Spectroscopic and Computational Evidence that [FeFe] Hydrogenases Operate Exclusively with CO-Bridged Intermediates

[FeFe] hydrogenases are extremely active H2-converting enzymes. Their mechanism remains highly controversial, in particular, the nature of the one-electron and two-electron reduced intermediates called HredH+ and HsredH+. In one model, the HredH+ and HsredH+ states contain a semibridging CO, while in the other model, the bridging CO is replaced by a bridging hydride. Using low-temperature IR spectroscopy and nuclear resonance vibrational spectroscopy, together with density functional theory calculations, we show that the bridging CO is retained in the HsredH+ and HredH+ states in the [FeFe] hydrogenases from Chlamydomonas reinhardtii and Desulfovibrio desulfuricans, respectively. Furthermore, there is no evidence for a bridging hydride in either state. These results agree with a model of the catalytic cycle in which the HredH+ and HsredH+ states are integral, catalytically competent components. We conclude that proton-coupled electron transfer between the two subclusters is crucial to catalysis and allows these enzymes to operate in a highly efficient and reversible manner.


S2
| Temperature dependent FTIR of CrHydA1 in the HsredH + state in H2O Figure S2 | Temperature dependent FTIR of CrHydA1 in the HsredH + state in H2O before spline curve fitting for background subtraction Figure S3 | Close up of the Hhyd bridging CO region in the temperature dependent FTIR of CrHydA1 in the HsredH + state in H2O Figure S4 | Temperature dependent FTIR of CrHydA1 in the HsredH + state in D2O Figure S5 | Temperature dependent FTIR of CrHydA1 in the HsredH + state in D2O Figure S6 | Temperature dependent FTIR of CrHydA1 in the HsredH + state in D2O before spline curve fitting for background subtraction Figure S7 | H2O/D2O dependent FTIR peak shifts of CrHydA1 HsredH + state at 40 K Figure S8 | Temperature dependent FTIR of DdHydAB in the HredH + state in H2O Figure S9 | Temperature dependent FTIR of DdHydAB in the HredH + state in H2O before spline curve fitting for background subtraction Figure S10 | Temperature dependent FTIR of DdHydAB in the HredH + state in D2O Figure S11 | Temperature dependent FTIR of DdHydAB in the HredH + state in D2O Figure S12 | Temperature dependent FTIR of DdHydAB in the HredH + state in D2O before spline curve fitting for background subtraction Figure S13 | H2O/D2O dependent FTIR peak shifts of DdHydAB HredH + state at 40 K Figure S14 | Structures of the μCO and μCO + states from DFT modeling Figure S15 | Structures of the μH and μH + states from DFT modeling Figure S16 | Structural comparison of the μCO + and μH + states from DFT modeling Figure S17 | Structural comparison of the μCO and μH states from DFT modeling Figure S18 | Comparison of H2O/D2O effect on DFT models of FTIR spectra of the HredH + state and the HsredH + state Figure S19 | NRVS of CrHydA1 and DdHydAB in the HsredH + and HredH + states with error bars Figure S20 | 57 Fe-PVDOS spectra from DFT calculations on the μCO + , μCO, Hhyd, μH + , and μH model states overlaid with the available NRVS experimental data Figure S21 | DFT calculations of the NRVS spectrum of the HsredH + state with experimental error bars Figure S22 | Comparison of H2O/D2O effect on DFT models of NRVS spectra of the HredH + state Figure S23 | DFT calculations of the NRVS spectrum of the HredH + state with experimental error bars Figure S24 | Comparison of H2O/D2O effect on DFT models of NRVS spectra of the HredH + state Figure S25 | 57 Fe-PVDOS spectra of the HredH + , HsredH + , and Hhyd states from NRVS experiments and DFT models          Figure S14 | Structures of the μCO (element colors, tube representation) and μCO + (green, thin tube) DFT models, overlaid together with their X-ray reference PDB 5BYQ (black, thin tube). Additionally, in ball representation are indicated the carbon nuclei locked to their original X-ray positions during DFT structural optimization, Fe sites, and the three H -to-D exchangeable protons. Single-letter amino acid labeling corresponds to the CrHydA1 enzyme sequence.

Supplementary Tables
Figure S15 | Structures of the μH (element colors, tube representation) and μH + (green, thin tube) DFT models, overlaid together with their X-ray reference PDB 5BYQ (black, thin tube). Additionally, in ball representation are indicated the carbon nuclei locked to their original X-ray positions during DFT structural optimization, Fe sites, and the three H -to-D exchangeable protons. Single-letter amino acid labeling corresponds to the CrHydA1 enzyme sequence.
Figure S16 | Structural comparison of the μCO + (element colors, tube representation) and μH + (red, thin tube) isoelectronic DFT models, overlaid together with their X-ray reference PDB 5BYQ (black, thin tube). Additionally, in ball representation are indicated the carbon nuclei locked to their original X-ray positions during DFT structural optimization, Fe sites, and the three H-to-D exchangeable protons. Single-letter amino acid labeling corresponds to the CrHydA1 enzyme sequence. Figure S17 | Structural comparison of the μCO (element colors, tube representation) and μH (red, thin tube) isoelectronic DFT models, overlaid together with their X-ray reference PDB 5BYQ (black, thin tube). Additionally, in ball representation are indicated the carbon nuclei locked to their original X-ray positions during DFT structural optimization, Fe sites, and the three H-to-D exchangeable protons. Single-letter amino acid labeling corresponds to the CrHydA1 enzyme sequence.
Figure S18 | Comparison of H2O/D2O effect on DFT models of FTIR spectra of the HredH + state and the HsredH + state. FTIR spectra were calculated using DFT (as described in the methods section) with a semibridging CO (μCO + /μCO D+ and μCO/μCO D ) model or a bridging hydride (μH + /D + and μH/D) model of the protonated (blue lines) and the deuterated (red lines) form of the HredH + and HsredH + states. The three H-to-D exchangeable protons in the DFT models are shown in Figures S14-S17. The baselines of the H/D spectra have been offset for clarity. The animated normal modes producing the bands shown here for μCO + , μCO, μH + , and μH are available as part of the Supporting Information.          Table S5. For the schematic representation, see Figure 1 of the main text.   [a] The CO/CN ligand labels follow those used in Figures 4, S14, and S15. Animations illustrating the calculated CO/CN modes are available in Supporting Information separately.
[b] The two strongly coupled vibrational modes calculated at 1934 and 1929 cm -1 which reduce to a single band at 1929 cm -1 upon broadening. Other minor couplings were omitted in this table.
[c] Note the reversed order of these frequencies with respect to those from the μH + model. [d] <Protein> fragment represents the 2 nd -shell protein ligands to the H-cluster, specifically residues P93, A94, C169, P194, Q195, M223, P224, K228, and M415 (CrHydA1 sequence) of the DFT model as shown in the figures; the total +1 <Protein> prototype fragment charge is due to the positively charged side chain of K228.

Supplementary Discussion
What's in a name:  [10][11]14 . The notion of a bridging hydride in the H-cluster originated from site-selective X-ray absorption and emission spectroscopy on a state referred to as sred, 7 which was generated by reducing CrHydA1 with either H2 or sodium dithionite at pH 8. The conditions typically yield the two-electron reduced state, which we refer to as the HredH + state. 9 In this state, [2Fe]H is thought to be in a homovalent Fe(I)Fe(I) state and [4Fe-4S]H is reduced to the 1+ state. This gives rise to an IR spectrum with a dominant feature at 1882 cm -1 and an EPR spectrum reminiscent of reduced [4Fe-4S] clusters. 6,15 More recently, the Haumann group has started referring to their sred state as the Hsred state. 16 The results in this publication exclude that bridging hydrides exist in the well-characterized states referred to as HredH + (a.k.a. Hred or red) and HsredH + (a.k.a. Hsred or sred). We cannot rule out that Haumann and co-workers have stabilized alternative states so far not characterized by IR spectroscopy, and that these states contain bridging hydrides. However, considering that the conditions used in our work and in the work from Haumann and co-workers in order to generate the HredH + and HsredH + state are similar (reduction with H2 or sodium dithionite at pH 8), this possibility seems unlikely. Furthermore, Haumann and co-workers contend that the IR spectra from the HredH + and HsredH + states indicate bridging hydride bound states. 14 In this respect, our work disagrees with this interpretation entirely.

Extended details on the DFT results and modeling
Method and Models. The PBE0 17-18 hybrid functional has been employed in this work at its higher computational cost, than the BP86 19-20 functional applied previously by some of us to model the Hhyd state. 1,[21][22] Benefits of the PBE0 application are, as presently found, (i) proper modeling of the redox balance between the [4Fe-4S]H and [2Fe]H subclusters at the two redox levels (Table S3 and S5), together with (ii) respectable quality results from the normal mode analysis. Notably, the models μCO and μCO + produced vanishingly small spin populations at the two [2Fe]H Fe(I) sites (rather than a broken-symmetry state with antiferromagnetic character), indicating the Fe(I)-Fe(I) metal-metal bonding as depicted in Figure 1B. In summary, PBE0 provided an empirically better overall match between the NRVS-observed and DFT-calculated 57 Fe-PVDOS spectra through the entire measured range (0-800 cm -1 ) when using the entire Hcluster model (L'); in contrast, BP86 was aimed by us earlier 1, 21-22 to the Fe-H hydride bands of Hhyd in the 600-800 cm -1 range only when using a model excluding the [4Fe-4S]H subcluster (L). As shown in Figures S20A-C and S25A-B, the present PBE0-based methodology seamlessly reproduces NRVS spectra of HredH + and HsredH + , as well as that of Hhyd.
HredH + and HsredH + vs Hhyd: 57 Fe-PVDOS. A point of transition between the high-intensity and the low-intensity or baseline (at higher vibrational energies) regions in NRVS spectra is known to aid in comparative analysis of related molecular systems. As an example, high-end of the Fe-S band region (360-400 cm -1 ) across the 57 Fe-enriched [3Fe-4S] 1+/0 and [4Fe-4S] 2+/1+ cluster variants in the Pf Fd D14C protein has been correlated with average oxidation level of iron sites in these iron-sulfur clusters. 23 Extending this approach to [FeFe] hydrogenase, high-end of the Fe-CO/CN region (600-640 cm -1 ) in the [2Fe]H subcluster can serve its 57 Fe-NRVS diagnostic purpose. We found that the high-end Fe-CO/CN vibrational energy is at ~610 cm -1 for Hhyd, while it is at ~620-640 cm -1 for HredH + /HsredH + ; the absolute position of the bands and their ~20-30 cm -1 shifts are consistent here between the observed ( Figure S25A) and calculated ( Figure S25B) 57 Fe-PVDOS spectra. Our analysis reveals that in the μCO + /μCO (as well as μH + /μH) DFT models of HredH + /HsredH + , the high-end Fe-CO/CN modes are coupled to the ADT fragment motion, while no such coupling is present in the Hhyd model; see the normal mode animations available as part of the Supporting Information. The representative models μCO + /μCO for the HredH + /HsredH + states display ADT vibrations at 639/636 cm -1 (S-C stretches coupled to the -NH2 +bridgehead bends) which supply only minor 57 Fe-PVDOS intensity and appear as highend shoulders of the Fe-CO/CN bands, as shown in Figure S25B.