Stepwise conversion of the Cys6[4Fe–3S] to a Cys4[4Fe–4S] cluster and its impact on the oxygen tolerance of [NiFe]-hydrogenase

The membrane-bound [NiFe]-hydrogenase of Cupriavidus necator is a rare example of a truly O2-tolerant hydrogenase. It catalyzes the oxidation of H2 into 2e− and 2H+ in the presence of high O2 concentrations. This characteristic trait is intimately linked to the unique Cys6[4Fe–3S] cluster located in the proximal position to the catalytic center and coordinated by six cysteine residues. Two of these cysteines play an essential role in redox-dependent cluster plasticity, which bestows the cofactor with the capacity to mediate two redox transitions at physiological potentials. Here, we investigated the individual roles of the two additional cysteines by replacing them individually as well as simultaneously with glycine. The crystal structures of the corresponding MBH variants revealed the presence of Cys5[4Fe–4S] or Cys4[4Fe–4S] clusters of different architecture. The protein X-ray crystallography results were correlated with accompanying biochemical, spectroscopic and electrochemical data. The exchanges resulted in a diminished O2 tolerance of all MBH variants, which was attributed to the fact that the modified proximal clusters mediated only one redox transition. The previously proposed O2 protection mechanism that detoxifies O2 to H2O using four protons and four electrons supplied by the cofactor infrastructure, is extended by our results, which suggest efficient shutdown of enzyme function by formation of a hydroxy ligand in the active site that protects the enzyme from O2 binding under electron-deficient conditions.


Table of content
. CO         Samples of H 2 -reduced MBH variants were exposed to air, leading to the re-oxidation of the enzymes.
(a) EPR spectra of native MBH (left), MBH C19G (middle), and MBH C120G (right) in purified heterodimeric form (dashed lines) and membrane-bound (solid lines) form.The upper and lower spectra are derived from oxidized and air re-oxidized samples.All spectra were recorded at 20 K.The spectra of oxidized samples are normalized to the signal intensity of the respective solubilized sample.The EPR spectra of oxidized native MBH and MBH C19G exhibit a complex coupled signal in the region of g = 2.5-1.9, which is characteristic for the paramagnetic proximal cluster interacting with both the active site in  .Spectra acquisition was accomplished with an excitation wavelength of 458 nm from an Ar + laser of 1 mW power.All spectra were normalized to the phenylalanine mode at 1004 cm -1 .The spectra of all three oxidized MBH variants are dominated by a broad band around 340 cm -1 (green box) that is characteristic for oxidized iron-sulfur clusters. 3,4The various substructures of the proximal cluster of the MBH C120G variant (Fig. 2) might be the reason for slight shifts and variations of these Fe-S modes in the corresponding MBH C120G spectrum.The weak features in the range between 355 and 375 cm -1 are presumably related to metal-sulfur vibrations of the active site. 3,5Upon reduction, the intensity of the main 340 cm -1 band decreased considerably, which can be explained by a different electronic structure of the reduced cluster when compared to its oxidized counterpart.This, in turn, resulted in a diminished resonance enhancement. 4,6While the band(s) around 340 cm -1 vanish completely in case of native MBH and MBH C120G , the spectrum of reduced MBH C19G still exhibits residual intensity in this range.This is in line with the presence of an HIPIP-like [4Fe-4S] cluster species that undergoes a 3 + -to-2 + transition, as opposed to standard ([4Fe-4S]) clusters mediating a 2 + -to-1 + transition.All three proteins required an overpotential of approx.80 mV to oxidize H 2 , which is typical for O 2tolerant membrane-bound [NiFe]-hydrogenases, 7 but apparently unrelated to the presence of the [4Fe-3S] cluster. 8The H 2 oxidation current increased until a potential of approx.50 mV was reached and then decreased because of oxidative inactivation, which is supposed to correlate with anaerobic Ni r -B formation.The current increased again when the potential was poised back to negative potentials and reached a maximum (at approx.50 mV), (continued on the next page).
before it started to decrease again as a result of the declining driving force.No significant difference in the voltammograms was observed for the MBH variants.The voltammograms of all three proteins, however, showed remarkable discrepancies in the currents measured in the forward and the reverse scan at 0 V, indicating either film loss or irreversible enzyme inactivation in the course of the experiment.This made it essentially impossible to distinguish between specific effects based on the amino acid exchanges and general stability effects.Therefore, an improved immobilization strategy was used to minimize current loss ( 9).(b) A PGE was first coated with multi-walled carbon nanotubes (MWNT), which were subsequently treated with 1-pyrenebutyric acid (Py).Upon chemical activation, the carboxylic groups of Py formed covalent bonds to surface lysines of the MBH derivatives.This immobilization technique enhanced the protein film stability and the electronic contact dramatically.While protein films of native MBH and the MBH C19G protein showed similar cyclic voltammograms in the presence of H 2 under otherwise anoxic conditions, the MBH C120G variant displayed a somewhat stronger oxidative inactivation at high potentials.(c) The enhanced protein film stability allowed for investigation of the effect of short O 2 pulses on the activity.At a potential of 0 V, which does not support direct O 2 reduction by the electrode, O 2 -saturated buffer was added to a final concentration of 145 µM, and the O 2 was then rapidly removed by flushing with the initial gas mixture (80 % O 2 , 20 % N 2 ).Indeed, O 2 caused an immediate drop in activity (20 -30 %) for all three MBH variants (20% for C19G, 30 % for C120G).After flushing (finished after a few seconds), native MBH (clearly) and MBH C120G (slightly) gained back activity even on the oxidative sweep (up to approx.50 mV), while the activity of MBH C19G dropped continuously, until the potential was raised again on the return sweep.However, at 0 V, the activity of MBH C19G reached less than 50% of the initial current, indicating the formation of inactive species as a result of the incubation with O 2 .The same treatment led to a loss of only about 10% of the initial activity of MBH C120G , while the native MBH variant showed no activity loss on the return scan.The enzyme-covered gold-electrode was covered with 3 mL of electrolyte solution (50 mM potassium phosphate buffer, pH 5.5).The actual experiment was composed of four different phases (I-IV).Prior to phase I, the electrolyte was bubbled with pure H 2 gas to achieve full saturation with H 2 and to remove any residual oxygen.Phase I was initialized by pulling the needle out of the buffer solution (without stopping the gas flow) and adjusting the electrode potential to 0 mV vs. SHE.While maintaining the H 2 gas flow, phase II comprised the injection of three different volumes (5 mL, 15 mL, and 25 mL) of air at different time points into the electrolyte.Due to constant gassing with H 2 , which is much lighter than the constituents of air, the injected air could not escape from the cell, resulting in apparently constant O 2 concentrations in the electrolyte solution.In the beginning of phase III, the H 2 -releasing needle was re-inserted into the electrolyte solution to flush the remaining O 2 .Upon O 2 removal, phase IV was started by applying two low-potential pulses at -400 mV after which the potential was set back to 0 mV.Panel (b) displays the current traces representing the H 2 oxidation activities of native MBH (top, dashed line), MBH C19G (middle, solid line), and MBH C120G (bottom, dotted line) during the four phases of the experiment.Phase I lasted until the current reached a kind of plateau.The air injections in phase II, led to distinct current decreases.In case of native MBH and MBH C120G , the current reached a constant level after each air injection.This was less obvious for MBH C19G , which, however, also showed a stepwise drop in current.As the electrochemical SEIRA setup was different from that of the chronoamperometry shown in Fig. 4, O 2 concentrations and the resulting current traces of the MBH variants were not identical in the two experiments.For instance, the MBH C19G variant showed a slower inactivation in the SEIRA setup, which led to a stepwise current decrease, as also observed for native MBH and MBH C120G .Notably, just partial reactivation of the MBH C19G variant was obtained upon flushing the O 2 in phase III, and the H 2 oxidation current increased by applying low-potential pulses in phase IV.This behavior was observed in both electrochemical setups, and it differed from native MBH and MBH C120G , whose currents were recovered just by removing O 2 .Low-potential pulses led to an only negligible further current increase for MBH C120G and no current increase for native MBH.Panel (c) shows the second derivatives of the SEIRA spectra in the region characteristic for the CO absorptions.The spectra were recorded at the end of each phase, marked by a black circle in panel (b).The spectra recorded in-situ during catalysis under pure H 2 (phase I) were in line with the corresponding IR results for the solubilized MBH variants (Fig. 3a).

Figure S1 .
Figure S1.Proximal Fe-S clusters of oxygen-tolerant and oxygen-sensitive [NiFe]-hydrogenases.The two proximal clusters of the oxygen-tolerant MBH from Cupriavidus necator (left) and the oxygensensitive DvMBH from Desulfovibrio vulgaris (right) are shown in ball/stick representation.Iron ions and sulfur atoms are presented as spheres in orange and yellow, respectively.The coordinating cysteines are depicted as sticks in green and blue for MBH and DvMBH, respectively, with the two additional cluster coordinating cysteine Cys19 and Cys120 of MBH in purple.The thiol group of Cys19 occupies the former position of the fourth sulfur atom in the cube-shaped [4Fe-4S] cluster, leading to the formation of the [4Fe-3S] cluster.The Cys120 stabilizes the open, trapezoidal shape of the cluster.

Figure S2 .
Figure S2.Proximal Cys 5 [4Fe-4S] cluster structures in reduced and oxidized MBH C19G .Panel (a) shows the structural model of the Cys 5 [4Fe-4S] cluster of MBH C19G in the oxidized state under H 2 -reducing conditions and panel (b) that of the super-oxidized state under oxidizing conditions.Panel (c) shows the 2mFo-DFc electron density map (blue mesh contoured at 1.2 σ) of MBH C19G in the oxidized state showing Fe3 in a double conformation (Fe3 and Fe3` position).Panel (d) shows the corresponding electron density map of MBH C19G in the super-oxidized state.Note, that Fe4 is not shifted toward the backbone nitrogen of Cys20.A fraction of Fe3 forms a covalent bond with Cys120, as shown in panel (e).Color codes of the ball and stick models: green, backbone carbons; blue, nitrogen; red, oxygen; yellow, sulfur; orange, iron.

Figure S3 .
Figure S3.Proximal Cys 5 [4Fe-4S] cluster structures in reduced and oxidized MBH C120G .Panel (a) shows the structural model of the Cys 5 [4Fe-4S] cluster of MBH C120G in the reduced state and panel (b) that of the oxidized state.Panel (c) shows the 2mFo-DFc electron density map (blue mesh, contoured at 1.0 σ) of reduced MBH C120G , showing Fe3 in a double conformation (positions Fe3 and Fe3`).A covalent bond was observed between Fe3 and an exo-sulfur.Panel (d) shows the result of the single-wavelength anomalous dispersion (SAD) experiment (purple meshes, contoured at 3.0 ), identifying a sulfur species at the former thiol group position of Cys120.Panel (e) shows that Fe4 (ca.50 %) of the oxidized cluster undergoes a redox-dependent shifting process and forms a bond with the backbone nitrogen of Cys20.Color codes of the ball and stick models: green, backbone carbons; blue, nitrogen; red, oxygen; yellow, sulfur; orange, iron.

Figure S4 .
Figure S4.Proximal Cys 4 [4Fe-4S] cluster structures in reduced and oxidized MBH C19G/C120G .Panels (a) and (b) display the structural models of the Cys 4 [4Fe-4S] cluster in the reduced and oxidized MBH C19G/C120G , respectively.Panel (c) shows the 2mFo-DFc electron density map (blue mesh, contoured at 1.0  level) of the reduced cluster, indicating that the former thiolate of Cys19 is replaced with sulfide S4.Panel (d) shows the result of an SAD experiment (purple meshes, contoured at 3.0 ), revealing a water molecule at the position of the former Cys120-derived thiolate.Panel (e) displays the 2mFo-DFc electron density map (blue mesh, contoured at 1.0  level) of the oxidized cluster, showing flexibilities of the Fe3, S1 and S3 positions.

Figure S6 .
Figure S6.Solvent accessibility of the proximal Fe-S cluster in the MBH variants.The solvent accessibility was calculated with the program CAVER 3.0, 1 and the proximal cluster was used as the initial starting point.The substitution of Cys120 with glycine allows water molecules (purple and blue spheres) to reach the proximal cluster via a tunnel that connects the protein surface with the cluster.The diameters of the corresponding tunnel bottlenecks in the reduced/oxidized structures of MBH C19G/C120G and MBH C120G were calculated with 1.24/1.22Å and 1.25/1.30Å, respectively.In case of MBH C19G and native MBH, the proximal clusters cannot be accessed by water because of the bulky Cys120.The cluster atoms and the coordinating amino acids are displayed in ball and stick representation.The protein backbone is shown in cartoon representation.Cavities are illustrated with PyMOL.2

Figure S7 .
Figure S7.[NiFe]-active site structures of reduced and oxidized MBH variants.The [NiFe] active site is shown as ball/stick representation with the nickel (Ni), iron (Fe), sulfur (S), carbon (C), oxygen (O)and nitrogen (N) as green, orange, yellow, white, red and blue spheres, respectively.Electron densities are represented as blue (2mFo-DFc) or green (mFo-DFc) meshes, which were contoured at the 1.0  and 3.0  level, respectively.Additional positive mFo-DFc difference density was found in the reduced MBH C19G/C120G and MBH C19G variants and interpreted as oxygen species.The corresponding occupancies were below 10 % and 40 -70 % in case of MBH C19G/C120G and MBH C19G , respectively.The Ni-Fe distances were generally 2.6 and 2.9 Å for the reduced and oxidized states, respectively.Only in case of reduced MBH C19G , the metal-metal distance was slightly larger (2.7 Å).

Figure S8 .Figure S9 .
Figure S8.EPR spectra of oxidized native MBH, MBH C19G , and MBH C120G measured at 80 K.The spectra of (a), native MBH (b), MBH C19G , and (c), MBH C120G are dominated by the rhombic signal characteristic for the Ni r -B state (orange, g x = 2.30, g y = 2.17, g z = 2.01).Traces of additional signals (marked by #) represent so far unknown active site species.In MBH C120G traces of Ni u -A (grey, g x = 2.30, g y = 2.23, g z = 2.01) and another species (denoted with +) were observed.

Figure S10 .
Figure S10.Resonance Raman (RR) spectra of single crystals of oxidized and reduced native MBH, MBH C19G and MBH C120G .Spectra acquisition was accomplished with an excitation wavelength of 458 nm from an Ar + laser of 1 mW power.All spectra were normalized to the phenylalanine mode at 1004 cm -1 .The spectra of all three oxidized MBH variants are dominated by a broad band around 340 cm -1 (green box) that is characteristic for oxidized iron-sulfur clusters.3,4The various substructures of the proximal cluster of the MBH C120G variant (Fig.2) might be the reason for slight shifts and variations of these Fe-S modes in the corresponding MBH C120G spectrum.The weak features in the range between 355 and 375 cm -1 are presumably related to metal-sulfur vibrations of the active site.3,5Upon reduction, the intensity of the main 340 cm -1 band decreased considerably, which can be explained by a different electronic structure of the reduced cluster when compared to its oxidized counterpart.This, in turn, resulted in a diminished resonance enhancement.4,6While the band(s) around 340 cm -1 vanish completely in case of native MBH and MBH C120G , the spectrum of reduced MBH C19G still exhibits residual intensity in this range.This is in line with the presence of an HIPIP-like [4Fe-4S] cluster species that undergoes a 3 + -to-2 + transition, as opposed to standard ([4Fe-4S]) clusters mediating a 2 + -to-1 + transition.

Figure S11 .
Figure S11.Temperature and microwave power dependence of the EPR signals of H 2 -reduced MBHC120G  .With increasing temperature, the sharp signal at g = 1.979, marked by a purple arrow, decreased more strongly than the broad species at g = 2.015 (orange arrow) at a fixed microwave power of 1 mW.The two signals also exhibit a different signal (normalized to the maximum intensity) saturation with varying microwave power at a fixed temperature of 6 K (inset).This suggests the presence of at least two different electronic/structural variants of the reduced Fe-S cluster with different relaxation behaviors, which is consistent with the crystal structure data (Fig.2c).

Figure S12 .
Figure S12.Cyclic voltammograms for native MBH, MBHC19G  and MBH C120G immobilized on bare and coated PGE electrodes.(a) The purified proteins were immobilized onto pyrolytic graphite electrodes (PGE), and the electrode potential was cycled at 0.5 mVs -1 between -0.4 and 0.25 V (all potentials are given versus the standard hydrogen electrode) in the presence of 606 µM H 2 (80 %).All three proteins required an overpotential of approx.80 mV to oxidize H 2 , which is typical for O 2tolerant membrane-bound [NiFe]-hydrogenases,7 but apparently unrelated to the presence of the [4Fe-3S] cluster.8The H 2 oxidation current increased until a potential of approx.50 mV was reached and then decreased because of oxidative inactivation, which is supposed to correlate with anaerobic Ni r -B formation.The current increased again when the potential was poised back to negative potentials and reached a maximum (at approx.50 mV), (continued on the next page).

Figure S13 .
Figure S13.Analysis of the MBH variants by chronoamperometry coupled with surface-enhanced infrared absorption (SEIRA) spectroscopy.Panel (a): The MBH variants were immobilized onto SAMmodified, SEIRA-compatible gold electrodes chemically deposited on top of a silicon prism (continued on the next page).

Figure S14 .
Figure S14.SEIRA spectra of the MBH variants taken before and after chronoamperometry.The spectra were recorded prior to phase I (solid lines), and after phase IV (dotted line) of the experiment described in Fig.S13.The intensity and shape of the amide I (1665 cm -1 ) and amide II (1550 cm -1 ) bands did not change significantly, indicating conformational stability of the protein on the SAM-modified gold electrode.Protein desorption was not observed.The lower overall band intensities for MBH C120G compared to native MBH and MBH C19G indicate poor immobilization, consistent with the lower current observed during chronoamperometry (Fig.S13).

Table S1 :
H 2 oxidation activity and protein yield of the purified MBH variants.

Table S2 .
Data collection and refinement statistics of MBH cluster variants.

Table
Ni r -B state the medial [3Fe-4S] + cluster.Upon reduction and subsequent re-oxidation with air, the complex EPR spectrum remained almost unchanged for the membranebound MBH samples, indicating full reversibility, when the MBH is still connected with its redox partners in the cytoplasmic membrane.Re-oxidation of the purified MBH samples, however, led to a considerable simplification of the spectra, which exhibited mainly uncoupled Ni r -B (g x = 2.30, g y = 2.17, g z = 2.01) and medial [3Fe-4S] + cluster (g┴ = 2.017; g║ = 2.001) signals.The EPR spectra of oxidized and re-oxidized purified MBH C120G as well as oxidized membrane-bound MBH C120G exhibit a very similar spectroscopic signature, comprising signals of uncoupled Ni r -B and the medial [3Fe-4S] + cluster.The low content of MBH C120G in the cytoplasmic membrane prevented its analysis after reoxidation.
(b) IR spectra of re-oxidized purified native MBH (top), MBH C19G (middle) and MBH C120G (bottom).Re-oxidation of purified native MBH and MBH C19G samples resulted in the recovery of mainly the Ni r -B state (~80%), with a characteristic CO stretching vibration at 1948 cm -1 .In addition, a second species (~20%), with a CO absorption band at 1930 cm -1 was observed, which is indicative for the irreversibly inactive Ni ia -S state of the active site.In case of the MBH C120G variant, the signal intensity of the Ni ia -S-related CO band was almost as large as that of the Ni r -B species.
While native MBH exhibited a mixture of mainly Ni a -C (1957 cm -1 , purple bar) and Ni a -SR' (1926 cm-1, red bar, with minor contributions of Ni a -SR'' at 1919 cm -1 ), the spectra of MBH C19G and MBH C120G were dominated mostly by Ni a -SR'.Notably, all MBH variants also showed significant contributions of the Ni ia -S state (1930 cm -1 , orange bar) already after phase I.In case of native MBH and MBH C19G , this inactive species increased slowly in the course of the experiment (phases I-IV).In the MBH C120G variant, however, it was already dominant in phase I, further supporting the inherent instability of this protein (continued on the next page)Treatment of the MBH variants with H 2 /O 2 mixtures in phase II, led to a decrease of bands related to catalytic intermediates.The decreasing amount of Ni a -SR' species led to a concomitant shift of the band at 1926 cm -1 to 1930 cm -1 , which is characteristic for the Ni ia -S species.Furthermore, an absorption band at 1948 cm -1 (blue bar) gained in intensity, indicating the formation of Ni r -B.In conclusion, our results suggest the presence of two inactivation pathways.Exposure of all three MBH variants to O 2 (phase II) resulted in a current decrease, which correlated with the formation of the Ni r -B state.In case of MBH C19G , removal of O 2 from the solution was not sufficient to lower the amount of Ni r -B state (indicated by residual intensity of the corresponding IR band after phase III), i.e. the enzyme stayed catalytically inactive.Partial recovery of catalytic current and the corresponding decrease of Ni r -B was only accomplished by applying low-potential pulses.Hence, the apparent O 2 intolerance ascribed to the MBH C19G variant seems to result from a kinetically hindered reactivation of enzyme species residing in the Ni r -B state.The second inactivation process relates to the formation of the Ni ia -S species, which continuously increased during the entire procedure for all three variants (even under reductive conditions as indicated by the blue shift of the peak maximum from phase I to III or IV towards 1930 cm -1 ).

Table S1 H
2 oxidation activity and protein yield of the purified MBH variants.

Table S2
Data collection and refinement statistics of MBH cluster variants.

Table S2 (
continued) Data collection and refinement statistics of MBH cluster variants.