A Morphing [4Fe‐3S‐nO]‐Cluster within a Carbon Monoxide Dehydrogenase Scaffold

Abstract Ni,Fe‐containing carbon monoxide dehydrogenases (CODHs) catalyze the reversible reduction of CO2 to CO. Several anaerobic microorganisms encode multiple CODHs in their genome, of which some, despite being annotated as CODHs, lack a cysteine of the canonical binding motif for the active site Ni,Fe‐cluster. Here, we report on the structure and reactivity of such a deviant enzyme, termed CooS‐VCh. Its structure reveals the typical CODH scaffold, but contains an iron‐sulfur‐oxo hybrid‐cluster. Although closely related to true CODHs, CooS‐VCh catalyzes neither CO oxidation, nor CO2 reduction. The active site of CooS‐VCh undergoes a redox‐dependent restructuring between a reduced [4Fe‐3S]‐cluster and an oxidized [4Fe‐2S‐S*‐2O‐2(H2O)]‐cluster. Hydroxylamine, a slow‐turnover substrate of CooS‐VCh, oxidizes the hybrid‐cluster in two structurally distinct steps. Overall, minor changes in CODHs are sufficient to accommodate a Fe/S/O‐cluster in place of the Ni,Fe‐heterocubane‐cluster of CODHs.


Table of Contents
Cloning and Expression. The gene CHY_RS00160 [15] annotated as coding for CODH-V in Carboxydothermus hydrogenoformans (CooS-VCh) was amplified by PCR with a pair of primers including BsaI restriction sites using Phusion DNA polymerase: 5'-GGAGATGGCGCCGCTACAAAAACGTCCATTCA-3' (forward) and 5'-GGAGATGTATCATTAAATACCCAGTTTGGCGC-3' (reverse). The resulting ~1.9 kbp DNA fragment was digested with BsaI and ligated into a BsaI-digested pASK-IBA 17-plus vector (IBA, Göttingen Germany). After transformation of the ligation products into Escherichia coli DH5 , a plasmid with the insert was analyzed by restriction digest and sequencing (Eurofins Genomics Germany GmbH), termed pPK-S5-strep. For expression of the gene, pPK-S5-strep was transformed into E. coli strain BL21 (DE3). Cultures were grown at 37 °C in modified TB media [16] supplemented with 50 µg/ml kanamycin, 1% (w/v) glucose, 0.2 mM FeSO4 and 0.2 mM Na2S in 5-L glass fermenters under stirring and access of air. After the OD600 reached a value of 0.4, the bottles were sealed with butyl rubber septa to restrict oxygen contact. The culture was further stirred until an OD600 of 0.7-0.8 was reached. Gene expression was stimulated by addition of 0.2 mg/l anhydrotetracycline and continued for 16 h at 30 °C. Cell pellets were stored at -80 °C until further use.
Purification. Frozen cells were resuspended in buffer S (50 mM Tris-HCl pH 8.0 and 300 mM NaCl) with 2 mg/L avidin and stirred for 30 min. N-octyl-β-D-maltoside was subsequently added until a final concentration of 0.5% (w/v) was reached. Cell lysis was achieved by sonication in an ice-cooled rosette cell. A clear, soluble fraction was obtained by ultracentrifugation at 95,800 g at 12 °C for 1 h. The supernatant was loaded onto a Strep-Tactin® Superflow® high capacity column (IBA, Göttingen Germany) equilibrated with buffer S, followed by extensive washing with ten column volumes of the same buffer. Protein was eluted with buffer E (buffer S + 2.5 mM desthiobiotin) in a volume of 20 mL and subsequently concentrated. Protein buffer was exchanged versus buffer G (20 mM Tris-HCl pH 8.0) on a PD-10 desalting column (GE Healthcare).
The N-terminal Strep-Tag was removed by treatment with strep-tagged TEV (tobacco etch virus) protease in 100-fold excess with 5 mM β-mercaptoethanol overnight at 25 °C. Strep-tag free CooS-VCh was isolated by collecting the flow-through fraction after loading on to the Strep-Tactin column. A PD-10 column was used to exchange buffer with 20 mM Tris-HCl pH 8.0. The protein was frozen in glass vials equipped with butyl rubber septum in liquid N2 and stored at -80 °C Fe and Protein Quantification. The non-heme iron content was determined using the published method. [17] Protein concentration was routinely measured according to Bradford [18] using the reagent from Roth (Roti®-Quant).
Spectroscopic Methods. UV-vis spectra of CooS-VCh were recorded on an Agilent 8453 photodiode array spectrophotometer. All measurements were performed anaerobically at room temperature using quartz cuvettes with a path length of 1 cm. Potassium ferricyanide was used to oxidize the protein and was subsequently removed by buffer exchange on PD-10 column. To obtain a reduced spectrum, the oxidized protein (18 µM in 50 mM Tris-HCl pH 8.0 and 100 mM NaCl) was titrated by Na-dithionite (DT) in 1 µM steps.
Electron Paramagnetic Resonance (EPR) Spectroscopy. Samples for EPR spectroscopy were prepared in the anoxic glove box. For the redox titration the oxidized/as-isolated protein (200 µM, Tris-HCl pH 8) was transferred into a dedicated redox cell equipped with a Pt redox electrode and continuous solution mixing. The protein solution was stepwise reduced by addition of Na-DT (1-10 µM).
Redox potential stabilization was achieved by including a redox mediator mixture in the solution. When the potential had stabilized, about 100 µl of protein solution was transferred into an EPR tube for EPR measurements. Continuous wave EPR spectra were recorded at 10 and 80 K on a home built X-band spectrometer featuring a Bruker ER 041 MR microwave source, a Bruker ER 4122-SHQ E resonator optimized for cw-measurements, a Stanford Research Systems SR810 lock-in amplifier, an AEG electromagnet, driven and controlled by a Bruker ESR 2388 power supply and a Bruker B-H 15 field controller. Temperature control was achieved using an Oxford Instruments ESR 910 cryostat attached to the resonator.

NH2OH-Reduction and CO-Oxidation Assay.
Hydroxylamine reduction activity was assayed as previously described by Wolfe et al. [19] monitoring the oxidation of reduced methyl viologen (MVred). In 2 mL quartz cuvettes, a buffer (100 mM HEPES pH 8.0 and 100 mM NaCl) was added and incubated for 2 min at 40 °C, before a base line spectrum was recorded. Subsequently added oxidized MV (final concentration: 10 mM) was reductively titrated with dithionite solution, until a final absorption of 1.2 at 578 nm (A578) was obtained. The concentration of hydroxylamine was varied from 0.5 to 100 mM. When no significant changes in absorbance were observed, the reaction was started by adding 1 µM CooS-VCh and monitored for 300 s. Initial rates were determined and specific activities calculated using the extinction coefficient of MVred at 578 nm (ε578 = 9.7 mM -1 cm -1 ). The steady-state kinetic constants Km and kcat were determined by fitting the experimental data with a nonlinear regression applying the Michaelis-Menten equation in GraFit 5. [20] CO-oxidation activity was measured as described previously. [16] NO Reductase Activity Assay. We used two different assays to monitor NO reductase activity of CooS-VCh: 1) detecting NADH oxidation by the enzyme hybrid cluster protein reductase (HCR) from E. coli by time-dependent absorbance measurement at 340 nm and 2) use of gas chromatography (GC) and mass spectrometry (MS) to detect NO consumption and N2O production from the gas phase of the reaction vessel. NO gas was purged through 1 M NaOH solution and quantified by formation of a NO-myoglobin complex with an ε at 421 nm (114 mM -1 cm -1 ). [21] 1) Time-dependent absorbance at 340 nm. The gene encoding HCR from E. coli BL21 (DE3) was cloned into a pET28 vector and HCR was aerobically expressed in E. coli BL21 (DE3) using an autoinduction medium. [22] HCR was purified by affinity chromatography using the N-terminal his-tag according to the manufacturer's recommendations (GE Healthcare). After the reconstitution, HCR possessed 1.9 Fe and 1.2 FAD per mole of protein.
We followed the reduction of CooS-VCh (0.2 µM) by adding HCR (2.5 µM) with 250 µM NADH as reducing agent, which was monitored by the decreasing absorption at 450 nm. The observed rate constant (kobs) of reduction of CooS-VCh by HCR of 50/min was determined by fitting a single-exponential function.
To measure NO reduction activity, 40 nM CooS-VCh and 160 nM HCR in 50 mM Tris-HCl pH 8.0 were mixed in a gas-tight cuvette without any gas phase. The reaction was started by adding ~240 µM NO solution and the decrease in NADH concentration was monitored at 340 nm. NADH consumption was quantified using an ε340nm of 6.22 mM -1 cm -1 .
2) Use of GC/MS. We adapted our assay [23] with a modification of the SIM mode (m/z=30 for NO and m/z=44 for N2O) to detect NO and N2O in the reaction vessel by MS. Two reduction methods were used: with HCR and NADH as in (1)  All crystals were flash-cooled in liquid N2 with 15% (v/v) 2R,3R-butanediol as a cryo-protectant.
Data Collection, Structure Determination and Refinement. Diffraction data from crystals cooled at 100 K were collected on beamlines BL 14.1 and 14.2 (BESSY, Berlin, Germany). [24] To position Fe and S atoms in the reduced and oxidized states and Xe atoms in the derivatized crystals, datasets were collected at the high-energy side of the K-absorption edge of Fe (λ = 1.74 Å), S (λ = 1.90 Å) and close to the LI-absorption edge of Xe (Xe-LI-edge: 2.27 Å, measured at λ = 1.90 Å) for the calculation of an anomalous difference Fourier map. Diffraction data were integrated and scaled using XDSAPP. [25] Initial phases were obtained from Patterson search techniques with AutoMR in Phenix [26] using the structure of CODH-IICh in the -320 mV state (PDB-ID: 3B53 [16] ) as a homologous search model. Several cycles of automated model building and refinement were carried out with AutoBuild in Phenix. [27] After iterative manual model building using Coot, [28] further refinements were performed using REFMAC5 [24] of the CCP4 suite [16] and phenix.refine [29] .
In the crystal batch used for Xe-derivatization, the length of the b-axis had doubled compared to the batch of crystals used for structure determination, resulting in four CooS-VCh molecules in the ASU. As the Br ions from the crystallization condition show weak anomalous scattering, not all anomalous scattering contributions derive from Xe atoms. 23 positions with anomalous scattering in the Xe-derivatized structure were modeled with Br ions, as the structure determined before Xe-derivatization showed Br ions at the same positions. In total, 64 Xe atoms were modeled according to their strong anomalous scattering contribution in the ASU ( Figure S7-A).
Data collection and refinement statistics of the reported structures are given in Table S1. Coordinates and structure factor amplitudes have been deposited in the Protein Data Bank under accession numbers 7B7Q.pdb (oxidized), 7B7T.pdb (reduced), 7B95.pdb (NH2OH-5 min), 7B97.pdb (NH2OH-30 min), and 7B9A.pdb (Xe-derivatized). 6 Table S1 continued. , Rfree factor was calculated from 5% of randomly selected data before refinement was carried out.

S5-oxd/Fe-peak S5-oxd/S-peak S5-red/Fe-peak S5-red/S-peak Data collection
c , Friedel pairs are treated as different reflection. asu, asymmetric unit Table S2. Structures similar to CooS-VCh, searched using DALI. [29] Only representative structures with Z-score higher than 20 with less than 3 Å of rmsd are selected in the  Table S3. Angles in the active site clusters of CooS-VCh. Average values from two molecules in asymmetric unit are used. Atomic numbers are same as in Figure   S6-C and -D. Figure S1. Phylogeny of CooS-type CODHs. Extended phylogeny of CooS-type CODHs, colored as in Figure 1. Taxa       The model is similarly oriented as C for comparison. Anomalous difference electron density for Xe is shown in the green box with 3.5 rmsd (blue mesh). Channel comparison of (B) HCP (pdb-ID:1OA1.pdb [31] ); (C) oxidized CooS-VCh and (D) CODH-IICh (pdb ID: 3B5B.pdb [32] ). All structures (B-C) are superposed and shown in the same orientation. Figure S8. Mirror tree cladogram for the history of Cys41 and Glu295. Selected part of the phylogeny shown in Figure S1 with character history for the sequence positions corresponding to Cys41 (characteristic for the presence of the [2Fe-2S]-cluster at cluster D) and Glu295 (exchange of cluster C coordinating Cys for Glu, characteristic for the binding motif of the hybrid-cluster). Colored balls indicate the type of amino acid present in the sequence (for terminal taxa) and most parsimonious amino acid (for ancestral nodes). Terminal nodes for sequences of sequence clusters I, II, III (cluster C coordinating motif) and IV were merged. CooS-VCh is indicated, other terminal taxa give UniProt Ids.