Discovery of novel [FeFe]-hydrogenases for biocatalytic H2-production

A semi-synthetic screening method for mining the biodiversity of [FeFe]-hydrogenases, expanding the toolbox for biocatalytic H2-gas production.


H2-production assays
In vivo E. coli cells treated with [2Fe] adt were transferred to an airtight vial and incubated at 37°C and 150 rpm for 1 hr. H2 production was then determined by analyzing the reaction headspace using a PerkinElmer Clarus 500 gas chromatograph (GC) equipped with a thermal conductivity detector (TCD) and a stainless-steel column packed with Molecular Sieve (60/80 mesh). The operational temperatures of the injection port, the oven and the detector were 100 °C, 80 °C and 100 °C, respectively. Argon was used as carrier gas at a flow rate of 35 mL min −1 .

In vitro
Following in vivo activation with [2Fe] adt , cells were transferred to the glovebox and harvested by centrifugation (13.000 rpm, 4 min). The cells were then washed two times with 1 mL Tris-HCl buffer (100 mM Tris, 150 mM NaCl, pH 7.5) and resuspended in 0.5 mL lysis buffer (30 mM Tris-HCl, 0.2 % (v/v) Triton X-100, 0.6 mg mL -1 lysozyme, 0.1 mg mL -1 DNAse, 0.1 mg mL -1 RNAse). Cell lysis was performed by three cycles of freezing/thawing in liquid N2 and the supernatant was recovered by centrifugation (13.000 rpm, 10 min). 0.38 mL supernatant was then diluted to 2 mL in potassium phosphate buffer (100 mM, pH 6.8) containing 10 mM methyl viologen, 20 mM sodium dithionite and 1 % (v/v) Triton X-100. Reactions were incubated in 37°C for 15 min. H2 production was then determined by analyzing the reaction headspace on GC (see above).

Protein film electrochemistry on cell lysates
Protein film electrochemistry experiments were run in an anaerobic glovebox, using a saturated calomel reference electrode (Fisher Scientific) and a 0.5 mm platinum wire (Sigma Aldrich) as counter. The functionalisation procedure consisted of preparing a solution of 1-3 mg/mL of MWCNTS in 1,2dinitropyrolidone followed by homogenisation by sonication for 15 min. Then, the GC surface (r= 1 or the enzyme onto the electrode. Electrochemical data was acquired using an Eco/Chemie PGSTAT10 and the GPES software (Metrohm/Autolab). Data were analysed using Qsoas (qsoas.org). 6

EPR and FTIR sample preparation
The 2 mL dense cell suspension generated via the in vivo artificial activation protocol followed by 1 hr incubation at 37°C were centrifuged and the cell pellet was washed with 1 mL Tris-HCl buffer (100 mM Tris, 150 mM NaCl, pH 7.5) three times under anaerobic conditions. For EPR samples the cells were resuspended with 400 µL TRIS-HCl buffer after the washing protocol and then transferred into EPR tubes. The tubes were capped and directly frozen in liquid N2. In case of the FTIR samples, four separate 2 mL sample preparations were combined, concentrated and resuspended in 400 µL Tris-HCl buffer and frozen in liquid N2 under anaerobic conditions.

Whole-cell EPR
Measurements were performed on a Bruker ELEXYS E500 spectrometer using an ER049X SuperX microwave bridge in a Bruker SHQ0601 cavity equipped with an Oxford Instruments continuous flow cryostat and using an ITC 503 temperature controller (Oxford Instruments). Measurement temperature was 10 K, using liquid helium as coolant, with the following EPR settings unless otherwise stated: microwave power 1 mW modulation amplitude 1 mT, modulation frequency 100 kHz. The spectrometer was controlled by the Xepr software package (Bruker).

Whole-cell FTIR
For in situ ATR FTIR spectroscopy, 1 µL cell suspension was deposited on the silicon crystal of an ATR cell in the beam path of a commercial FTIR spectrometer (Bruker). All experiments were performed at ambient temperature (~24 °C) and pressure (~1 atm), in the dark, and on hydrated films of physiological pH values (pH ~8). The cell suspension was dried under 100% N2 gas and re-hydrated with A. bidest in the humidified gas stream (aerosol), similar to what was reported for purified protein earlier. 7 Reduction of [FeFe]-hydrogenase in the cells was induced by adding 1% H2 to the N2 gas stream (1.5 L min -1 ). In the absence of H2, Hox recovered due to auto-oxidation. This latter process was rapid in the case of Cr-HydA1, while extended incubation under N2 was required in the case of Tam-HydA.
Transitions were followed with a spectral precision of 2 cm -1 and 1,000 averages of interferometer scans per spectrum. Difference spectra were calculated by subtraction of a N2 spectrum from an H2 spectrum. In the CO/CNregime of the H-cluster, negative bands are assigned to Hox whereas positive bands represent Hred. 8 Table S1. In vivo condition screening

Supplementary tables and figures
The robustness of the screening protocol was probed using Cr-HydA1, and the results are summarized in Table S1. Two different plasmid constructs were tested, using a standard T7 promoter or a low expression trc promoter. The two constructs were expressed in three different media, LB, TB or M9, to probe the importance of the growth medium. In all cases successful enzyme activation was observed, as determined by the detection of H2-production.
Cells containing a Cr-HydA1 expressing plasmid were grown in 100 mL volume in different media. In one set of the experiments 0.5 mM IPTG was added at the time of the inoculation, and the cells were grown at 37˚C until OD600 = 0.2. In the other set of experiments the cells were pre-grown until OD600 = 0.2, before the protein overproduction was induced with 0.5 mM IPTG and incubated for 2 hours at 37˚C. In both cases, the cells were then concentrated to 2 mL with addition of fresh, 0.4% glucose supplemented media, and an in vivo enzyme activation was performed with addition of 100 µg [2Fe] adt (final concentration 80 M) under anaerobic conditions. H2-production was measured after 1 h incubation at 37˚C by analyzing the reaction headspace on GC (see above).        [2Fe] adt -Tam-HydA ( Figure S4). EPR spectra of [2Fe] pdt added to BL21(DE3) cells not expressing any [FeFe] hydrogenase (magenta spectrum) and [2Fe] pdt -Cr-HydA1 (green spectrum) shown for reference.
EPR spectra were recorded at 10 K, 1 mW microwave power at a microwave frequency of 9.28 GHz. Here, Tam-HydA and Cr-HydA1 both adopt Hox.