulgaris (Hildenborough). The partially oxidized samples, used for NMR redox titrations, were obtained by first removing the hydrogen from the reduced sample with nitrogen and then adding controlled amounts of air into the NMR tubes.

NMR experiments

All NMR spectra were obtained on a Bruker Avance 600 spectrometer at 288K and were calibrated using the water signal as internal reference. The proton chemical shifts reported are relative to tetramethylsilane. To assign the heme substituents signals in the reduced form, 2 H 2 O NOESY (50-100ms mixing time) and TOCSY (60ms mixing time) spectra were acquired using standard pulse techniques collecting 2048(t 2 ) × 512(t 1 ) data points to cover a sweep width of 8 kHz, with 64 scans per increment. To map the oxidation of the individual hemes throughout the redox titrations, a series of 2D-exchange spectroscopy (EXSY) experiments were collected for each pH value (covering the range 5.0-8.5) at several degrees of oxidation. All the 2D-EXSY NMR experiments were acquired with a mixing time of 25 ms and recording 2048(t 2 ) × 256(t 1 ) data points to cover a sweep width of 42kHz, with at least 256 scans per increment.

Ring-current shifts calculation

The heme substituent chemical shifts were calculated by correcting the heme proton reference shifts (9.36 ppm for heme meso protons, 6.13 for heme thioether methines, 3.48 for heme methyls, and 2.12 for heme thioether methyls) [START_REF] Pessanha | Redox characterization of Geobacter sulfurreducens cytochrome c 7 : physiological relevance of the conserved residue F15 probed by sitespecific mutagenesis[END_REF] with the ring current shifts calculated from the domain C crystal structure, following the procedure described by Turner et al. and Messias et al. [START_REF] Messias | Solution structure of Desulfovibrio vulgaris (Hildenborough) ferrocytochrome c 3 : structural basis for functional cooperativity[END_REF]16].

Redox titrations followed by visible spectroscopy

Redox titrations of domain C using 10µM protein solutions in 80mM phosphate buffer (pH 7 and 8) with NaCl (250mM final ionic strength) were performed inside an anaerobic chamber at 288K as previously described [START_REF] Pokkuluri | Structure of a novel c 7 -type three-heme cytochrome domain from a multidomain cytochrome c polymer[END_REF], with redox mediators at a final concentration of 1.5µM. The reduced fraction of the proteins was determined by the method described by Paquete et al. [17]. The experiments were performed at least two times, and the reduction potentials were found to be reproducible within ±5 mV.

Overview of the method used

To describe adequately the redox properties of the heme groups in a multiheme protein it is necessary to probe individually the redox behavior of each center. NMR spectroscopy is well suited for this purpose since the proton chemical shift variations for each heme substituent are proportional to the degree of oxidation of that particular heme [START_REF] Santos | NMR studies of electron transfer mechanisms in a protein with interacting redox centres: Desulfovibrio gigas cytochrome c 3[END_REF][19][START_REF] Santos | Two-dimensional NMR studies of electron transfer in cytochrome c 3[END_REF]. When the intermolecular electron transfer rate is slow and the intramolecular electron transfer rate is fast on the NMR timescale, four oxidation stages connected by steps of one electron transfer can be defined each containing the microstates with 0, 1, 2, or 3 oxidized hemes. The heme methyl substituents are the most suitable ones to probe the degree of oxidation of a particular heme because as the protein oxidation progresses, their resonances shift towards empty regions of the NMR spectrum. As previously described, the minimum number of parameters that fully account the redox properties of the centers in a triheme protein is ten [START_REF] Pessanha | Thermodynamic characterization of triheme cytochrome PpcA from Geobacter sulfurreducens: evidence for a role played in e -/H + energy transduction[END_REF]21]. These are the three heme reduction potentials, the deprotonation energy of the protonatable center, three redox interactions between the heme groups (which measure the influence of oxidation stage of one heme on the reduction potential of a neighboring one), and three interactions between the hemes and the protonatable center (redox-Bohr interactions -which account for the pH effect on the heme reduction potentials). Starting from the fully reduced protein (oxidation stage 0), and by following the pH dependence of the heme methyl proton chemical shifts throughout the remaining oxidation stages, the thermodynamic parameters relative to the fully reduced protonated protein can be determined [START_REF] Pessanha | Thermodynamic characterization of triheme cytochrome PpcA from Geobacter sulfurreducens: evidence for a role played in e -/H + energy transduction[END_REF][START_REF] Turner | NMR studies of cooperativity in the tetrahaem cytochrome c 3 from Desulfovibrio vulgaris[END_REF]. Given that the NMR data only gives relative parameters, the total protein reduced fractions were measured at pH 7 and 8 using redox titrations followed by visible spectroscopy to determine the absolute values [START_REF] Turner | NMR studies of cooperativity in the tetrahaem cytochrome c 3 from Desulfovibrio vulgaris[END_REF]. The experimental uncertainty associated with the NMR data was estimated from the line width of each NMR signal at half height, and the visible data points were given an uncertainty of 3% of the total optical signal.

Assignment of domain C heme signals in the reduced state

The structure of domain C [START_REF] Pokkuluri | Structure of a novel c 7 -type three-heme cytochrome domain from a multidomain cytochrome c polymer[END_REF] showed that hemes I and III have bis-histidinyl axial coordination (His-His) while heme IV is coordinated by one histidine and one methionine (His-Met) (Figure 1). Thus, as expected, the 1D-1 H-NMR spectra of domain C, both in the oxidized and reduced forms, are typical of low spin hemes (Figure 2A,B). The protein is diamagnetic when reduced (Fe(II), S=0) and paramagnetic when oxidized (Fe(III), S=1/2). The 1 H-NMR spectrum of the reduced domain C (Figure 2A,B) clearly shows that a methionine residue is an axial heme ligand in solution. The typical pattern of heme axial methionine resonances [START_REF] Mcdonald | Proton magnetic resonance evidence for methionine-iron coordination in mammalian-type ferrocytochrome c[END_REF] includes a three-proton intensity peak at approximately -3 ppm, and up to four resolved oneproton intensity peaks in the low-frequency region of the spectrum.

The heme proton resonances in the reduced protein were identified by following the assignment strategy for heme protons in multiheme ferrocytochromes described by Turner et al. [16] and are indicated in Supplementary Table. This assignment was tested by comparing the observed heme proton chemical shifts with those calculated from the crystal structure. Of the 6 possible permutations for the three sets of heme protons with respect to the crystal structure, one was clearly preferred (cf. Supplementary Table andFigure 3) since all three hemes simultaneously had their smallest root mean square deviation (rmsd) from the calculated chemical shifts. The rmsd of the 36 shifts was 0.18 ppm, with deviations of 0.02 (heme I), 0.07 (heme III), 0.09 (heme IV). The shifts correlate very well, even for the protons subject to the larger ring current effects, as it is the case of the protons 10H I , 20H III , 12 1 3; a heme c numbered accordingly with IUPAC-IUB nomenclature is indicated in Supplementary Figure ). This assignment was further tested by examining the interheme NOE connectivities, measured from the 2D-NOESY spectra, which were then compared with the distances obtained from the crystal structure. All NOE connectivities between protons that are closer than 3Å were observed in the 2D-NOESY spectra, confirming that both crystal and solution structures are similar.

Order of oxidation of the heme groups

As mentioned above, the heme groups in domain C are low spin in oxidized and reduced states. This is convenient for NMR studies since widely different well-resolved spectra are obtained for both oxidation states (Figure 2A,B), facilitating the assignment of the heme signals and the determination of their relative order of oxidation. The redox titration of the heme groups followed by 2D-EXSY NMR experiments at pH 8.0 is indicated in Figure 2C-E. These spectra show that domain C exhibits fast intramolecular and slow intermolecular electron exchange on the NMR time scale since signals from a particular heme methyl can be followed throughout the four oxidation stages. The paramagnetic chemical shifts of heme methyl groups 7 1 IV were used to calculate the oxidation fraction of each heme at different stages of oxidation (Table 1). From Table 1 it is possible to obtain the order of oxidation of the hemes: heme III is the heme that becomes more oxidized in the first redox step (stage 1), the largest fractional oxidation of heme IV is obtained by the second step (stage 2), followed by heme I (stage 3). Indeed, in the last step heme I oxidation fraction increases by 59%, whereas hemes III and IV only increase 28% and 17%, respectively. Given that heme I has bis-histidinyl axial coordination, this data clearly indicates that the heme with His-Met axial coordination (heme IV) is not the heme with the highest reduction potential. As described in Materials and Methods section, in order to determine the absolute values for the heme reduction potentials, heme-heme redox interactions, and redox-Bohr interactions, it is necessary to monitor the heme oxidation patterns at different pH values by 2D-EXSY NMR and combine this information with that obtained from the redox titrations followed by visible spectroscopy.

Mapping the heme redox properties of domain C

The pH dependence of the paramagnetic chemical shifts of each heme methyl group in the pH range 5.0-8.5, and the data obtained for redox titrations followed by visible spectroscopy at pH 7 and 8 were used to monitor the thermodynamic properties of domain C. The fittings of both NMR and visible data are reported in Figures 4 and5, respectively. The thermodynamic parameters are listed in Table 2. The parameters show that the microscopic reduction potentials of each heme are different: heme III has the lowest redox potential in the reduced and protonated protein (-136 mV), followed by hemes IV (-125 mV) and I (-106 mV). The interaction energy (redox interaction) for each pair of hemes is positive, indicating that the oxidation of a particular heme renders the oxidation of its neighbors more difficult. On the other hand, the interaction energies between the hemes and the acid-base center (redox-Bohr interactions) are negative, i.e. the oxidation of the hemes facilitates the deprotonation of the acid-base center, and vice versa. The observed redox-Bohr effect in the redox titration curves (Figure 5) and the dependence of the heme methyl NMR paramagnetic shifts (Figure 4) are not significant in the physiological pH range, implying that the redox-Bohr interactions are small. The oxidation profile of the heme groups at pH 7 is shown by solid lines in Figure 6, where oxidized fraction of each heme is plotted against the solution potential. The crossovers between the individual curves clearly indicate that the affinity of each redox center is tuned by the neighboring heme groups. The positive redox interactions between the hemes modulate the affinity of each redox center during the oxidation of the protein such that their apparent midpoint reduction potentials E app (i.e. the point at which the oxidized and reduced fractions of each heme group are equally populated: -71, -146, -110 mV, respectively for hemes I, III and IV) are different in relation to those for the fully reduced protein (Table 2). This effect can be easily seen by comparing the dashed and the solid lines in Figure 6. The dashed lines were generated considering no redox-interactions between the heme groups, whereas the solid ones were obtained with the thermodynamic parameters listed in Table 2. The effect of the positive redox interactions between the hemes is particularly pronounced on the reduction potentials of hemes I and IV. Indeed, since heme III dominates the first stage of oxidation (where hemes I and IV are mostly reduced), its reduction potential is little affected by the oxidation of hemes I and IV and, consequently, the E app of heme III (-146 mV) is less affected in relation to that of the fully reduced protein (-136 mV). However, with the subsequent oxidation of the protein, the oxidation fractions of hemes I and IV also increase which lead to a strong modulation of the redox behavior of heme III (see plateau on the heme III redox curve in Figure 6). On the other hand, with the progressive oxidation of hemes I and IV, and since a large fraction of heme III is oxidized, their oxidation curves are also modulated and both E app values increase significantly in comparison with those of the fully reduced protein (cf. dashed lines and solid lines of the respective heme in Figure 6). Consequently, the midpoint reduction potentials separation is extended significantly to 75 mV in comparison with that in the fully reduced protein (30 mV) and is physiologically relevant (compare the E app values for the dashed and solid lines in Figure 6).

Origins for the low reduction potential of heme IV (His-Met) in domain C

The core of domain C (see Fig. 1) is formed by three hemes where the Fe-Fe distances between hemes I -III, I -IV and III -IV are 11.4 , 17.8, 12.0 Å, respectively ([10], PDB code 1rwj). The solvent exposure of the hemes I, III and IV is 79Å 2 , 173Å 2 , and 167Å 2 , respectively. The surroundings of heme I is more hydrophobic than that of the other hemes. The propionic acids of heme I form hydrogen bonds with the peptide nitrogens of residues 44 and 45; heme III propionic acid forms hydrogen bond with the peptide nitrogen of residue 70; and heme IV propionic acid forms a hydrogen bond with peptide nitrogen of residue Met51 (sixth axial ligand of heme IV) and forms a salt bridge with Arg48 (it was erroneously reported to heme III propionic acid in [START_REF] Pokkuluri | Structure of a novel c 7 -type three-heme cytochrome domain from a multidomain cytochrome c polymer[END_REF]). In Pokkuluri et al. [START_REF] Pokkuluri | Structure of a novel c 7 -type three-heme cytochrome domain from a multidomain cytochrome c polymer[END_REF], domain C structure was compared with that of the triheme cytochrome c 7 PpcA. It was later discovered [START_REF] Morgado | Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes[END_REF] that the structure of PpcA was influenced by a deoxycholic acid molecule used for crystallization and, therefore, domain C is compared with PpcB [START_REF] Morgado | Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes[END_REF] in the present study. The Fe-Fe distances between hemes I -III, I -IV and III -IV in PpcB monomer-A (PDB code, 3bxu) are 11.7, 18.2, 12.6 Å, respectively. The iron to iron distances and also the relative orientations of the heme groups of the two proteins are similar (Fig. 7A). Though the heme core of domain C is like that of PpcB, the protein conformation surrounding the hemes is different as a result of the insertions and deletions in the sequences between the two proteins (Fig. 7B). Thus domain C is similar to c 7 cytochromes in size with a conserved three heme core, but they differ in their polypeptide conformation and in the axial coordination of heme IV (the sixth axial ligand for heme IV is indicated in Fig. 7B).

For PpcB only a quantitative macroscopic redox characterization was previously performed [START_REF] Morgado | Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes[END_REF] in contrast to the present study of domain C. All PpcB hemes have bis-His coordination [START_REF] Morgado | Structural insights into the modulation of the redox properties of two Geobacter sulfurreducens homologous triheme cytochromes[END_REF] and we determined the macroscopic reduction potential of PpcB to be -137 mV, which is shifted to lower reduction potential when compared with that determined in this work for domain C (E app -103 mV). Based exclusively on these macroscopic values it can be hypothesized that the higher macroscopic potential of domain C is due to the heme that has His-Met coordination (heme IV). However, the detailed microscopic characterization carried out in this work for domain C showed that this is not the case. The individual reduction potential values of the hemes are -71, -146 mV, and -110 mV respectively for hemes I, III and IV at pH 7 clearly showing that the His-Met coordinated heme IV does not have the highest reduction potential. Therefore the origin of higher E app of domain C is not just due to one heme with His-Met coordination.

It was surprising that heme IV did not have the highest reduction potential in domain C since it was observed that in aqueous solution a His-Met coordinated heme had 150 mV higher reduction potential than a His-His coordinated heme [24]. However, the results obtained in domain C are consistent with the observations of heme potentials in the cytochrome subunit of the photosynthetic reaction center from Rhodopseudomonas viridis, where three of the hemes have His-Met coordination and one heme has His-His coordination. Although the heme-heme redox interactions couldn't be experimentally obtained for the cytochrome subunit, the redox studies hinted that the heme with His-His coordination does not have the lowest reduction potential [START_REF] Dracheva | Electrogenic steps in the redox reactions catalyzed by photosynthetic reaction-centre complex from Rhodopseudomonas viridis[END_REF][START_REF] Fritzsch | Assignment of cytochrome hemes in crystallized reaction centers from Rhodopseudomonas viridis[END_REF]. Voigt and Knapp [START_REF] Voigt | Tuning heme redox potentials in the cytochrome c subunit of photosynthetic reaction centers[END_REF] performed a detailed theoretical study in this cytochrome to show how the heme potential can be tuned by electrostatic interactions of the heme with its protein surroundings and with the neighbouring hemes. The experimental results obtained in this work for domain C and the theoretical predictions made for the cytochrome subunit of photosynthetic reaction center are in contrast to the results obtained for mutants of a cytochrome c 3 [START_REF] Takayama | Strategic roles of axial histidines in structure formation and redox regulation of tetraheme cytochrome c 3[END_REF]. In the study of cytochrome c 3 , Takayama et al. [START_REF] Takayama | Strategic roles of axial histidines in structure formation and redox regulation of tetraheme cytochrome c 3[END_REF] replaced the native His residues to Met in the sixth axial ligand position of each heme. The measured microscopic reduction potential of the heme with His-Met coordination in two mutants was found to be much higher (about +200 mV) than the corresponding bis-His heme in the native protein. In these mutants only the ligand of a heme was changed within the context of the same polypeptide surrounding reflecting the change in potentials caused only by the ligand exchange.

Our results show that the microscopic reduction potential for the heme with His-Met axial coordination in domain C is lower than another heme with His-His axial coordination, indicating that the polypeptide chain surrounding the heme can be the largest factor in determining the heme reduction potential. The protein fold around the hemes controls the intrinsic reduction potentials [29] by controlling the nature of interactions between the heme and the protein and the solvent exposure of the heme. In addition, the close proximity of the redox centres to each other results in the establishment of strong redox interactions between the closest pairs of hemes (Table 2) which modulate the redox properties of the heme groups during the oxidation of the protein (Figure 6).

Functional implications of the cooperative redox interaction networks

Multiheme cytochromes with high heme content, such as dodecaheme cytocromes of Gs, are thought to work as capacitors that enhance the electron-storage capacity of the bacterial periplasm. This large electron-storage capacity is thought to permit continued electron flow from the inner membrane to the periplasm and thus to generate energy that might be used by Geobacter species to move toward zones where external terminal electron acceptors are available, as suggested by Núñez et al. [START_REF] Esteve-Núñez | Fluorescent properties of c-type cytochromes reveal their potential role as an extracytoplasmic electron sink in Geobacter sulfurreducens[END_REF]. Our results demonstrate that an important advantage of having the redox centers closely and strategically arranged along the polypeptide chain is to extend the range of the protein working redox potentials allowing it to face different electron acceptors. , 12 1 CH 3 III and 7 1 CH 3 IV were chosen to monitor each heme oxidation through the four-oxidation stages (see text). The heme fractions of oxidation, x i , in each stage of oxidation were calculated accordingly to the equation x i = (δ i -δ 0 )/(δ 3 -δ 0 ), where δ i , δ 0 , and δ 3 are the observed chemical shifts of the heme methyl in stage i, 0, and 3, respectively. Experiments were perfor med at 288K at pH 7 (circles) and pH 8 (squares). The solid lines represent the best fit of the experimental data using the parameters reported in Table 2.

Figure 6. Oxidized fractions of the individual hemes of domain C at pH 7.

Solid lines were calculated as a function of the solution reduction potential using the parameters reported in Table 2. The dashed lines were generated assuming that no redox interactions exist between each pair of hemes. The arrow indicates, from the left to the right, the E app values of each heme (the point at which the oxidized and reduced fractions are equally populated). For both solid and dashed lines the order of the E app values is the same: E app III < E app IV < E app I . ΔE app correspond to the E app separation between hemes I and III for each set of lines.

Biochemical

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Biochemical

  Journal Immediate Publication. Published on 08 Apr 2009 as manuscript BJ20082428 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082428 A c c e p t e d M a n u s c r i p t 6 RESULTS AND DISCUSSION

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Biochemical

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Biochemical

  Journal Immediate Publication. Published on 08 Apr 2009 as manuscript BJ20082428 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082428 A c c e p t e d M a n u s c r i p t 9

Biochemical

  Journal Immediate Publication. Published on 08 Apr 2009 as manuscript BJ20082428 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082428 A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 13 29 Zheng, Z. and Gunner, M. R. (2008) Analysis of the electrochemistry of hemes with E m s spanning 800 mV. Proteins, in press Biochemical Journal Immediate Publication. Published on 08 Apr 2009 as manuscript BJ20082428 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082428 A c c e p t e d M a n u s c r i p t

Figure 1 .

 1 Figure 1. Structure of domain C [10]. Heme axial ligands are shown in black: H23 and H36 (Heme I), H26 and H64 (Heme III), M51 and H80 (Heme IV).

Figure 2 .

 2 Figure 2. Domain C 1D and 2D NMR spectra. (A-B) Lower field regions of the 1D-NMR proton spectra of domain C in the reduced (upper spectrum) and oxidized (lower spectrum) forms. Arrows indicate the protons of the heme axial methionine. (C-E) Portion of 2D-EXSY NMR spectra of partially oxidized domain C. Cross peaks connecting the signals of the heme methyls belonging to hemes I, III, and IV in the different oxidation stages are indicated by dotted, dashed and solid lines, respectively. The crosspeaks connecting different oxidation stages (0-3) are boxed. The Roman and Arabic numbers indicate the heme groups and the oxidation stages, respectively. All spectra were obtained at 288K and pH 8.

Figure 3 .

 3 Figure 3. Comparison between the calculated and observed chemical shifts for all heme substituents of reduced domain C. The heme substituents of heme I, III, and IV are represented by squares, triangles and circles, respectively. The solid line has a unit slope.

Figure 4 .

 4 Figure 4. pH dependence of the 1 H NMR chemical shift of one heme methyl from each of the three hemes (7 1 CH 3 I , 12 1 CH 3 III , and 7 1 CH 3 IV ) of domain C. Stages of oxidation 1-3 are represented by the symbols: stage 1 (Δ); stage 2 (□); stage 3 (○). The full lines represent the best fit of the data using the parameters reported in Table2. The chemical shift of the heme methyl groups in the fully reduced stage 0 are not plotted since they are unaffected by the pH.

Figure 5 .

 5 Figure 5. Domain C redox titrations followed by visible spectroscopy.Experiments were perfor med at 288K at pH 7 (circles) and pH 8 (squares). The solid lines represent the best fit of the experimental data using the parameters reported in Table2.

Figure 7 .

 7 Figure 7. Comparison of domain C and PpcB. (A) Heme core architecture of domain C [10] (black) and PpcB [10] (grey). The two structures were overlapped with all three heme ring atoms. (B) Structure-guided alignment of the amino acid sequences of domain C of the four domain poly-c 7 -type cytochrome GSU1996 and cytochrome c 7 PpcB (monomer A) from G. sulfurreducens. Identical residues are shown in bold; the heme binding motif is indicated by the corresponding heme shown below. The sixth axial

  Figure 1

Figure 2 Biochemical 20 Figure 3

 2203 Figure 2

  Figure 4

  Figure 5

  Figure 6

  Figure 7

Table 1 . Redox-dependent heme methyl chemical shifts in domain C at pH 8.0.

 1

	The heme methyls 7 1 CH 3	I

Table 2 . Thermodynamic parameters determined for domain C.

 2 Diagonal terms (in bold) represent the oxidation energies of the three hemes and the deprotonating energies for the protonatable center in the fully reduced and protonated protein.The off-diagonal elements represent the redox and redox-Bohr interactions energies between the four centers. Standard errors are given in parenthesis.

	Biochemical Journal Immediate Publication. Published on 08 Apr 2009 as manuscript BJ20082428
	Oxidation stage	Chemical shift (ppm)		x i		Σ x i
		I	III	IV	I	III	IV
	0	3.44	3.86	3.62	0	0	0	0
	1	5.61	24.06	11.54	0.08	0.64	0.25	0.97
	2	14.79	26.57	30.08	0.41	0.72	0.83	1.96
	3	31.05	35.38	35.41	1	1	1	3

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© 2009 The Authors Journal compilation © 2009 Portland Press Limited

ACKNOWLEDGEMENTS

Dr. David L. Turner is acknowledged for the conception of the thermodynamic program. We acknowledge LabRMN at FCT-UNL and Rede Nacional de RMN for access to the facilities. Rede Nacional de RMN is supported with funds from FCT, Projecto de Re-equipamento Científico, Portugal.

FUNDING

This work was supported by Fundação para a Ciência e a Tecnologia -Portugal [POCI/QUI/60060/2004, PPCDT/QUI/60060/2004] and U.S. Department of Energy's Office of Science, Biological and Environmental Research GENOMICS:GTL -USA [DE-AC02-06CH11357]. L.M. is supported by Fundação para a Ciência e a Tecnologia grant SFRH/BD/37415/2007. Biochemical Journal Immediate Publication. Published on 08 Apr 2009 as manuscript BJ20082428 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20082428