Spectroscopical Investigations on the Redox Chemistry of [FeFe]-Hydrogenases in the Presence of Carbon Monoxide

[FeFe]-hydrogenases efficiently catalyzes hydrogen conversion at a unique [4Fe–4S]-[FeFe] cofactor, the so-called H-cluster. The catalytic reaction occurs at the diiron site, while the [4Fe–4S] cluster functions as a redox shuttle. In the oxidized resting state (Hox), the iron ions of the diiron site bind one cyanide (CN−) and carbon monoxide (CO) ligand each and a third carbonyl can be found in the Fe–Fe bridging position (µCO). In the presence of exogenous CO, A fourth CO ligand binds at the diiron site to form the oxidized, CO-inhibited H-cluster (Hox-CO). We investigated the reduced, CO-inhibited H-cluster (Hred´-CO) in this work. The stretching vibrations of the diatomic ligands were monitored by attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR). Density functional theory (DFT) at the TPSSh/TZVP level was employed to analyze the cofactor geometry, as well as the redox and protonation state of the H-cluster. Selective 13CO isotope editing, spectro-electrochemistry, and correlation analysis of IR data identified a one-electron reduced, protonated [4Fe–4S] cluster and an apical CN− ligand at the diiron site in Hred´-CO. The reduced, CO-inhibited H-cluster forms independently of the sequence of CO binding and cofactor reduction, which implies that the ligand rearrangement at the diiron site upon CO inhibition is independent of the redox and protonation state of the [4Fe–4S] cluster. The relation of coordination dynamics to cofactor redox and protonation changes in hydrogen conversion catalysis and inhibition is discussed.


Introduction
Hydrogenases [1] are remarkably efficient catalysts for hydrogen conversion with significant potential in renewable energy applications [2][3][4].The chemistry at the transition metal cofactor is based on a sophisticated interplay between redox and protonation changes, as well as protein-cofactor interactions [5][6][7] and is of prime interest for the design of biomimetic, synthetic hydrogen conversion catalysts [8][9][10].Accordingly, hydrogenase proteins have been extensively characterized by X-ray crystallography [5], protein film electrochemistry [11][12][13], and numerous spectroscopic techniques [14].However, the relations between coordination dynamics at the active site and redox chemistry are still under debate [15].
[FeFe]-hydrogenases are found in archaea, bacteria, and unicellular algae [16].They show truly bidirectional hydrogen conversion (i.e., catalysis of H2 oxidation and proton reduction at similar rates) [17].In the cell, [FeFe]-hydrogenases typically serve as "electron valves" that prevent the accumulation of excess reducing equivalents by the release of H2, e.g., in photosynthetic algae such as Chlamydomonas reinhardtii [18].Their unique catalytic cofactor, the so-called H-cluster, comprises a canonical [4Fe-4S] cluster linked by a cysteine thiolate to a diiron complex, [FeFe] [19,20].The pendant amine base of an aminodithiolate group (adt-NH) serves as proton relay between the diiron site and the adjacent amino acid residues [21][22][23].In the active-ready, oxidized state (Hox), the [FeFe] site binds two terminal carbon monoxide (CO) and cyanide (CN -) ligands and a single carbonyl (µCO) in the Fe-Fe bridging position (Figure 1).The natural presence of CO and CN -ligands at the active site of [FeFe]-hydrogenases facilitates cofactor-specific investigation by infrared spectroscopy [24][25][26][27].The structure ( protein data bank entry 4XDC) [28] of the Clostridium pasteurianum enzyme (CPI) is poised in the oxidized resting state (Hox).The CN -ligand at Fep is stabilized by a hydrogen bond (blue dashes) to the protein backbone [29], whereas the diatomic ligands at Fed are located in a hydrophobic pocket.The aminodithiolate (adt) group may be hydrogen-bonded to C299 (blue dashes).The asterisk marks a likely protonation site at the [4Fe-4S] cluster.Catalytic protons are exchanged via adt-NH and C299 in the putative proton transfer pathway to the diiron site.The circle marks the open coordination site at Fed where exogenous CO initially binds to form the CO-inhibited, oxidized state (Hox-CO).(B) Proposed structure of the H-cluster in the Hox-CO state (the [4Fe-4S] cluster is omitted for clarity).The orientation of the diatomic ligands in various cofactor states is under debate.
Protein crystallography on oxidized [FeFe]-hydrogenases has shown that the diiron site adopts an unusual inverted-pyramid geometry with a µCO ligand and an open coordination site at Fed (Figure 1A) [19,20].In Hox, the H-cluster has been assigned to a formal [4Fe-4S] 2+ -[FeFe I,II ] redox configuration [30].Exogenous CO or O2 compete with H2 at the open coordination site of the oxidized cofactor [31].However, while the reaction with O2 finally causes cofactor degradation, CO binding at the H-cluster in Hox induces the reversible formation of the CO-inhibited, oxidized state (Hox-CO) (Figure 1B) [31][32][33].Little is known about CO binding to the reduced cofactor, which is the central topic of the present study.
The [FeFe]-hydrogenase from C. reinhardtii (HYDA1) is particularly well-suited for spectroscopic studies, because it exclusively binds the H-cluster and no further iron-sulfur clusters [34].In recent work, we have extensively characterized HYDA1 protein films by attenuated total reflection Fourier- Protein crystallography on oxidized [FeFe]-hydrogenases has shown that the diiron site adopts an unusual inverted-pyramid geometry with a µCO ligand and an open coordination site at Fe d (Figure 1A) [19,20].In Hox, the H-cluster has been assigned to a formal [4Fe-4S] 2+ -[FeFe I,II ] redox configuration [30].Exogenous CO or O 2 compete with H 2 at the open coordination site of the oxidized cofactor [31].However, while the reaction with O 2 finally causes cofactor degradation, CO binding at the H-cluster in Hox induces the reversible formation of the CO-inhibited, oxidized state (Hox-CO) (Figure 1B) [31][32][33].Little is known about CO binding to the reduced cofactor, which is the central topic of the present study.

H-cluster
The [FeFe]-hydrogenase from C. reinhardtii (HYDA1) is particularly well-suited for spectroscopic studies, because it exclusively binds the H-cluster and no further iron-sulfur clusters [34].
In recent work, we have extensively characterized HYDA1 protein films by attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR) in combination with gas exposure, spectro-electrochemistry, and 13 CO isotope editing [35][36][37][38][39][40].The cofactor geometries and isotope editing patterns, as well as the redox and protonation states, were assigned by quantum chemical calculations (density functional theory, DFT).Previous work suggests that rearrangement of the ligands at [FeFe] relative to Hox is involved in the formation of Hox-CO and the reduced H-cluster species Hred and Hsred [35,36].Furthermore, protonation at the [4Fe-4S] cluster in a one-electron reduced species with a µCO ligand (Hred´) was implied [38,39].These results tempted an assignment of H-cluster intermediates with a µCO to the catalytic cycle of H 2 conversion and of species lacking a µCO to regulatory reactions [41].The apparent mobility of the ligands at the distal iron seems to be crucial for H 2 sensing and CO inhibition.However, whether CO binding affects the ligand geometry in the reduced H-cluster has not been addressed.
In this study, 13 CO isotope editing and ATR FTIR spectro-electrochemistry on HYDA1 protein films was employed to poise the H-cluster in the CO-inhibited, oxidized state (Hox-CO) or one-electron reduced state (Hred´-CO).DFT facilitated an assignment of the experimental IR band patterns to underlying cofactor geometries.In comparison with Hox, our analysis suggests that Hred´-CO and Hox-CO show similar ligand reorientation at Fe d with an apical CN -ligand rather than an apical CO ligand.The experimental and computational results indicate that Hred´-CO comprises a reduced and protonated [4Fe-4S] cluster, similar to its non-inhibited counterpart, Hred´ [39].These findings imply that ligand rearrangement upon CO-inhibition is independent of the redox and protonation state of the [4Fe-4S] cluster.The importance of proton-coupled electron transfer to the [4Fe-4S] cluster for stabilization of the µCO geometry is emphasized.

ATR-FTIR Spectro-Electrochemistry and 13 CO Isotope Editing
Purified [FeFe]-hydrogenase HYDA1 apo-protein was maturated in vitro with a synthetic diiron complex (Fe 2 (µ-adt)(CO) 4 (CN) 2 , adt = (SCH 2 ) 2 NH) [42,43].The catalytically competent enzyme was injected onto a thin gold mesh that was used to cover the silicon crystal of an ATR cell.This set-up facilitates both in situ FTIR spectro-electrochemistry and monitoring changes in the protein film upon exposure to varying atmospheres in the sample headspace, in particular 13 CO isotope editing (see below).Increasingly reducing potentials (−100 to −800 mV vs. normal hydrogen electrode, NHE) were applied to CO-inhibited films of HYDA1 protein (Figure 2).In agreement with earlier studies, the reduction of the CO-inhibited cofactor was identified by a CO/CN -band pattern similar to Hox-CO but shifted by 5-10 cm −1 to lower frequencies [22].Reminiscent of the spectral differences between Hox and Hred´ [39], the relatively small frequency shifts suggested that a one-electron reduced state with a similar cofactor geometry as in Hox-CO was formed, including a low-frequency µCO band at 1793 cm −1 .In the following, we will refer to this state as Hred´-CO.Previous studies have revealed that the summed IR band intensities of the oxidized and reduced H-cluster species can be considered constant [38,39].Accordingly, the overall band intensity of each cofactor species corresponds to its fractional population in the sample.Determination of the population of states thus facilitated an assignment of the redox midpoint potential (E m ) for the Hox-CO → Hred´-CO transition (Figure 3).Using the Nernst equation, this approach yielded E m values of −365 ± 10 mV and −53 0 ± 30 mV versus NHE at pH 5 and pH 8, respectively (∆E m = 165 ± 30 mV).
Figure 2. Formation of the one-electron reduced state, CO-inhinbited state Hred´-CO at pH 5 and pH 8.The spectra show the gradual population of Hred´-CO (red band frequency labels) at the expense of Hox-CO (black band frequency labels) for a step-wise decrease of redox potential at pH 5 (left panel) or pH 8 (right panel).Spectra were normalized to uniform integral band intensity.Minor bands are due to small reduced H-cluster species Hred and Hsred populations (e.g., at 1891 cm −1 and 1882 cm −1 ) and unrelated to the CO-inhibited states.Prior to isotope editing, the self-oxidation activity of HYDA1 was employed to accumulate Hox in the presence of N2.Thereafter, exposure to 12 CO or 13 CO gas in the dark or in combination with white light illumination resulted in the selective enrichment of Hox-CO isotopomers 1-4 with different 13 CO labeling patterns (Figure 4), as reported previously [35].The determined IR frequencies and intensities of the CO/CN -ligands at the H-cluster are listed in Table 1 and Table S1.The spectra show the gradual population of Hred´-CO (red band frequency labels) at the expense of Hox-CO (black band frequency labels) for a step-wise decrease of redox potential at pH 5 (left panel) or pH 8 (right panel).Spectra were normalized to uniform integral band intensity.Minor bands are due to small reduced H-cluster species Hred and Hsred populations (e.g., at 1891 cm −1 and 1882 cm −1 ) and unrelated to the CO-inhibited states.

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Figure 2. Formation of the one-electron reduced state, CO-inhinbited state Hred´-CO at pH 5 and pH 8.The spectra show the gradual population of Hred´-CO (red band frequency labels) at the expense of Hox-CO (black band frequency labels) for a step-wise decrease of redox potential at pH 5 (left panel) or pH 8 (right panel).Spectra were normalized to uniform integral band intensity.Minor bands are due to small reduced H-cluster species Hred and Hsred populations (e.g., at 1891 cm −1 and 1882 cm −1 ) and unrelated to the CO-inhibited states.Prior to isotope editing, the self-oxidation activity of HYDA1 was employed to accumulate Hox in the presence of N2.Thereafter, exposure to 12 CO or 13 CO gas in the dark or in combination with white light illumination resulted in the selective enrichment of Hox-CO isotopomers 1-4 with different 13 CO labeling patterns (Figure 4), as reported previously [35].The determined IR frequencies and intensities of the CO/CN -ligands at the H-cluster are listed in Table 1 and Table S1.Prior to isotope editing, the self-oxidation activity of HYDA1 was employed to accumulate Hox in the presence of N 2 .Thereafter, exposure to 12 CO or 13 CO gas in the dark or in combination with white light illumination resulted in the selective enrichment of Hox-CO isotopomers 1-4 with different 13 CO labeling patterns (Figure 4), as reported previously [35].The determined IR frequencies and intensities of the CO/CN -ligands at the H-cluster are listed in Table 1 and Table S1.  1CO labeling patterns (right panel, 13 CO in red).Hox-CO species 1 is formed upon exposure of the oxidized enzyme (Hox) to 12 CO gas in the dark.The exposure of isotopomer 1 to 13 CO gas in the dark yields species 2, further illumination with white light under 13 CO results in isotopomer 3. The exposure of isotopomer 3 to 12 CO in the dark results in the formation of isotopomer 4. Note that the frequencies of the CN -ligands are barely affected by 13 CO isotope editing, while the Hox → Hox-CO conversion is associated with difference signals in the CN -regime as well (2050-2100 cm −1 ).
Potential jump experiments were applied to convert Hox-CO 13 CO isotopomers 1-4 into the corresponding Hred´-CO species.The resulting in situ difference spectra are shown in Figure 5.For all isotopomers, the Hox-CO → Hred´-CO transition was accompanied by downshifts of CO/CN - band frequencies of about 6-15 cm −1 .Interestingly, the symmetrical stretching vibrations of the distal CO ligands (d1 and d2) in most isotopomers showed changes in band intensity besides frequency shifts.Minor populations of non-inhibited reduced states (i.e., Hred and Hsred) were subtracted to gain pure spectra of Hox-CO and Hred´-CO (Figure 6).The IR frequencies and intensities of the four Hred´-CO isotopomers are compiled in Table 1 and Table S1.   CO labeling patterns (right panel, 13 CO in red).Hox-CO species 1 is formed upon exposure of the oxidized enzyme (Hox) to 12 CO gas in the dark.The exposure of isotopomer 1 to 13 CO gas in the dark yields species 2, further illumination with white light under 13 CO results in isotopomer 3. The exposure of isotopomer 3 to 12 CO in the dark results in the formation of isotopomer 4. Note that the frequencies of the CN -ligands are barely affected by 13 CO isotope editing, while the Hox → Hox-CO conversion is associated with difference signals in the CN - regime as well (2050-2100 cm −1 ).
Potential jump experiments were applied to convert Hox-CO 13 CO isotopomers 1-4 into the corresponding Hred´-CO species.The resulting in situ difference spectra are shown in Figure 5.For all isotopomers, the Hox-CO → Hred´-CO transition was accompanied by downshifts of CO/CN -band frequencies of about 6-15 cm −1 .Interestingly, the symmetrical stretching vibrations of the distal CO ligands (d 1 and d 2 ) in most isotopomers showed changes in band intensity besides frequency shifts.Minor populations of non-inhibited reduced states (i.e., Hred and Hsred) were subtracted to gain pure spectra of Hox-CO and Hred´-CO (Figure 6).The IR frequencies and intensities of the four Hred´-CO isotopomers are compiled in Table 1 and Table S1.Pure IR spectra of Hred´-CO isotopomers 1-4.Spectro-electrochemical experiments yielded pure IR spectra for Hox-CO (dotted lines) and Hred´-CO (solid lines) after the subtraction of the minor IR contributions of other H-cluster species, such as Hred and Hsred, and the other isotopomers of Hox-CO/Hred´-CO (compare Figure 5).The CO/CN − stretching frequencies for Hred´-CO are annotated.

CO binding in the Presence of H2
Our results clearly indicate the enrichment of Hred´-CO in films where HYDA1 was first inhibited by exogenous CO and reduced afterwards.In order to address the question whether CO binding to the reduced H-cluster results in alternative CO-inhibited species, HYDA1 was first reduced with H2 (open circuit potential) and thereafter exposed to a CO atmosphere (1% CO in 99% H2).This approach resulted in the concomitant increase of Hox-CO and Hred´-CO in the film, but further CO-inhibited species were not observed (Figure 7).The complete loss of Hred´ within less than 10 s after addition of CO resembles the complete loss of Hox under N2.However, small fractions of Hred and Hsred remained detectable even after several minutes of CO exposure.This hints at differences in CO sensitivity between Hred´ and Hred/Hsred.(3) (4) Figure 6.Pure IR spectra of Hred´-CO isotopomers 1-4.Spectro-electrochemical experiments yielded pure IR spectra for Hox-CO (dotted lines) and Hred´-CO (solid lines) after the subtraction of the minor IR contributions of other H-cluster species, such as Hred and Hsred, and the other isotopomers of Hox-CO/Hred´-CO (compare Figure 5).The CO/CN − stretching frequencies for Hred´-CO are annotated.

CO binding in the Presence of H 2
Our results clearly indicate the enrichment of Hred´-CO in films where HYDA1 was first inhibited by exogenous CO and reduced afterwards.In order to address the question whether CO binding to the reduced H-cluster results in alternative CO-inhibited species, HYDA1 was first reduced with H 2 (open circuit potential) and thereafter exposed to a CO atmosphere (1% CO in 99% H 2 ).This approach resulted in the concomitant increase of Hox-CO and Hred´-CO in the film, but further CO-inhibited species were not observed (Figure 7).The complete loss of Hred´within less than 10 s after addition of CO resembles the complete loss of Hox under N 2 .However, small fractions of Hred and Hsred remained detectable even after several minutes of CO exposure.This hints at differences in CO sensitivity between Hred´and Hred/Hsred.7 of 13 Figure 7. CO inhibition of pre-reduced HYDA1.Kinetic traces in (A) and corresponding FTIR spectra in (B).[FeFe]-hydrogenase was first exposed to 100% H2 to accumulate the reduced species Hred, Hsred, and Hred´.No external potential was applied.Upon injection of 1% CO into the gas stream, the H-cluster rapidly forms about equal populations of Hox-CO and Hred´-CO.While Hred´ is lost within ~10 s, Hred and Hsred decrease to reach a stable population of around 5% after ~1 min.* The high-frequency band at 2026 cm −1 has been to assigned to Hsred and is unrelated to the CO-inhibited species.

Assignment of H-Cluster Species by Density Functional Theory
DFT was employed to generate geometry-optimized H-cluster model structures and to calculate CO/CN -vibrational frequencies by normal mode analysis, as previously reported (whole cofactor structures, TPSSh/TZVP functional/basis-set combination) [35].Correlation analysis of experimental and calculated IR frequencies and intensities was carried out (Equations ( 1)-( 3)).This procedure revealed that the IR spectra of Hox-CO and Hred´-CO for the four 13 CO isotopomers were much better described by H-cluster structures with an apical cyanide ligand (aCN) instead of an apical carbonyl ligand (aCO) at Fed (Figure 8, Figures S1 and S2; Tables S1-S4).Linear regressions of calculated versus experimental data consistently revealed about two-fold smaller errors of offset and slope parameters and a larger R 2 value, which indicates a significantly better fit quality for the aCN structures.The root-mean-square deviation (rmsd) for the calculated IR frequencies (corrected for systematic theory-inherent deviations) for all 13 CO patterns with an aCN ligand was about three-fold smaller (mean of ~7 cm −1 vs. ~20 cm −1 ).For the band intensities, about two-fold smaller rmsd values were observed for the aCN structures (mean of ~7% vs. ~13%).The computational results thus facilitated a unique assignment of the 13 CO labeling patterns in the four isotopomers of Hox-CO and Hred´-CO (Table 1).We have previously assigned protonation at a sulfur atom of a cysteine ligand (C417 in HYDA1) of the [4Fe-4S] cluster in Hred´ [39].A comparison of structures with or without such a proton at the [4Fe-4S] cluster resulted in a significantly improved match between the calculated and experimental IR data for Hred´-CO.The calculation of apparent relative probabilities of H-cluster structures from the R 2 and rmsd values from the IR data correlations further supported an apical CN − in Hox-CO and Hred´-CO and a surplus proton at the [4Fe-4S] cluster in Hred´-CO (Figure 8).[FeFe]-hydrogenase was first exposed to 100% H 2 to accumulate the reduced species Hred, Hsred, and Hred´.No external potential was applied.Upon injection of 1% CO into the gas stream, the H-cluster rapidly forms about equal populations of Hox-CO and Hred´-CO.While Hred´is lost within ~10 s, Hred and Hsred decrease to reach a stable population of around 5% after ~1 min.* The high-frequency band at 2026 cm −1 has been to assigned to Hsred and is unrelated to the CO-inhibited species.

Assignment of H-Cluster Species by Density Functional Theory
DFT was employed to generate geometry-optimized H-cluster model structures and to calculate CO/CN -vibrational frequencies by normal mode analysis, as previously reported (whole cofactor structures, TPSSh/TZVP functional/basis-set combination) [35].Correlation analysis of experimental and calculated IR frequencies and intensities was carried out (Equations ( 1)-( 3)).This procedure revealed that the IR spectra of Hox-CO and Hred´-CO for the four 13 CO isotopomers were much better described by H-cluster structures with an apical cyanide ligand (aCN) instead of an apical carbonyl ligand (aCO) at Fe d (Figure 8, Figures S1 and S2; Tables S1-S4).Linear regressions of calculated versus experimental data consistently revealed about two-fold smaller errors of offset and slope parameters and a larger R 2 value, which indicates a significantly better fit quality for the aCN structures.The root-mean-square deviation (rmsd) for the calculated IR frequencies (corrected for systematic theory-inherent deviations) for all 13 CO patterns with an aCN ligand was about three-fold smaller (mean of ~7 cm −1 vs. ~20 cm −1 ).For the band intensities, about two-fold smaller rmsd values were observed for the aCN structures (mean of ~7% vs. ~13%).The computational results thus facilitated a unique assignment of the 13 CO labeling patterns in the four isotopomers of Hox-CO and Hred´-CO (Table 1).We have previously assigned protonation at a sulfur atom of a cysteine ligand (C417 in HYDA1) of the [4Fe-4S] cluster in Hred´ [39].A comparison of structures with or without such a proton at the [4Fe-4S] cluster resulted in a significantly improved match between the calculated and experimental IR data for Hred´-CO.The calculation of apparent relative probabilities of H-cluster structures from the R 2 and rmsd values from the IR data correlations further supported an apical CN − in Hox-CO and Hred´-CO and a surplus proton at the [4Fe-4S] cluster in Hred´-CO (Figure 8).8 of 13 Figure 8. Correlation analysis for Hox-CO and Hred´-CO.The calculated IR frequencies stem from density functional theory (DFT) calculations (corrected for systematic deviations; Equations ( 1) and (2); Figures S1 and S2, Tables S1-S3).H-cluster structures: apical CO at Fed (aCO, open symbols), apical CN -at Fed (aCN, solid symbols).The lines show the diagonal for ideal correlation ((A), Hox-CO: mean rmsd of 20 cm −1 for aCO or 8 cm −1 for aCN.(B), Hred´-CO (including a proton at the [4Fe-4S] cluster): mean rmsd of 19 cm −1 for aCO or 7 cm −1 for aCN, Table S3).Insets: apparent probability of aCO and aCN structures (Table S4).

Discussion
FTIR spectro-electrochemistry and quantum chemical calculations have resulted in a conclusive characterization of the reduced, CO-inhibited state Hred´-CO.We report the full vibrational spectrum of the H-cluster including the CO and CN -bands of four 13 CO labeled isotopomers of Hred´-CO while previously only CO stretching frequencies were reported [22].Four isotopomers were generated by selective 13 CO isotope editing [35] and clearly assigned by the computational results.Furthermore, we monitored the Hox-CO → Hred´-CO conversion as a function of both redox potential and pH.The decrease of the redox midpoint potential by about 60 mV per pH unit indicates that the formation of Hred´-CO is accompanied by protonation of the H-cluster.This is reminiscent of the Hox → Hred´ transition for which ATR FTIR spectro-electrochemistry and DFT data have identified a cysteine ligand at the [4Fe-4S] cluster as most likely protonation site in Hred´ [38,39].Our present computational results suggest a similar protonation in Hred´-CO and support the tentative assignment of a one-electron reduced [4Fe-4S] cluster [22].Accordingly, the presence of an additional CO ligand at the diiron site seems not to affect the protonation/reduction behavior of the [4Fe-4S] cluster.We have proposed earlier that the proton stabilizes a reduced [4Fe-4S] cluster in Hred´ and prevents formation of Hred and Hsred with a reduced diiron site [41].Carbon monoxide is expected to bind preferably at the oxidized diiron site [17], which agrees with the observation that only Hred´-CO and no CO-inhibited species with a reduced diiron site are observed.
Our analyses clearly favor an apical CN -at Fed in Hox-CO and Hred´-CO.The crystal structure of CO-inhibited [FeFe]-hydrogenase from Clostridium pasteurianum (CPI) was interpreted to bind exogenous CO in an apical orientation at Fed [19,32].Furthermore, changes in the electron density distribution between dark-adapted and illuminated CPI crystals apparently supported this view, however also showed differences at various other positions of the cofactor (e.g., a nearby methionine and all around Fed) [44].The limited resolution of the protein crystallography prohibits a unique distinction between CO and CN -.Therefore, the crystallographic assignment of cofactor geometry in the CO-inhibited state remains ambiguous.We find that an apical CN -describes the IRband frequency and intensity patterns for all 16 possible 13 CO isotopomers of Hox-CO and four 13 CO isotopomers of Hred´-CO consistently better than an apical CO [35].The secondary amine of the dithiolate ligand at the diiron site (adt-NH) may form a hydrogen bond with an apical, negatively charged cyanide, thereby stabilizing the CO-inhibited cofactor.Intramolecular stabilization between  1) and ( 2); Figures S1 and S2, Tables S1-S3).H-cluster structures: apical CO at Fe d (aCO, open symbols), apical CN -at Fe d (aCN, solid symbols).The lines show the diagonal for ideal correlation ((A), Hox-CO: mean rmsd of 20 cm −1 for aCO or 8 cm −1 for aCN.(B), Hred´-CO (including a proton at the [4Fe-4S] cluster): mean rmsd of 19 cm −1 for aCO or 7 cm −1 for aCN, Table S3).Insets: apparent probability of aCO and aCN structures (Table S4).

Discussion
FTIR spectro-electrochemistry and quantum chemical calculations have resulted in a conclusive characterization of the reduced, CO-inhibited state Hred´-CO.We report the full vibrational spectrum of the H-cluster including the CO and CN -bands of four 13 CO labeled isotopomers of Hred´-CO while previously only CO stretching frequencies were reported [22].Four isotopomers were generated by selective 13 CO isotope editing [35] and clearly assigned by the computational results.Furthermore, we monitored the Hox-CO → Hred´-CO conversion as a function of both redox potential and pH.The decrease of the redox midpoint potential by about 60 mV per pH unit indicates that the formation of Hred´-CO is accompanied by protonation of the H-cluster.This is reminiscent of the Hox → Hred´transition for which ATR FTIR spectro-electrochemistry and DFT data have identified a cysteine ligand at the [4Fe-4S] cluster as most likely protonation site in Hred´ [38,39].Our present computational results suggest a similar protonation in Hred´-CO and support the tentative assignment of a one-electron reduced [4Fe-4S] cluster [22].Accordingly, the presence of an additional CO ligand at the diiron site seems not to affect the protonation/reduction behavior of the [4Fe-4S] cluster.We have proposed earlier that the proton stabilizes a reduced [4Fe-4S] cluster in Hred´and prevents formation of Hred and Hsred with a reduced diiron site [41].Carbon monoxide is expected to bind preferably at the oxidized diiron site [17], which agrees with the observation that only Hred´-CO and no CO-inhibited species with a reduced diiron site are observed.
Our analyses clearly favor an apical CN -at Fe d in Hox-CO and Hred´-CO.The crystal structure of CO-inhibited [FeFe]-hydrogenase from Clostridium pasteurianum (CPI) was interpreted to bind exogenous CO in an apical orientation at Fe d [19,32].Furthermore, changes in the electron density distribution between dark-adapted and illuminated CPI crystals apparently supported this view, however also showed differences at various other positions of the cofactor (e.g., a nearby methionine and all around Fe d ) [44].The limited resolution of the protein crystallography prohibits a unique distinction between CO and CN -.Therefore, the crystallographic assignment of cofactor geometry in the CO-inhibited state remains ambiguous.We find that an apical CN -describes the IR-band frequency and intensity patterns for all 16 possible 13 CO isotopomers of Hox-CO and four 13 CO isotopomers of Hred´-CO consistently better than an apical CO [35].The secondary amine of the dithiolate ligand at the diiron site (adt-NH) may form a hydrogen bond with an apical, negatively charged cyanide, thereby stabilizing the CO-inhibited cofactor.Intramolecular stabilization between adt and the apical ligand has been suggested for Hhyd [37] and previously.Electrostatic attraction is not expected with a carbonyl ligand and can explain the pronounced CO sensitivity of [FeFe]-hydrogenases.The absence of hydrogen bonding to CN -may be part of the reason why HYDA1 with a pdt ((SCH 2 ) 2 CH 2 ) instead of an adt dithiolate is apparently not inhibited by CO [22,45].
Our findings imply that diatomic ligand rearrangement accompanies Hox-CO and Hred´-CO formation.The reduction of the H-cluster after CO inhibition (i.e., starting from Hox-CO) readily results in Hred´-CO and no further CO/CN -reorientation needs to take place.However, when the H-cluster was first poised in Hred´and exposed to CO thereafter, only Hred´-CO and Hox-CO and no structural isomers of the CO-inhibited species were formed.CO/CN -ligand rearrangement, hence, is independent of the redox and protonation state of the [4Fe-4S] cluster and primarily governed by the structural and electronic properties of the diiron site, as suggested earlier [15].This conclusion is corroborated by the observation that small yet significant fractions of Hred/Hsred remain detectable after prolonged CO exposure.In these states, CO binding and ligand rearrangement may be retarded due to the stabilization of an altered diiron site geometry with an apical CO ligand [36,46], whereas redox species with a vacant Fe d site such as Hox and Hred´react instantaneously with CO [17].We note that the ligand rearrangement at Fe d was questioned due to a potential salt bridge of the distal CN - to a lysine residue in the active site niche [47][48][49].However, at room temperature, such an interaction may be overcome by the vibrational dynamics of the cofactor-protein system [15], finally resulting in hydrogen-bonding between CN -and adt-NH in the CO-inhibited states.
In conclusion, the H-cluster appears to be optimized to prevent ligand rearrangement at the diiron site during rapid H 2 conversion [41].The stabilization of a cofactor geometry with a µCO ligand in the catalytic cycle is achieved by site-selective reduction and protonation at the [4Fe-4S] cluster in the first redox step.Preventing ligand rearrangement, for example, in enzymes with tailored cofactor variants, may improve the CO tolerance of [FeFe]-hydrogenases in hydrogen fuel production applications.

Sample Preparation
[FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii was heterologously expressed in E. coli, isolated as an apo-protein (including only the [4Fe-4S] cluster), and activated in vitro with an adt-NH containing a synthetic diiron complex as previously described [42,43].

Fourier-Transform Infrared Spectroscopy
Infrared spectroscopy was conducted at room temperature (24 • C) inside an anaerobic chamber (Coy, less than 2 ppm O 2 ) on a FTIR spectrometer (Tensor27, Bruker, Esslingen, Germany) equipped with a HgCdTe photodiode (Kolmar Technologies, Newburyport, MA, USA) and an ATR unit (DuraSamplIR II, Smiths Detection, London, UK).The stainless-steel ATR crystal plate was covered with non-conductive Kapton tape, leaving a hole for the Si crystal.A small strip of 9 µm thick Au mesh was deposited on the Si crystal, and 1 µL of HYDA1 protein (about 500 µM) was pipetted onto the Si crystal.ATR-FTIR spectra were recorded with a spectral resolution of 2 cm −1 and various co-additions of interferometer scans (a typical time resolution of ~5 s was achieved with 25 scans).Further details on the experimental set-up, data processing, and evaluation procedures (including baseline subtraction and spectral fit approaches) can be found in [38,39].
Before spectro-electrochemistry, 13 CO isotope editing was performed to accumulate Hox-CO isotopomers 1-4, as previously reported [35,36].Species 1 shows no 13 CO ligands and is formed upon contact of oxidized protein (Hox) with 12 CO gas.Species 2 carries a single 13 CO ligand at Fe d and accumulates in the presence of 13 CO gas in the dark.For Species 3, the sample film is additionally irradiated with white light, which allows the exchanging of both CO ligands at Fe d to 13 CO, as well as

Figure 1 .
Figure 1.Crystal structure of the H-cluster in [FeFe]-hydrogenase.(A)The structure ( protein data bank entry 4XDC)[28] of the Clostridium pasteurianum enzyme (CPI) is poised in the oxidized resting state (Hox).The CN -ligand at Fep is stabilized by a hydrogen bond (blue dashes) to the protein backbone[29], whereas the diatomic ligands at Fed are located in a hydrophobic pocket.The aminodithiolate (adt) group may be hydrogen-bonded to C299 (blue dashes).The asterisk marks a likely protonation site at the [4Fe-4S] cluster.Catalytic protons are exchanged via adt-NH and C299 in the putative proton transfer pathway to the diiron site.The circle marks the open coordination site at Fed where exogenous CO initially binds to form the CO-inhibited, oxidized state (Hox-CO).(B) Proposed structure of the H-cluster in the Hox-CO state (the [4Fe-4S] cluster is omitted for clarity).The orientation of the diatomic ligands in various cofactor states is under debate.

AFigure 1 .
Figure 1.Crystal structure of the H-cluster in [FeFe]-hydrogenase.(A) The structure ( protein data bank entry 4XDC) [28] of the Clostridium pasteurianum enzyme (CPI) is poised in the oxidized resting state (Hox).The CN -ligand at Fe p is stabilized by a hydrogen bond (blue dashes) to the protein backbone [29], whereas the diatomic ligands at Fe d are located in a hydrophobic pocket.The aminodithiolate (adt) group may be hydrogen-bonded to C299 (blue dashes).The asterisk marks a likely protonation site at the [4Fe-4S] cluster.Catalytic protons are exchanged via adt-NH and C299 in the putative proton transfer pathway to the diiron site.The circle marks the open coordination site at Fe d where exogenous CO initially binds to form the CO-inhibited, oxidized state (Hox-CO).(B) Proposed structure of the H-cluster in the Hox-CO state (the [4Fe-4S] cluster is omitted for clarity).The orientation of the diatomic ligands in various cofactor states is under debate.

Figure 3 .
Figure 3. Hox-CO → Hred´-CO redox transition as function of potential.The conversion of Hox-CO (black symbols) to Hred´-CO (red symbols) was probed at pH 5 (solid symbols) or pH 8 (open symbols).The lines show fit curves using the Nernst equation.At pH 5 a midpoint potential (Em) of −360 ± 10 mV was determined, while at pH 8, Em was shifted to −530 ± 30 mV.The larger error for the Em value at pH 5 was due to the increasing formation of Hsred at the expense of Hred´-CO at low potentials, which caused further changes in the Hox-CO and Hred´-CO populations (compare Figure 2).

Figure 2 .
Figure 2. Formation of the one-electron reduced state, CO-inhinbited state Hred´-CO at pH 5 and pH 8.The spectra show the gradual population of Hred´-CO (red band frequency labels) at the expense of Hox-CO (black band frequency labels) for a step-wise decrease of redox potential at pH 5 (left panel) or pH 8 (right panel).Spectra were normalized to uniform integral band intensity.Minor bands are due to small reduced H-cluster species Hred and Hsred populations (e.g., at 1891 cm −1 and 1882 cm −1 ) and unrelated to the CO-inhibited states.

Figure 3 .
Figure 3. Hox-CO → Hred´-CO redox transition as function of potential.The conversion of Hox-CO (black symbols) to Hred´-CO (red symbols) was probed at pH 5 (solid symbols) or pH 8 (open symbols).The lines show fit curves using the Nernst equation.At pH 5 a midpoint potential (Em) of −360 ± 10 mV was determined, while at pH 8, Em was shifted to −530 ± 30 mV.The larger error for the Em value at pH 5 was due to the increasing formation of Hsred at the expense of Hred´-CO at low potentials, which caused further changes in the Hox-CO and Hred´-CO populations (compare Figure 2).

Figure 3 .
Figure 3. Hox-CO → Hred´-CO redox transition as function of potential.The conversion of Hox-CO (black symbols) to Hred´-CO (red symbols) was probed at pH 5 (solid symbols) or pH 8 (open symbols).The lines show fit curves using the Nernst equation.At pH 5 a midpoint potential (E m ) of −360 ± 10 mV was determined, while at pH 8, E m was shifted to −530 ± 30 mV.The larger error for the E m value at pH 5 was due to the increasing formation of Hsred at the expense of Hred´-CO at low potentials, which caused further changes in the Hox-CO and Hred´-CO populations (compare Figure2).

Figure 4 .
Figure 4. CO inhibition of Hox and preparation of Hox-CO isotopomers 1-4.Shown are in situ attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) difference spectra of Chlamydomonas reinhardtii (HYDA1) in the CO/CN -frequency regime of the H-cluster (left panel) and corresponding diiron site structures with indicated13 CO labeling patterns (right panel,13 CO in red).Hox-CO species 1 is formed upon exposure of the oxidized enzyme (Hox) to12 CO gas in the dark.The exposure of isotopomer 1 to13 CO gas in the dark yields species 2, further illumination with white light under13 CO results in isotopomer 3. The exposure of isotopomer 3 to12 CO in the dark results in the formation of isotopomer 4. Note that the frequencies of the CN -ligands are barely affected by13 CO isotope editing, while the Hox → Hox-CO conversion is associated with difference signals in the CN -regime as well (2050-2100 cm −1 ).

Figure 4 .
Figure 4. CO inhibition of Hox and preparation of Hox-CO isotopomers 1-4.Shown are in situ attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) difference spectra of Chlamydomonas reinhardtii (HYDA1) in the CO/CN -frequency regime of the H-cluster (left panel) and corresponding diiron site structures with indicated13 CO labeling patterns (right panel,13 CO in red).Hox-CO species 1 is formed upon exposure of the oxidized enzyme (Hox) to12 CO gas in the dark.The exposure of isotopomer 1 to13 CO gas in the dark yields species 2, further illumination with white light under13 CO results in isotopomer 3. The exposure of isotopomer 3 to12 CO in the dark results in the formation of isotopomer 4. Note that the frequencies of the CN -ligands are barely affected by13 CO isotope editing, while the Hox → Hox-CO conversion is associated with difference signals in the CN - regime as well (2050-2100 cm −1 ).

Figure 6 .
Figure6.Pure IR spectra of Hred´-CO isotopomers 1-4.Spectro-electrochemical experiments yielded pure IR spectra for Hox-CO (dotted lines) and Hred´-CO (solid lines) after the subtraction of the minor IR contributions of other H-cluster species, such as Hred and Hsred, and the other isotopomers of Hox-CO/Hred´-CO (compare Figure5).The CO/CN − stretching frequencies for Hred´-CO are annotated.

Figure 7 .
Figure 7. CO inhibition of pre-reduced HYDA1.Kinetic traces in (A) and corresponding FTIR spectra in (B).[FeFe]-hydrogenase was first exposed to 100% H 2 to accumulate the reduced species Hred, Hsred, and Hred´.No external potential was applied.Upon injection of 1% CO into the gas stream, the H-cluster rapidly forms about equal populations of Hox-CO and Hred´-CO.While Hred´is lost within ~10 s, Hred and Hsred decrease to reach a stable population of around 5% after ~1 min.* The high-frequency band at 2026 cm −1 has been to assigned to Hsred and is unrelated to the CO-inhibited species.
[35]sition of CO ligands at [FeFe]: p, proximal Fe; µ , Fe-Fe bridging; d, distal Fe (1 and 2).Ligand position and isotopic labeling assignments are based on experimental and computational evidence in the present study and earlier reports (compare Figure2)[35].
[35]sition of CO ligands at [FeFe]: p, proximal Fe; µ, Fe-Fe bridging; d, distal Fe (1 and 2).Ligand position and isotopic labeling assignments are based on experimental and computational evidence in the present study and earlier reports (compare Figure2)[35].