An iron-sulfur cluster as a new metal centre in a flavodiiron protein

doi:10.1101/2024.04.03.587993

An iron-sulfur cluster as a new metal centre in a flavodiiron protein

2024 · doi:10.1101/2024.04.03.587993

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Abstract

Syntrophomonas wolfei contains two distinct multiple domain flavodiiron proteins (FDPs), of Classes H and E, presumably acting as oxygen reductases to protect this anaerobic bacterium from oxidative stress due to exposure to environments containing, even if only transiently, oxygen. The Class E FDP was predicted to have, besides the two core domains characteristic of this type of enzymes, an extra C- terminal domain putatively harbouring an iron-sulfur centre. Bioinformatic analyses showed that, thus far, Class E FDPs are only present in three other bacteria of the Syntrophomonas genus: Syntrophomonas palmitatica , Syntrophomonas zenhnderi and Thermosyntropha lipolytica. In this work, we extensively characterized the enzyme from Syntrophomonas wolfei (wild type, site directed mutants and truncated domains) and showed unequivocally, using EPR and Resonance Raman spectroscopies, that indeed it contains a [3Fe- 4S] 1+/0 centre, a novelty in the field of FDPs. Structure prediction using Alphafold indicated some similarities to [3Fe4S] 1+/0 containing ferredoxins. The reduction potentials of each cofactor were determined: +70 mV, -5/-70 mV and -90 mV for the FeS, diiron centre and flavin, respectively.

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An iron-sulfur cluster as a new metal centre in a flavodiiron protein | bioRxiv /* */ /* */  π* and π --> π* transitions [ 26 , 27 ], indicating an unaltered isoalloxazine ring. However, when the spectrum of the flavin isolated from FDP_E was compared with the commercial FMN and with the FMN extracted from E. coli FDP (pink spectrum) it shows a deviation mainly in the 330-370 nm band. [ 26 , 27 ] Upon reduction with excess of sodium dithionite, a spectrum of a neutral (blue) semiquinone was obtained ( Figure 4 panel C) with maxima at 400 nm, a shoulder at ≈ 420nm and a very broad band with a maximum at 560 nm as described before [ 20 ]. As for the FDP_E, the complete reduction of this sample was not possible, even upon the addition of a large excess of sodium dithionite. The fluorescence properties of this molecule were also studied. The emission spectra of FDP_E, partially reduced FDP_E (containing the “430 nm” species) and the flavin isolated from FDP_E (prepared as above) were almost identical with a single band with a maximum at 535 nm using both λ Ex = 455 nm (Figure S3 A) and λ Ex = 430 nm (FigureS3 B), whereas the excitation spectra (following the λ Em =520 nm, Figure S3 C) of these samples, presented broad bands at 460 nm and 360 nm, characteristic of oxidized FMN [ 26 , 27 ]. These similarity may indicate that the chemical nature of these species is identical among themselves and to other flavins. Reverse phase HPLC revealed that this molecule has a higher hydrophobicity than the FMN, FAD or the riboflavin used as standards, and suggest heterogeneity of the extracted cofactor, as three elution peaks were observed, making impossible its direct identification by this technique (Figure S4). FeS cluster properties The presence of an FeS cluster as part of a flavodiiron protein constitute a feature restricted to this class of FDPs and was characterized, by the first time, in the present work. As already mentioned in the amino acids sequence analysis’ section, the C-terminal FeS domain shares homology with a protein family “Fer4_19” that may harbour either a [3Fe-4S] 1+/0 or a [4Fe-4S] 2+/1+ cluster. In Figure 5 panel A the EPR spectrum at 12 K of the as purified (oxidized) form of FDP_E (blue spectrum) showed a single species with a signal reminiscent of those of a [3Fe-4S] 1+ cluster with a rhombic EPR signal with g-values very close to 2: 2.030, 2.019 and 2.003 ( Table 1 ); the signal is observable up to 30 K, temperature at which it starts broadening, again a characteristic of oxidized [3Fe-4S] centres. Also, because of the presence of the flavin, we observed the contribution of a radical species with g ≍ 2, that must arise from its semiquinone form. The reduction of FDP_E with sodium dithionite in the presence of methyl viologen ( Figure 5 panel A) completely bleached the [3Fe-4S] 1+ EPR signal, being observed only a radical species signal with an isotropic signal at g ≍ 2, probably related with the presence of methyl viologen radical in the sample. The spectrum of FDP_E_Cter is identical to the one of the intact FDP_E acquired in the same conditions ( Figure 5 panel A, pink spectra), which points to the presence of the same species in both proteins, the [3Fe-4S] 1+/0 centre. The absence of an EPR signal in the reduced FDP_E and FDP_E_Cter samples indicated that there was no [4F-e4S] 2+/1+ centre in the sample or that this centre would have a reduction potential lower than that of methyl viologen (-440 mV at pH 7). View this table: View inline View popup Download powerpoint Table 1 g-values for the EPR signals of the [3Fe-4S] 1+/0 cluster in the oxidized samples of the different protein variants, obtained by theoretically simulating the spectra using SpinCount [ 23 ]. Download figure Open in new tab Figure 5. (A) EPR spectra of 150 µM of FDP_E (blue), E_H441C (yellow), E_441A (green) and FDP_E_Cter (pink) in the oxidized and reduced (spectra 10x multiplied) forms . The black line corresponds to the simulated spectra. (B) Redox titration of the FeS cluster of FDP_E . Temperature, 7 K; Microwave frequency, 9.39 GHz; Modulation amplitude, 1.0 mT; Microwave power, 2 mW. Spectral simulations and image preparation were performed using SpinCount [ 23 ]. To rule out the hypothesis that the FeS in FDP_E was originally a [4Fe4S] 2+/1+ cluster, coordinated by the conserved cysteines and the histidine from the “Fer4_19” motif, that later undergoes degradation during the purification and manipulation procedures, giving rise to the 3Fe centre observed by EPR, we produced and purified two site-directed mutants of FDP_E. In these mutants the conserved histidine (residue 441) was replaced either by a cysteine (E_H441C) or by an alanine (E_H441A) to evaluate the possible role of the conserved histidine as a putative fourth ligand of the FeS cluster. The EPR spectra of E_H441A and E_H441C variants in the “as purified” samples ( Figure 5 green and yellow spectra respectively) revealed EPR signals very similar to the one of the wild type protein ( Table 1 ). As for the wild type protein, the reduction with sodium dithionite and methyl viologen led to a bleaching of the EPR spectra, with no indication for the presence of a tetranuclear cluster, being only observed the radical species at g ≈ 2. The presence of the [3Fe-4S] 1+/0 cluster as a single species in FDP_E, E_H441A and E_H441C variants and FDP_E_Cter was verified also by Resonance Raman (RR) spectroscopy. The spectra of all samples exhibit a band centred at 345 cm −1 ( Figure 6 ), indicative of FeS bridging vibrational mode of an oxidized 3Fe cluster. Note that no other bands were detected, apart from ice lattice modes [ 28 ], also present in the spectrum of the buffer (Figure S5). The spectra lack the characteristic pattern of [3F-e4S] 1+/0 clusters, particularly the terminal FeS vibrational modes, derived from cysteinyl S ligands (2 nd most intense bands typically found at ca. 366 cm −1 )[ 29 – 32 ]. Despite the incomplete assignment of characteristic RR features for a [3Fe-4S] 1+/0 cluster the detected signal is not attributable to binuclear or tetranuclear centres. The E_H441A and E_H441C variants have identical spectra to the wild type and C-terminal domain proteins indicating that the histidine residue cannot act as a non-cysteinyl ligand to the cluster and that its substitution by cysteine also does not lead to a [4Fe- 4S] 2+/1+ configuration. These results support that FDP_E houses a [3Fe-4S] 1+/0 cluster, which agrees with the finding that Class E FDPs bear only three cysteines in the proposed cluster binding motif located in the C- terminal domain (CxHxxxCx 33 CP). Download figure Open in new tab Figure 6. Resonance Raman spectra of FDP-E wild type (blue), C-terminal domain (pink) and variants E_H441A (green) and E_H441C (yellow) in 20 mM Tris-HCl pH 7.5, measured with 405 nm excitation. The FDP_E was also anaerobically titrated and the EPR signal at g ≈2 was used to determine the cluster’s reduction potential. The experimental points were manually adjusted to aNernst equation for a monoelectronic transition, yielding a potential of 70 ± 10 mV ( Figure 5 panel B). This potential is more positive when compared with the generality of the reduction potentials described for this type of centres, normally varying between –100 and –400 mV [ 5 , 33 ], but is similar, for example, to those reported for the [3Fe-4S] 1+/0 centres of several succinate:quinone oxidoreductases (varying between +60 and +140 mV) [ 34 , 35 ]. FDP_E electron transfer partner The bioinformatic analysis of the S. wolfei genome did not reveal any canonical physiological electron donor for the FDPs (such as NADH:oxidoreductases or rubredoxins) [ 36 ]. Therefore, in an attempt to perform a kinetic characterization of FDP_E, we tested its reduction with the electron donors from other FDP’s classes: the S. wolfei FDP_H_Cter [ 9 ], the C. difficile FDP_F_Cter [ 24 ] and the E. coli flavorubredoxin reductase (NADH:flavorubredoxin oxidoreductase, NROR) [ 20 ] together with its rubredoxin domain, which has proved to be efficient for other FDPs whose electron donors are not known [ 37 ]. However, none of these systems was able to transfer electrons to FDP_E (data not shown). In addition, and given the structural homology of the C-terminal domain of FDP_E with ferredoxins (see “Amino acid sequence analysis and structural predictions” section) we also tested a commercially available Ferredoxin NADP + Reductase, but also without success. Therefore, we could not determine whether this enzyme catalyses the reduction of any of the regular FDP substrates such as oxygen or nitric oxide. Conclusion In summary, this work contributes to the characterization, by the first time, of one member of the class E FDPs. This protein is encoded in the genome of the anaerobic bacterium Syntrophomonas wolfei subsp . wolfei str. Goettingen G311 and by spectroscopic techniques was verified the presence of the three in silico predicted domains: the metallo-β-lactamase and the flavodoxin-like domains that constitute the typical FDP core and the C-terminal domain which containing a [3Fe- 4S] 1+/0 centre. Although the spectroscopic features of the metallo-β-lactamase like domain are in line with the features already described for the analogous domains in the other FDPs, the features of the flavin of the core domain are quite unusual. The results obtained in the characterization of the flavodoxin domain points to the presence of a flavin moiety covalently bound to the polypeptide chain, as it is not extractable under mild conditions. Furthermore, a species never previously reported, to the best of our knowledge, has an UV-Visible spectrum with a pronounced peak at 430 nm. Moreover, the more unexpected feature of FDP_E flavin is the lower HPLC retention time in comparison with FMN, FAD and riboflavin molecules pointing either for the presence of a flavin molecule with some type of modification or to its degradation upon thermal denaturation of FDP_E, as suggested from the several peaks detected by reverse phase HPLC. The characterization of the C-terminal domain of FDP_E revealed, for the first time in this family of proteins, the presence of a [3Fe-4S] +1/0 centre. This domain showed structural and spectroscopic similarities with [3F-e4S] 1+/0 containing ferredoxins, suggesting the role of these centres as key players in the establishment of a new type of electron transfer centre from the C-terminal domain of FDP_E to the core domains. Materials and Methods Protein Expression and Anaerobic Purification Syntrophomonas wolfei subsp . wolfei str. Goettingen G311 Swol_2363, encoding a class E FDP, was retrieved from NCBI and synthetized at GenScript Inc. USA. A truncated form comprising the C-terminal domain (residues 401 to 508) was also produced. The recombinant proteins were denominated as FDP_E and FDP_E_Cter, respectively. Additionally, two site-directed mutants were produced where the histidine (residue 441) localized in the conserved motif CXHX 3 C-X 32 CP presumably involved in the binding of a FeS cluster were substituted by an alanine (named as E_H441A) or a cysteine (named as E_H441C). In all syntheses were used codons optimized for expression in E. coli and the genes were cloned in a pET24a(+) vector, using NdeI and HindIII restriction sites. The plasmids were used to transform E . coli BL21(DE3) Gold competent cells (Nzytech). 300 mL of Luria Bertani medium containing 50 μg/mL kanamycin was inoculated with a single colony of E. coli BL21(DE3) Gold cells containing pET24a(+) FDP_E, FDP_E_Cter, E_H441A or E_H441C plasmids. Upon 16 h at 37 °C and 150 rpm, 60 mL of cell cultures of FDP_E, E_H441A and E_H441C was used to inoculate 2 L of M9 minimal medium, in 2 L Erlenmeyer flasks, containing 50 μg/mL kanamycin and supplemented with 0.1 mM of FeCl 2 . These cultures were grown at 37 °C and 160 rpm, until an optical density at 600 nm of approximately 0.4. At this point, 0.1 mM of FeCl 2 was added and protein expression was induced with 100 μM of IPTG; cells were grown for additional 16 h at 25°C and 120 rpm. The expression of FDP_E_Cter was performed differently: 30 mL of pre inoculum culture was used to inoculate 1 L of LB medium, in 2 L Erlenmeyer flasks, containing 50 μg/mL kanamycin and 0.1 mM of FeCl 2. This culture was grown at 37 °C and 150 rpm until an optical density at 600 nm of 1.2. At this point, the protein expression was induced with 500 μM of IPTG and a new addition of 0.1 mM FeCl 2 was made; cells were grown for more 16 h at 25°C and 120 rpm. All the cells were harvested by centrifugation (7 000 g 10 min) resuspended in 20 mM Tris-HCl buffer pH 7.5 and disrupted by 3 cycles in a French-Press apparatus at 16000 psi (Thermo) in the presence of DNAse (Applichem). From this step forward all the purification procedures were realized under anaerobic conditions inside an anaerobic chamber (Coy Lab Products). We purified the holoenzyme (wild type and mutants) and the C-ter construct anaerobically, to avoid the eventual degradation of a [4Fe-4S] 2+/1+ if it were present, as this type of clusters are frequently oxygen sensitive. We found no differences between the proteins purified aerobic- or anaerobically. The crude extracts were centrifuged at 25 000 g for 30 min and at 138 000 g for 2 h at 4 °C to remove cell debris and membrane fraction, respectively. The supernatants were maintained overnight at 4 °C anaerobically in an anaerobic encapsulated flask. The soluble extracts were loaded onto a Q-Sepharose Fast-Flow column (65 mL, GE Healthcare) previously equilibrated with 20 mM Tris- HCl buffer pH 7.5 containing 18 % glycerol (buffer A). The proteins were eluted with a linear gradient from buffer A to 20 mM Tris-HCl pH 7.5 containing 18 % glycerol and 500 mM NaCl. The eluted fractions were analyzed by 15 % SDS-PAGE and UV-Visible spectroscopy. Fractions with the desired protein were pooled and concentrated. The selected fractions were loaded into a Fractogel column (20 mL, Merck Millipore) previously equilibrated with buffer A and eluted with a linear gradient from A to 20 mM Tris-HCl pH 7.5 containing 18 % glycerol and 400 mM NaCl. The FDP_E_Cter was subjected to a third chromatographic step on a size exclusion S200 column (120 mL, GE Healthcare) equilibrated with 20 mM Tris–HCl, pH 7.5 containing 18 % glycerol and 150 mM NaCl. Fractions containing pure proteins were pooled and concentrated. Protein, metal and flavin quantification All the proteins (FDP_E, FDP_E_Cter, E_H441A and E_H441C) were quantified using the Bicinchoninic Acid Kit (Thermo) and bovine serum albumin as standard. Iron content was determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) at Laboratório de Análises, Chemistry Department, CQFB/REQUIMTE, FCT-UNL). For flavin quantification, an FDP_E was incubated for 45 min at 95 °C, centrifuged at 16 000 g for 10 min and the supernatants were used to measure the absorbance spectrum; the flavin content was quantified using ɛ 446nm = 12.5 mM −1 .cm −1 [ 27 ]. The flavin type was analyzed by reverse phase HPLC at the ITQB Small Molecule Analysis facility, as previously described [ 24 ]. Protein quaternary structure determination The quaternary structures were determined by size exclusion chromatography using a 25 mL Superdex 200 10/300 GL Increase column (GE Healthcare) for FDP_E, as previously described in [ 9 , 24 ], and a 25 mL Superdex S75 10/300 GL column in the case of FDP_E_Cter. A mixture containing conalbumin (75 kDa), ovalbumin (44 kDa), carbonic anydrase (29 kDa), ribonuclease a (13.7 kDa), aprotinin (6.5 kDa), and dextran blue (2000 kDa) as a void volume marker was used as the standard in the Superdex S-75 10/300 GL column. UV-Visible spectroscopy FDP_E and FDP_E_Cter samples were prepared inside an anaerobic chamber (Coy Lab Products) to a final concentration of 30 μM in 50 mM Tris-HCl pH 7.5 containing 18% and in the presence of an O 2 scavening system composed by: 10 mM glucose, 375 nM glucose oxidase and 750 nM catalase. Once the UV-Visible initial spectra (corresponding to the oxidized state) were obtained the samples were reduced by sequential additions of a buffered sodium dithionite solution (50 mM Tris-HCl pH 9). The reduction potential of FDP_E flavin was determined by UV-Visible spectroscopy, using a Shimadzu UV-1603 spectrophotometer. 30 μM of FDP_E in 50 mM Tris–HCl, pH 7.5 containing 18% glycerol and in the presence of the O 2 scavenging system (above mentioned) was anaerobically titrated by consecutive additions of a buffered solution of a sodium dithionite solution (50 mM Tris-HCl pH 9) in the presence of a mixture of redox mediators (1 μM of each, assay final concentration): potassium ferricyanide (E’ 0 = +430mV), N,N dimethyl-p-fenilenodiamine (E’ 0 = +340mV), 1,2- naphtoquinone-4-sulphonic acid (E’ 0 = +215 mV), 1,2 naphtoquinone (E’ 0 = +180 mV), trimethylhydroquinone (E’ 0 = +115 mV), 1,4 naphtoquinone (E’ 0 = +60 mV), 5-hydroxy-1,4-naphthoquinone (E’ 0 = +30 mV), duroquinone (E’ 0 = +5 mV), menadione (E’ 0 = 0 mV), plumbagin (E’ 0 = -40 mV), resorufin (E’ 0 = -51 mV), indigo trisulphonate (E’ 0 = -70 mV), phenazine (E’ 0 = -125 mV), 2-hydroxy-1,4-naphthoquinone (E’ 0 = - 152 mV), anthraquinone-2-sulphonate (E’ 0 = -225 mV), phenosafranine (E’ 0 = -255 mV), safranine (E’ 0 = -280 mV) and neutral red (E’ 0 = -325 mV). A combined Pt electrode (Ag/AgCl in 3.5 M KCl, as reference) was used and calibrated at 20 °C against a saturated quinhydrone solution (pH 7). The reduction potentials were calculated in relation to the standard hydrogen electrode. The experimental points represented are the result of three independent redox titrations. The experimental data was manually adjusted with a Nernst equation. Electron paramagnetic resonance (EPR) spectroscopy Electron paramagnetic resonance (EPR) spectroscopy was performed using a Bruker EMX spectrometer equipped with an Oxford Instruments ESR-900 continuous-flow helium cryostat and a perpendicular mode rectangular cavity. The as isolated protein samples were prepared anerobically to final concentrations of 150 µM (for FDP_E, E_H441A and E_H441C) or 100 µM (in the case of FDP_E_Cter). A reduced sample for all the proteins was prepared anaerobically by incubation with sodium dithionite (10x excess) and methyl viologen (5x excess). EPR spectra were obtained at 12 K and were simulated using the program SpinCount [ 23 ] The reduction potentials of the diiron and the FeS centres were determined by EPR spectroscopy. 100 µM of FDP_E samples were prepared under anaerobic conditions in 50 mM Tris–HCl, pH 7.5 containing 18% glycerol and in the presence of the O 2 scavening system. These samples were titrated by consecutive additions of a buffered sodium dithionite solution (50 mM Tris-HCl pH 9) in the presence of a mixture of redox mediators (30 μM of each, assay final concentration) similar to the one mentioned for UV-Visible spectroscopy with the addition of two mediators: phenazine methosulfate (E’ 0 = +80mV) and methylene blue (E’ 0 = +11mV). The electrode and the respective calibration are the same as mentioned in the above section. The experimental points represented correspond to the normalized intensity of the signal at g =1.85 for the diiron and g ≈ 2 for the FeS centres and result from two independent redox titrations (independent normalization of the two datasets). The experimental data was manually adjusted to the appropriate Nernst equations: monoelectronic Nernst equation for the FeS centre, and a Nernst equation for two consecutive monoelectronic transitions for the diiron centre. Ressonance Raman spectroscopy Resonance Raman (RR) spectra were acquired with a Raman spectrometer (Horiba LabRam HR- 800) equipped with a 1200 lines/mm grating and a liquid-nitrogen-cooled CCD detector. An Olympus 20x objective was used for laser focusing onto the sample and light collection in the backscattering geometry. Spectra of FDP_E, FDP_E_Cter, E_H441A and E_H441C variants were measured using a 405 nm diode laser (Toptica Photonics AG) at −190 °C, from 3 μL aliquots of the samples (0.2–0.5 mM) placed in a microscope stage (Linkham THMS 600). Experiments were performed with a laser power of 9 mW and 40- 60s accumulation times. Up to 16 spectra were co-added in each measurement to improve the signal-to-noise ratio (S/N). Fluorescence spectroscopy The flavin molecule isolated from FDP_E was analysed by steady-state fluorescence in a Varian Cary Spectrometer. Sample concentration was 10 μM to avoid saturation of the emission spectra. Excitation spectrum was recorded between 250 and 500 nm at λ Em = 520 nm, whereas the emission spectrum was recorded between 500 and 800 nm with excitation at λ Exc = 455 nm and λ Exc = 430 nm, with a slit width of 10 nm in both cases, in order to excite the flavin molecule. Author Contributions The project was coordinated and designed by MT and FF. FF and MCM performed the experiments and analysed the data. The Raman Spectroscopic studies were realized and analysed by CS. All authors contributed to the manuscript writing and approved the final version of the article. Funding This work was financially supported by the Portuguese Fundação para a Ciência e Tecnologia (FCT), PTDC/BIA-BQM/27959/2017 and PTDC/BIA- BQM/0562/2020 projects, MOSTMICRO-ITQB R&D Unit (references UIDB/04612/2020 and UIDP/04612/2020), LS4FUTURE Associated Laboratory (LA/P/0087/2020). MCM is recipient of FCT PhD grant SFRH/BD/143651/2019. We also acknowledge the ITQB Analytical Services for the reverse phase HPLC assays. References 1. ↵ Martins MC , Romão C V , Folgosa F , Borges PT , Frazão C & Teixeira M ( 2019 ) How superoxide reductases and flavodiiron proteins combat oxidative stress in anaerobes . Free Radic Biol Med 140 , 36 – 60 . 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