Hydride state accumulation in native [FeFe]-hydrogenase with the physiological reductant H2 supports its catalytic relevance

Small molecules in solution may interfere with mechanistic investigations, as they can affect the stability of catalytic states and produce off-cycle states that can be mistaken for catalytically relevant species. Here we show that the hydride state (Hhyd), a proposed central intermediate in the catalytic cycle of [FeFe]-hydrogenase, can be formed in wild-type [FeFe]-hydrogenases treated with H2 in absence of other, non-biological, reductants. Moreover, we reveal a new state with unclear role in catalysis induced by common low pH buffers.


Table S1
Overview of NaDT concentrations involved in characterization of Hhyd in previous studies.

Figure S1
Absolute and difference ATR-FTIR spectra of HydA1 during the pH 4 adjustment and under H2 and N2.

Figure S2
Gas interaction kinetics of HydA1 at pH 4. Plotted is the difference in peak area associated with each redox state over time.

Figure S3
Absolute ATR-FTIR spectra of HydA1 exposed to different buffers.

Figure S4
Absolute and difference ATR-FTIR spectra of HydA1 equilibrated with mixed buffer at pH 8 and pH 4.

Figure S5
Absolute ATR-FTIR spectra of HydA1 equilibrated with mixed buffer at pH 8 (10 mM Tris buffer) and pH 4 with 100 mM capronate buffer.

Supporting References
Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2022

Materials and Methods:
Protein purification and the absence of NaDT Chlamydomonas reinhardtii HydA1 was expressed, purified and reconstituted with minor modifications as previously reported. 1, 2 The codon-optimized gene coding for CrHydA1 was cloned in frame with a Strep-tag II on a pET11a vector by GeneScript, that was then used BL21(DE3) E. coli strain. The enzyme was aerobically expressed in its apo-form and purified using pre-packed StrepTrap columns on an Äkta-Ready system (Cytiva). After semi-enzymatic [4Fe4S] cluster reconstitution, the enzyme was matured using a [Fe2(adt)(CO)4(CN)2] 2-(adt = azadithiolate, -SCH2NHCH2S -) synthetic mimic as follows: a mixture containing 100uM reconstituted CrHydA1, 600uM Fe2(adt)(CO)4(CN)2 2-, 2mM sodium dithionite was prepared in a 100mM Tris/HCl pH 8, 200mM KCl buffer; the mixture was incubated for 90 minutes at room temperature and excess mimic and dithionite were removed using a PD10 desalting column, pre-equilibrated with a 100mM Tris/HCl pH 8, 200mM KCl buffer. To ensure complete removal of residual dithionite, the buffer was further exchanged to 10mM Tris/HCl pH 8 via four concentration and dilution cycles using Amicon® Ultra 0.5 Centrifugal Filter Units (Millipore), as recommended by the manufacturer.
ATR-FTIR spectroscopy 1µl enzyme solution (1mM CrHydA1) in 10 mM Tris buffer (pH 8) was deposited on the ATR crystal in the anaerobic atmosphere of a Braun Glove box. The ATR unit (BioRadII from Harrick) was sealed with a custom build PEEK cell that allowed for gas exchange and illumination (inspired by Stripp et al. 3,4 ) mounted in a FTIR spectrometer (Vertex V70v, Bruker). The sample was dried under 100% nitrogen gas and rehydrated with a humidified aerosol (100 mM Tris-HCl (pH 8) or 100 mM propionate/acetate/formate/capoate buffer (pH 4)) as described before 5 . Spectra were recorded with 2 cm -1 resolution, a scanner velocity of 80 Hz and averaged of varying number of scans (mostly 1000 Scans). All measurements were performed at ambient conditions (room temperature and pressure, hydrated enzyme films). where NaDT has been removed by gel filtration. Traces of a rhombic EPR signal attributable to the Hhyd state are visible in one "NaDT free" sample, no signal attributable to Hhyd is observed by FTIR in the absence of NaDT. C FTIR spectra are reported for H2 and D2 treated auto-oxidized samples of WT CrHydA1 and CaI. Traces of a partial Hhyd FTIR signature are visible in the spectra, collected at pH and pD = 8.

Figure S1 Absolute and difference ATR-FTIR spectra of HydA1 during the pH 4 adjustment and under H2 and N2. (A)
Absolute spectra of HydA1 (10mM Tris pH 8) exposed to a 100 mM propionate buffer (pH 4) via the aerosol. Formation of the new species Hoxc is followed over time. (B) Difference spectra of the same process as in (A). (C) Absolute spectra of HydA1 equilibrated at pH 4 (100mM propionate, compare (A) and (B)) exposed to H2 and N2. (D) Absolute spectra of HydA1 at pH 8 (100 mM Tris) exposed to H2 and N2. All spectra read from top to bottom. Note that a small population of Hox-CO is present in all spectra (small peak at 2013 cm -1 ). Over the time course of 20 h its population changes only slightly (e.g. compare (B)).

Figure S2 Gas interaction kinetics of HydA1 at pH 4. Plotted is the difference in peak area associated with each redox state over time.
Hox is scaled by 0.5. top: The sample exposed to N2 after being equilibrated under H2 (compare Fig.S1 C) adjusts to the new gas atmosphere (auto-oxidation) within ca. 20 seconds. bottom: The sample exposed to H2 after being equilibrated under N2 (compare Fig.S1 C) adjusts to the new gas atmosphere (H2 uptake) within ca. 2 seconds.

Figure S3
Absolute ATR-FTIR spectra of HydA1 exposed to different buffers. HydA1 exposed to different pH 4 buffers (100 mM) composed of (formate, acetate, propionate, oxalate, succinate, citrate-phosphate). Only for acetate, propionate and formate population of Hoxc is observed. The dashed spectra represent HydA1 at pH 8 (100 mM Tris). The small peak at 2013 cm -1 indicates small contributions of Hox-CO in some samples.