Biochimica et Biophysica Acta 1844 (2014) 1238–1247

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Biochimica et Biophysica Acta

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Characterisation of Desulfovibrio vulgaris haem b synthase, a radical SAM family member

Susana A.L. Lobo a, Andrew D. Lawrence b,CéliaV.Romãoa,MartinJ.Warrenb, Miguel Teixeira a,⁎, Lígia M. Saraiva a,⁎ a Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal b Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK article info abstract

Article history: An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genus Received 28 February 2014 and in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogen Received in revised form 28 March 2014 III and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted into Accepted 31 March 2014 haem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we re- Available online 5 April 2014 port the biochemical characterisation of the Desulfovibrio vulgaris AhbD enzyme that catalyses the last step of the pathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S-adenosylmethionine (SAM) Keywords: ′ Desulfovibrio and converts iron-coproporphyrin III via two oxidative to yield haem b,methionineandthe5- Alternative haem biosynthesis deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD contains Iron-coproporphyrin III two [4Fe–4S]2+/1+ centres and that binding of the substrates S-adenosylmethionine and iron-coproporphyrin III S‐adenosylmethionine induces conformational modifications in both centres. Amino acid sequence comparisons indicated that iron-sulfur protein D. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-like motif and two -rich se- quences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model of D. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron- coproporphyrin III is discussed. © 2014 Elsevier B.V. All rights reserved.

1. Introduction oxidase (HemF or HemN). Subsequently the macrocyle is oxidised by the removal of six hydrogens to give protoporphyrin IX in a reaction All modified tetrapyrroles, which include some of the major cofac- catalysed by protoporphyrinogen oxidase (HemG or HemY) and finally tors of life such as haem, sirohaem, haem d1, and cobala- iron is inserted into the core of the substrate template by ferrochelatase mins () are synthesised along a branched biosynthetic (HemH) to give haem b [1,2]. However, in sulfate reducing bacteria of pathway. Recently, the discovery of a new branch of the tetrapyrrole the Desulfovibrio genus and in methanogenic Archaea, haem b is pro- pathway revealed that haem b can be made by one of two distinct duced along a distinct branch of the modified tetrapyrrole pathway routes. In the classic haem b synthesis pathway, found in eukaryotes where sirohaem acts as an intermediate precursor (Fig. 1) [3–5]. and many bacteria, four enzymatic steps are required to transform Along this route uroporphyrinogen III is transformed into sirohaem uroporphyrinogen III, the primogenitor of all modified tetrapyrroles, by methylation at C2 and C7, oxidation of the macrocycle and into haem b.Thefirst step of the classic pathway is catalysed by ferrochelation. Sirohaem is then converted into haem b by the enzymes uroporphyrinogen decarboxylase (), which mediates the sequen- of the alternative haem biosynthetic pathway (Ahb). Initially, sirohaem tial of the four acetic acid side chains of the substrate to is acted upon by the AhbA and AhbB proteins that promote the decar- give coproporphyrinogen III. Next, the two propionic acid side chains at- boxylation of the acetic acid chains attached to C12 and C18 yielding tached to C3 and C8 undergo oxidative decarboxylations to generate 12,18-didecarboxysirohaem (Fig. 1). This decarboxylated form of protoporphyrinogen IX in a reaction catalysed by coproporphyrinogen sirohaem is modified by AhbC, which catalyses the loss of the acetic acid side chains at C2 and C7 in an S-adenosylmethionine (SAM)- dependent reaction, to form iron-coproporphyrin III (Fe-Cp III) (Fig. 1). The final step is also a SAM-dependent reaction promoted by Abbreviations: SAM, S‐adenosylmethionine; HPLC, high-performance liquid chroma- AhbD that converts the two propionate side chains attached to C3 and tography; MS, mass spectrometry; Ahb, alternative haem biosynthetic proteins; Fe-Cp C8 of Fe-Cp III into vinyl groups forming haem b (Fig. 1) [3]; therefore, III, iron-coproporphyrin III ⁎ Corresponding authors. the conversion of sirohaem into haem b via the alternative haem path- E-mail addresses: [email protected] (M. Teixeira), [email protected] (L.M. Saraiva). way involves several reactions that are linked to radical SAM chemistry.

http://dx.doi.org/10.1016/j.bbapap.2014.03.016 1570-9639/© 2014 Elsevier B.V. All rights reserved. S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247 1239

Fig. 1. Alternative haem b biosynthesis pathway in which sirohaem is converted to haem b via the AhbA, AhbB, AhbC and AhbD enzymes.

Radical SAM enzymes are a vast family of proteins with a wide diver- predicted the tridimensional structure of AhbD and propose the puta- sity of functions participating, for example, in the biosynthesis of cofac- tive pockets for the binding of the iron–sulfur centres and the SAM tors, vitamins, DNA precursors, and antibiotics. One of the features of and iron-coproporphyrin III substrates. these enzymes is the presence of, at least, one [4Fe–4S]2+/1+ cluster co- ordinated by only three cysteine residues arranged in a conserved

Cx3Cx2C motif located at the N-terminal region of the protein [6–10]. 2. Materials and methods The catalytically active species of the cluster is the reduced [4Fe–4S]1+ form that promotes the reductive cleavage of SAM to yield 2.1. Cloning and expression of recombinant D. vulgaris AhbD and a highly reactive 5′-deoxyadenosyl (dAdo) radical. Due to the oxy- gen lability of this iron–sulfur cluster these reactions usually occur The gene encoding the D. vulgaris Hildenborough AhbD (gene locus under anaerobic conditions. This large family of enzymes also includes DVU0855) was amplified in a PCR reaction using genomic DNA and two the oxygen-independent coproporphyrinogen oxidase, HemN, from the different pairs of oligonucleotides to produce a His-tagged and a non- canonical haem b pathway [11,12]. Radical SAM enzymes may have ad- labelled protein. The DNA fragment amplified with the pair 5′-CAC ditional iron–sulfur centres, dubbed “auxiliary clusters” [13]. For exam- ATA TGG GAG CGC ATC CCA CTG CCC-3′ and 5′-CAC TCG AGG CGG ple, the synthase, BioB, contains an extra [2Fe–2S]2+/1+ cluster GAG GGC GTC ATA C-3′ was cloned into NdeI/XhoI pET23b+ vector [14], whereas the methylthiotransferases RimO and MiaB bind an (Novagen), which allowed production of a C-terminal 6x-HisTag fused auxiliary [4Fe–4S]2+/1+ cluster coordinated by three invariant , protein. The DNA fragment amplified with primers 5′-CGG GCG GCC which is proposed to be involved in activation of sulphide or ATA TGG GAG CG-3′ and 5′-CAT GTC GGT GGA GAA TTC CCC TGC-3′ methylsulfide [15,16]. In the molybdenum biosynthetic was cloned into NdeI/EcoRI pET28a+ vector (Novagen), excised with enzyme MoaA, the additional [4Fe–4S]2+/1+ centre is also coordinated the same restriction enzymes and cloned into pET23b+ to generate a by three cysteines [17] while in the anaerobic sulfatase maturating protein with no fusion tags, designated AhbD. All recombinant plasmids enzyme AnSME from Clostridium perfringens the two auxiliary were sequenced to confirmtheabsenceoferrorsinthegenesequences [4Fe–4S]2+/1+ centres are coordinated by four cysteine residues [18]. and then transformed in E. coli BL21 Star(DE3)pLysS cells (Invitrogen). In this work, the AhbD enzyme involved in the last step of the For protein expression, E. coli cells carrying the recombinant plasmids alternative haem b biosynthetic pathway in Desulfovibrio vulgaris weregrownin1lflasks fully filled with LB medium, to which the appro- Hildenborough was biochemically and spectroscopically characterised priate antibiotics were added. Cells growing at 37 °C that reached an and shown to be a member of the radical SAM superfamily of proteins OD600 of approximately 0.5 were supplemented with 400 μM isopropyl having two [4Fe–4S]2+/1+ centres. Based on modelling studies, we β-D-1-thiogalactopyranoside (IPTG) and 30 μM Fe (in the form of 1240 S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247

FeSO4.7H2O), and after an overnight growth, at 20 °C, were harvested by (2.1 × 150 mm; Advanced Chromatography Technologies Ltd) attached centrifugation. to an Agilent 1100 series HPLC equipped with a diode array detector and coupled to a micrOTOF-Q II (Bruker) mass spectrometer. The tetrapyr- 2.2. Cell free lysates preparation role derivatives were detected at 390 nm and separation was achieved by applying a binary gradient at a flow rate of 0.2 ml min−1 with 0.1% D. vulgaris cells were grown anaerobically in lactate/sulfate medium trifluoroacetic acid (TFA) as solvent A and acetonitrile as solvent B.

[19], at 37 °C, in 1 l closed flasks until an OD600 correspondent to the sta- The column was first equilibrated with 20% solvent B and after sample tionary phase (OD600 ~1.2), at which point cells were collected by cen- injection the concentration of solvent B was increased to 100%. The trifugation and kept at −80 °C. total duration of each run was 50 min [3].

In an anaerobic chamber (Belle Technology, b2 ppm O2), D. vulgaris For the analysis of SAM derived products, reaction mixtures contain- and the E. coli BL21 Star(DE3)pLysS cells overexpressing D. vulgaris ing ~40 μM D. vulgaris AhbD, 1 mM sodium dithionite and 15 μMFe-Cp AhbD were resuspended in degassed 50 mM Tris–HCl pH 8 (buffer A), III in a final 0.5 ml volume of buffer A were anaerobically prepared, and lysed by sonication and centrifuged for 20 min at ~30,000 ×g for remov- the reaction was initiated by adding 1 mM SAM. After overnight incuba- al of unbroken cells. The resulting cell free lysates of D. vulgaris and of tion, 100 μl aliquots were taken and immediately quenched by addition

E. coli overexpressing AhbD were used in the activity assays as described of an equal volume of 100 mM H2SO4. The mixtures were then centri- below. fuged for 20 min at 16,000 ×g,and50μl of the supernatants containing the SAM related products were analysed by HPLC–MS. As control, a mix 2.3. Protein purification of SAM, 5′-deoxyadenosine (dAdo), 5′-methylthioadenosine (MTA) and S-adenosyl-L-homocysteine (SAH) was also prepared and the products For protein isolation, E. coli BL21 Star(DE3)pLysS cells overexpress- were separated and analysed by mass spectrometry as previously de- ing D. vulgaris AhbD were resuspended in buffer A and disrupted by scribed [3],ataflow rate of 0.2 ml/min and with a gradient of acetoni- passing three times through a French Press, at 900 Psi under aerobic trile in 0.1% TFA as follows: 0–1 min isocratic 0% B (acetonitrile), 1– conditions, after which the suspensions were centrifuged for 30 min 15 min ramp to 10% B, 15–20 min ramp to 30% B, 20–25 min ramp to at ~30,000 ×g. The resulting supernatant was submitted to an argon at- 50% B, 25–30 min ramp to 100% B, 30–35 min isocratic 100% B, 35– mosphere and further manipulated in a Coy model A-2463 anaerobic 40 min ramp to 0% B and 40–50 min to 0%. The elution profile was mon- chamber containing a gas mixture of 95% argon plus 5% hydrogen. itored by UV–visible spectroscopy at 260 nm. The supernatant was applied to a 3 ml Ni2+ saturated Chelating Sepharose resin (GE, Healthcare), previously equilibrated in buffer A 2.5. Binding assay with 500 mM NaCl and 10 mM imidazole (binding buffer). A step gradi- ent of imidazole in the same buffer was applied and the protein was The binding of Fe-Cp III to AhbD was analysed by monitoring the eluted at 250 mM imidazole, concentrated in an Amicon Stirred Ultrafil- changes of the UV–visible spectrum of the reduced Fe-Cp III (15 μM) tration Cell using a 10 kDa membrane (Millipore), and submitted to upon successive additions of reduced AhbD protein (2 to 36 μM). The re- buffer exchange by means of a PD10 column (GE Healthcare) that duced form of Fe-Cp III was obtained by previous incubation with used buffer A as running buffer. The purity of the protein was evaluated 2 molar equivalents of sodium dithionite in buffer A, while reduction by SDS-PAGE and the protein concentration was determined by the of AhbD was achieved by addition of 8 molar equivalents of sodium Pierce Bicinchoninc acid Protein Assay kit (Thermo Scientific) using dithionite. The shift of the Soret band and the variation of the absor-

Sigma protein standards. UV–visible spectra were recorded inside the bance at 550 nm were monitored. The association constant (KA)and anaerobic chamber in a Shimadzu UV-1800 spectrophotometer. the maximal number of binding sites (Bmax) values were determined

The molecular mass and quaternary structure of the protein were by fitting the data to the equation Y =Bmax*X /(X + KA) by nonlinear determined by passage in a Superdex 200 10/300GL column (GE, regression using GraphPad Prism software version 5.0 for Windows, Healthcare), equilibrated with 50 mM sodium-phosphate pH 7 buffer GraphPad Software, San Diego, California, USA (www.graphpad.com). containing 150 mM NaCl. The iron content of the pure protein was de- In this equation Y was calculated from the absorbance at 550 nm or termined by the 2,4,6-tripyridyl-1,2,3-triazine (TPTZ) method [20]. the shift of the Soret band maximum and X represents the concentration of the protein. The titration data were normalised between 0 and 100 in 2.4. Analysis of the reaction products by high-performance liquid relation to the smallest and the largest values of each data set. chromatography–mass spectrometry (HPLC–MS) 2.6. EPR studies Independent assays were performed using the purified protein as well as the cell free lysates of D. vulgaris and of E. coli BL21Star(DE3) Four samples prepared under anaerobic conditions were analysed pLysS cells overexpressing D. vulgaris AhbD. by EPR spectroscopy, all containing the same concentration of AhbD Anaerobically purified D. vulgaris AhbD (15 μM) was mixed with (160 μM): sample 1, constituted by the as-purified AhbD; sample 2, 15 μM Fe-Cp III, 1 mM SAM and 0.5 mM sodium dithionite in a final vol- consisting of purified enzyme reduced with sodium dithionite ume of 1 ml and left to react for 3 h and overnight, at room temperature, (2 mM); sample 3, a mixture of AhbD with SAM (1 mM) and sodium under anaerobic conditions. dithionite (2 mM); and sample 4, a mixture of AhbD, SAM (1 mM), Reaction mixtures of 4 ml in buffer A containing D. vulgaris cell ly- Fe-Cp III (175 μM) and sodium dithionite (2 mM). All samples were pre- sate, 20 μM Fe-Cp III (Frontier Scientific), 0.1 mM SAM, 10 mM sodium pared in buffer A with a final volume of 300 μl, loaded into EPR tubes, dithionite and 0.5 mM NADH were prepared and anaerobically incubat- sealed with caps and immediately frozen in liquid nitrogen. EPR spectra ed overnight, at room temperature. Reaction mixtures of 4 ml in buffer A were acquired at several temperatures and microwave powers in a with E. coli cell lysate, 20 μM Fe-Cp III, 0.5 mM SAM and 10 mM sodium Bruker EMX spectrometer equipped with an Oxford Instruments con- dithionite were also prepared and incubated overnight. tinuous flow helium cryostat. Spectra simulation was done with the In all cases, after the completion of the reaction, the tetrapyrrole in- SPINCOUNT program (http://www.chem.cmu.edu/groups/hendrich/ termediates were purified by a haem extraction protocol [21] and facilities/index.html). Several independent protein preparations from analysed by reverse phase chromatography and mass spectrometry. distinct cell cultures were studied yielding consistently the same results. To this end, the final extraction solvent, ethyl acetate, was evaporated The reduction potential of AhbD was determined by means of an an- and the intermediates were resuspended in a mixture of 90% acetoni- aerobic redox titration (under constant flux of argon) of the purified trile–10% methanol (HPLC grade) and resolved on an Ace 5 AQ column AhbD, monitored by EPR. To this end, 200 μM AhbD resuspended in S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247 1241 buffer A was titrated by stepwise addition of buffered sodium dithionite (10, 50 and 100 mM), in the presence of a standard redox mediators mixture (100 μM): 1,2 naphtoquinone (E′0 = 180 mV), 1,4- naphtoquinone (E′0 = 60 mV), methylene blue (E′0 = 11 mV), mena- dione (E′0=0mV),indigotetrasulfonate(E′0=−30 mV), plumbagin (E′0=−40 mV), resorufin(E′0=−51 mV), indigo trisulfonate (E′0=−70 mV), indigo disulfonate (E′0=−110 mV), phenazine (E′0=−125 mV), 2-hydroxy-1,4-naphtoquinone (E′0=−152 mV), anthraquinone sulfonate (E′0=−225 mV), phenosafrine (E′0= −255 mV), safranine (E′0=−280 mV), neutral red (E′0= −325 mV), benzyl viologen (E′0=−345 mV) and methyl viologen (E′0=−440 mV). A mixture of catalase (130 U/ml), glucose oxidase (4 U/ml) and glucose (3 mM) was also added to the sample mixture to further ensure that anaerobic conditions were maintained. Combined silver/silver chloride and platinum electrodes calibrated against a satu- rated quinhydrone solution at pH 7 were used. Potentials are reported relative to the standard hydrogen electrode. The experimental data were fitted to a single one-electron Nernst curve (n =1).

Fig. 3. Titration of Fe-Cp III with D. vulgaris AhbD. UV–visible spectra of reduced Fe-Cp III 3. Results (15 μM) obtained upon successive additions of AhbD (spectral shifts are indicated by arrows) and respective titration curve (inset) considering the shift of the Soret band max- 3.1. Biochemical and UV–visible spectroscopic analysis of D. vulgaris AhbD imum (black squares) and the absorbance increase at 550 nm (open diamonds); the values were normalised to their final stage. The solid line corresponds to the fitting of the curves with an equation for a 1:1 binding stoichiometry and a binding constant of fi The recombinant D. vulgaris AhbD was puri ed under anaerobic con- 8 μM(seeMaterials and methods). ditions in order to protect any putative oxygen-sensitive clusters. Nev- ertheless, the stability of the AhbD clusters was tested by exposure of seen in control samples that contained all components except AhbD. the protein to oxygen and no degradation was observed. The protein We observed that the Soret band of Fe-Cp III shifted from 405 nm to was eluted from an analytical gel filtration chromatography as a mono- 413 nm, concomitantly with the development of bands at 528 mer with an apparent molecular mass of ~40 kDa, in agreement with its and 550 nm. These alterations are consistent with the binding of the gene-derived amino acid sequence. The as-isolated protein exhibits a Fe-Cp III to the protein and the formation of a low-spin hexa- brown colour with a visible spectrum displaying two absorption bands coordinated Fe-Cp III. Furthermore, the trace resultant from the subtrac- at 325 and 415 nm (Fig. 2). These features are similar to those of the tion of the final titration spectrum from that of the iron-coproporphyrin oxidised form of iron–sulfur cluster containing proteins. Indeed, metal alone exhibits maxima at 424, 518 and 550 nm. These maxima are sim- analysis also showed that AhbD contains 6 ± 2 mol of iron per mol of ilar to those previously observed for D. desulfuricans ATCC2774 protein, suggesting the presence of more than one iron–sulfur cluster. bacterioferritin, which is naturally isolated with iron-coproporphyrin as the haem cofactor and that has two haem-iron axial ligands [22,23]. 3.2. D. vulgaris AhbD binds Fe-Cp III We also evaluated the affinity of the Fe-Cp III substrate to D. vulgaris AhbD by performing titration experiments of AhbD with Fe-Cp III which In the alternative biosynthetic pathway operative in D. vulgaris, were monitored by following the alteration of the Soret band and the AhbD converts iron-coproporphyrin III into haem b. In this work, we absorbance increase at 550 nm (see Materials and methods). The data have analysed the binding of the substrate by following changes in the allowed the determination of an association constant (KA), consistent visible spectrum of Fe-Cp III upon addition of AhbD. The mixing of with a single (1:1) binding stoichiometry, of 8 ± 1 μM(Fig. 3). AhbD with oxidised Fe-Cp III caused no alterations in the spectrum of Fe-Cp III. However, addition of reduced AhbD to the sodium dithionite reduced iron-tetrapyrrole induced shifts in all the porphyrin- 3.3. D. vulgaris AhbD promotes cleavage of SAM and converts Fe-Cp III into associated absorption bands (Fig. 3). Note that these shifts were not haem b

Since the step promoted by AhbD is proposed to be a SAM- dependent reaction, the products of SAM cleavage were analysed by HPLC–MS after incubation of the purified protein with SAM and sodium dithionite. Under these conditions, the formation of 5′-deoxyadenosine (dAdo) product was detected (Fig. 4A). The reaction mixture containing the same concentration of all components but done in the presence of the Fe-Cp III substrate was also analysed. We observed that the addition of the second substrate led to an approximately 10-fold increase of the amount of the dAdo formed, indicating that the metalloporphyrin po- tentiates the SAM cleavage (Fig. 4C). The data show that D. vulgaris AhbD performs a reaction that is typical of the radical SAM enzymes, namely the reductive cleavage of SAM into methionine and the dAdo radical. As with other members of this family of proteins [24] the SAM derived products were observed in the absence of the second substrate. The products formed during the conversion of Fe-Cp III into haem b catalysed by D. vulgaris AhbD were also determined by HPLC–MS. The reaction was performed separately with D. vulgaris free cell lysates, Fig. 2. UV–visible spectrum of the as-isolated D. vulgaris AhbD (5 μM) in 50 mM Tris–HCl E. coli cell extracts overproducing an untagged form of D. vulgaris pH 8, recorded under anaerobic conditions. AhbD and purified D. vulgaris AhbD. The reaction mixtures consisting 1242 S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247

a quasi-isotropic signal with g-values at around 2.02 (2.03, 2015, 2.005) as determined by simulating the spectrum using the program SPINCOUNT (Fig. 6Aa), characteristic of an oxidised [3Fe–4S]1+/0 clus- ter. This resonance accounts 10–15% of the spectral intensity of the dithionite reduced sample, under the same non saturating conditions, and most likely results from a minor oxidative degradation of one or two of the tetranuclear centres, a common feature in low-reduction po- tential [4Fe–4S]2+/1+ centres. Reduction of AhbD with sodium dithionite led to development of a more complex signal with g-values ranging from g ~2.06 to 1.92, which are typical of reduced bi- or tetranuclear iron–sulfur centres (Fig. 6Ab). These resonances were de- tected without significant line broadening only up to a temperature of ~30 K. Hence, we concluded that are not originated from binuclear cen- tres but from tetranuclear iron–sulfur clusters which agrees with the cysteine motifs present in the AhbD amino acid sequence (see below). The resonances were deconvoluted by varying the temperature and mi- crowave power. When, at 10 K, the microwave power was increased a

resonance with gmax = 2.083 developed. This is was clearly discernible at 40 mW (Fig. 6Ba) while at lower g-values no further resonances were detected. Also, resonances at low magnetic fields, which can originate from cluster spin states higher than ½, were not observed even at high microwave powers and low temperatures down to 4.6 K (data not shown). The experimental data at 10 K could be rationalised as resulting from two reduced tetranuclear centres, with distinctly dis-

cernible gmax values and quite different relaxation properties: a fast relaxing species, only observable at low temperatures and high micro- wave power, with g-values at 2.083, 1.921 and 1.84, and a slower relaxing one with g-values at 2.057, and 1.925 (Fig. 6Ba). Within the limitations resulting from the distinct relaxation properties, these spe- cies are present essentially in equimolar amounts. To further characterise D. vulgaris AhbD, the redox potential of the iron–sulfur centres was determined by performing a redox titration followed by EPR. The results were analysed with a one-electron Nernst curve and allow determining a single reduction potential value of −390 ±10mV(Fig. 7). The data also indicate that the reduction potentials of the two centres are similar as the individual reduction potentials of the two iron–sulfur centres could not be deconvoluted. Moreover, the lack of a plateau observed under the tested conditions shows that the metal centres of AhbD have very low reduction potentials, whose upper limit is −390 mV. Fig. 4. HPLC–MS analysis of the cleavage derived products of SAM promoted by D. vulgaris We have also analysed the EPR spectra of dithionite-reduced AhbD AhbD. From top to bottom: HPLC trace of the standards 5′-deoxyadenosine (dAdo) and samples upon addition of the substrates. In the presence of SAM, S-adenosylhomocysteine (SAH); (A) AhbD incubated with SAM in the presence of sodium some modifications of the spectra occurred and the data could again dithionite; (B) AhbD and SAM done in the absence of the reducing agent (control sample); (C) AhbD incubated with SAM and Fe-Cp III under reducing conditions; (D) AhbD mixed be interpreted as resulting from two main components, with g-values with SAM and Fe-Cp III without the reducing agent (control reaction). HPLC traces were only slightly distinct from those of the reduced sample (Fig. 6Ac and acquired at 390 nm after an overnight incubation of the reaction mixture. In reaction mix- Bb). The minor alterations observed upon addition of SAM only are tures A-D SAM was eluted as two peaks with the same m/z value of 399. not unexpected as EPR spectroscopy, although sensitive to minute mod- ifications of the paramagnetic centre electronic structure, does not nec- of Fe-Cp III, SAM and the purified AhbD protein or the D. vulgaris free cell essarily reflect specific changes of, e.g., the coordination of the iron ions lysates overproducing AhbD were incubated overnight, and the extract- in the clusters. ed samples separated and analysed. Peaks typically corresponding to Major differences were observed when the substrates SAM and haem b (m/z 616), a mono-vinyl derivate of Fe-Cp III (m/z 662) and Fe-Cp III were both added: two sets of resonances are clearly affected, the unreacted form of Fe-Cp III (m/z 708) were detected indicating but to differing extents. By varying the microwave power, two major that the conversion was incomplete (Fig. 5A and D). However, when species were defined, one that was more similar to the previously E. coli cell free extracts overexpressing AhbD were used in the presence identified slow relaxing centre, with g-values at 2.064 and 1.92, and of SAM, a complete conversion of Fe-Cp III into haem b was observed a second one from a faster relaxing centre with very broad reso- after an overnight incubation (Fig. 5B), while control reactions per- nances and g-values of 2.13, and ~1.92, underneath the gmed of the formed in the absence of SAM contained essentially only Fe-Cp III first species (Fig. 6Ad and Bc). (Fig. 5C). Above ~40 K, the spectra display solely a signal with g-values of Altogether, these results show that AhbD converts Fe-Cp III into 2.035, 2.023, and 2.01, quite distinct from that of the [3Fe–4S]1+ centre haem b and that the reaction occurs in two consecutive steps. detected in the oxidised protein; this species is also observed in the 10 K spectra (Fig. 6A and B, marked by an asterisk). This species, which has a 3.4. D. vulgaris AhbD contains two [4Fe–4S]2+/1+ centres slow relaxation rate (it is not detected at high microwave powers) and of unknown origin, was observed in all spectra of the reduced protein The nature of the iron–sulfur clusters in AhbD was studied in detail (including in the presence of the substrates), and in different prepara- by EPR spectroscopy. The EPR spectrum of the as-isolated AhbD exhibits tions, albeit accounting always to less than 10% of the total spectral S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247 1243

Fig. 5. HPLC–MS analysis of tetrapyrrole derivatives derived from reaction mixtures of cell free extracts or purified D. vulgaris AhbD. The reaction mixtures contained: (A) D. vulgaris cell free extracts, Fe-Cp III, SAM and sodium dithionite; (B) E. coli cell free extracts overexpressing D. vulgaris AhbD, Fe-Cp III, SAM and sodium dithionite; (C) purified D. vulgaris AhbD, Fe-Cp III and sodium dithionite; (D) purified D. vulgaris AhbD, Fe-Cp III, SAM and sodium dithionite. HPLC traces were acquired at 390 nm after an overnight incubation of the reaction mixture. Fe- Cp III eluted from the HPLC column as two peaks (marked with an asterisk) with the same m/z value of 708.

intensity at 10 K. Since this species corresponds to a minor component, alternative haem b pathway, namely the D. vulgaris AhbC that converts its origin was not further investigated. 12,18-didecarboxysirohaem into Fe-Cp III (Figs. 1 and 8). Altogether, the data show that D. vulgaris AhbD contains two [4Fe– Fig. 8 compares the amino acid sequence of D. vulgaris AhbD with 4S]2+/1+ centres that are perturbed upon addition of the substrates those of enzymes involved in the synthesis of tetrapyrroles and decar- SAM and Fe-Cp III. It should be noted that all experiments were per- boxylation reactions (Fig. 8). D. vulgaris AhbD shares 25% identity, 39% formed in the presence of excess sodium dithionite, and for this reason similarity with Paracoccus denitrificans NirJ, which participates in reoxidation of the clusters was not observed upon addition of SAM; the the synthesis of haem d1.InrelationtoE. coli HemN, the oxygen- iron of Fe-Cp III will be in the 2+ state and is therefore EPR silent in the independent coproporphyrinogen III oxidase of the classical haem b conditions used, which is consistent with our previous observation that pathway that performs the oxidative decarboxylation of the propionate the ferrous Fe-Cp III adopts a low-spin (S = 0) configuration when side chains of rings A and B of coproporphyrinogen III to vinyl groups bound to the protein. The largest changes at one of the clusters probably producing protoporphyrinogen IX, a low degree of similarity is seen reflect the binding of SAM to the non-cysteine coordinated iron of the (10% identity, 20% similarity) [3,26,30]. The low degree of similarity cluster in the protein N-terminal domain, as has been shown for several among these proteins suggests that despite the small structural differ- other radical SAM enzymes (e.g. [15,25–28]. The major effects after ad- ences of the several tetrapyrrole substrates their conversion requires dition of the Fe-Cp III substrate suggest that it induces alterations in the enzymes with highly specific structures. conformation of the AhbD protein, and that the Fe-Cp III binding pocket A comparative analysis of the amino acid sequence of D. vulgaris is probably close to the iron–sulfur clusters. AhbD against the NCBI protein database shows that the protein belongs to the radical SAM enzymes superfamily. The protein has a radical 3.5. Amino acid sequence analysis of D. vulgaris AhbD SAM motif in the N-terminal region and a SPASM-like motif in the C-terminus (Fig. 8). The SAM motif contains a cysteine rich region

A BLAST search against Desulfovibrio spp. and Archaea genomes (C31x3C35xHC38) typical of SAM proteins, namely the Cx3CxXC se- showed that homologs of D. vulgaris AhbD are widespread in quence, where X is an aromatic residue; these three cysteines bind Desulfovibrio spp., sharing 27%–89% identity and 42%–92% similarity the catalytic [4Fe–4S]2+/1+ centre, leaving the fourth iron free to in- when considering the first 100 retrieved amino acid sequences. For ex- teract with SAM (e.g., [6,18,31]). The N-terminal region of D. vulgaris ample, AhbD from D. vulgaris and D. desulfuricans ATCC 27774 have 70% AhbD has also a GGD triad in place of the GGE motif, which is present in identity and 77% similarity between them. Orthologs of D. vulgaris AhbD several radical SAM proteins and is involved in the binding of SAM [31]. also occur in methanogenic Archaea (e.g., Methanosarcina barkeri AhbD, The C-terminal region of AhbD houses a second cysteine rich region

50% identity, 68% similarity). In agreement with our data, recent studies C326x2C329x5C335x2C338, characteristic of a SPASM motif, whose showed that the M. barkeri AhbD also catalyses the conversion of Fe-Cp name is derived from the following biochemically characterised mem- III into haem and the authors proposed that it contains [4Fe–4S]2+/1+ bers of this subfamily: subtilosin A maturase AlbA, pyrroloquinoline centres [29]. Interestingly, D. vulgaris AhbD shares ~26% identity and quinone E PqqE, anaerobic Sulfatase maturating enzyme, and 44% similarity with the enzyme involved penultimate step of the peptide maturase [32–34]. The SPASM domain is quite 1244 S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247

Fig. 6. EPR spectra of D. vulgaris AhbD: oxidised and reduced forms and upon incubation with SAM and Fe-Cp III. (A) EPR spectra of AhbD: as prepared, oxidised form (a); sodium-dithionite reduced (b); sodium-dithionite reduced AhbD mixed with SAM (c); sodium-dithionite reduced AhbD incubated with SAM and Fe-Cp III (d). Spectra were recorded at 10 K and 2 mW. (B) Power dependence of the EPR spectra of AhbD: sodium-dithionite reduced form (a); sodium-dithionite reduced AhbD mixed with SAM (b); sodium-dithionite reduced AhbD protein mixed with SAM and Fe-Cp III (c). Spectra were recorded at 10 K and 2, 20, 40 and 63 mW, as indicated. Asterisk denotes the unknown species (b10%). conserved in members of the radical SAM family and is described 4. Discussion to bind auxiliary [4Fe–4S]2+/1+ centres. The biochemical and spectroscopic studies presented above indicate that in D. vulgaris In this work, D. vulgaris AhbD is shown to be a monomeric cytoplas- AhbD the SPASM motif is also involved in the binding of one auxiliary mic protein and a member of the radical SAM superfamily. The [4Fe–4S]2+/1+ cluster, most possibly coordinated by four cysteine resi- protein harbours two [4Fe–4S]2+/1+ centres, one located at the N- dues. For most of the enzymes, the function of the additional cluster re- terminal domain and coordinated by three cysteines and the other at mains, so far, unclear [13]. the C-terminus putatively ligated by four cysteines. Under reductive conditions and in the presence of SAM, AhbD promotes the sequential decarboxylation of the two propionate side chains attached to C3 and C8 of Fe-Cp III to form vinyl groups, i.e., generating haem b. Using two of the more similar structural protein analogues of D. vulgaris AhbD, namely the AnSME from C. perfringens [18] and MoaA of S. aureus [17], we predicted a three dimensional model for the structure of AhbD (Fig. 9). AnSME, which has two auxiliary [4Fe–4S]2+/1+ clusters each coordinated by four cysteines located at the C-terminus, shares with D. vulgaris AhbD 15% identity, 31% similar- ity, while MoaA that has one additional [4Fe–4S]2+/1+ coordinated by only three cysteines shares 14% identity and 34% similarity with the D. vulgaris enzyme (Fig. 8). The D. vulgaris AhbD structure model has β/α motifs arranged in a TIM barrel-like motif, the common feature of most radical SAM enzymes [6], although some of the beta strands ap- pear as loops probably due to the low amino acid sequence similarity. As predicted from the amino acid sequence, the N-terminal region cor- responds to the SAM radical protein domain, which includes the three cysteines that bind the catalytic [4Fe–4S]2+/1+ cluster, the two glycine Fig. 7. Redox titration of the [4Fe–4S] 2+/1+ clusters of D. vulgaris AhbD. Titration was residues, Gly75 and Gly76, and the aspartate residue Asp77 that inter- monitored by EPR spectroscopy, measuring the peak-to peak intensity of the g =1.91res- onance, at 10 K. The solid line corresponds to a one-electron Nernst curve, with a reduction acts with the SAM molecule. The C-terminal domain contains four cys- potential of −390 mV. teines (Cys326, Cys329, Cys335 and Cys356) that are predicted to be S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247 1245

Fig. 8. Amino acid sequence alignment of D. vulgaris Hildenborough AhbD (gene locus DVU0855) with D. desulfuricans ATCC 27774 AhbD (Ddes0287), Methanosarcina barkeri AhbD

(MbarA1458) D. vulgaris Hildenborough AhbC (DVU0857), Methanosarcina barkeri AhbC (MbarA1793), D. desulfuricans ATCC 27774 AhbC (Ddes0289), P. denitrificans haem d1 biosynthetic enzyme NirJ (Pden2494), Clostridium perfringens AnSME (CPF_0616), Staphylococcus aureus MoaA (SA2063) and HemN (b2955). The two domains of D. vulgaris AhbD namely, the AdoMet radical domain and the auxiliary iron–sulfur binding SPASM domain are indicated. The amino acid residues of the AdoMet radical motif for the binding of the con- served iron–sulfur centre CX3CX2C are shaded in black and marked with stars. Residues of the GG(E/D)P motif involved in the binding of SAM are shaded in dark grey and indicated with open circles. Cysteine residues of the SPASM motif are shaded in light grey. The alignment was constructed using the amino acid sequence of D. vulgaris AhbD to search for similar sequences against all microbial genomes using NCBI Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov/), performed in ClustalX 2.1 [37] and edited in GeneDoc [38]. 1246 S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247

Fig. 9. 3D model of D. vulgaris AhbD. (A) Cartoon representation of the predicted D. vulgaris AhbD structure. (B) Tridimensional structure of C. perfringens AnSME displayed in the same orientation as in panel A. (C) Surface representation of D. vulgaris AhbD model. (D) Surface representation of D. vulgaris AhbD model rotated by 90° from the view in panel C. The figure depicts a channel (marked with an arrow) and the predicted binding site of the SAM molecule which is located at the end of the channel. The model was generated by the I-TASSER server [39–41] using as template the structure of C. perfringens AnSME (PDB ID: 4K36). The root-mean-square deviation between the two models is 0.987 Å. In panels A and B, the monomer is coloured in gradient, starting from blue (N-terminal) to red (C-terminal) and the region of the conserved cysteines involved on the iron–sulfur clusters is boxed and zoomed. The amino acid residues, the SAM molecule and the iron–sulfur clusters are represented as sticks. Pictures were built with the PyMOL program [42]. the ligands of the auxiliary [4Fe–4S]2+/1+ cluster equivalent to auxiliary exchange of electrons with yet unknown physiological partners. Inter- centre II in the C. perfringens AnSME (Fig. 9A). Both iron–sulfur clusters estingly, in the place where C. perfringens AnSME binds the auxiliary are expected to be close to the protein surface, which may allow cluster I, the D. vulgaris enzyme has still two cysteine residues S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247 1247

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