The large subunit of the regulatory [NiFe]-hydrogenase fromRalstonia eutropha- a minimal hydrogenase?

Article abstract of DOI:10.1039/d0sc01369b

Chemically synthesized compounds that are capable of facilitating the reversible splitting of dihydrogen into protons and electrons are rare in chemists' portfolio. The corresponding biocatalysts - hydrogenases - are, however, abundant in the microbial world. [NiFe]-hydrogenases represent a major subclass and display a bipartite architecture, composed of a large subunit, hosting the catalytic NiFe(CO)(CN)₂cofactor, and a small subunit whose iron-sulfur clusters are responsible for electron transfer. To analyze in detail the catalytic competence of the large subunit without its smaller counterpart, we purified the large subunit HoxC of the regulatory [NiFe]-hydrogenase of the model H₂oxidizerRalstonia eutrophato homogeneity. Metal determination and infrared spectroscopy revealed a stoichiometric loading of the metal cofactor. This enabled for the first time the determination of the UV-visible extinction coefficient of the NiFe(CO)(CN)₂cofactor. Moreover, the absence of disturbing iron-sulfur clusters allowed an unbiased look into the low-spin Fe²⁺of the active site by M?ssbauer spectroscopy. Isolated HoxC was active in catalytic hydrogen-deuterium exchange, demonstrating its capacity to activate H₂. Its catalytic activity was drastically lower than that of the bipartite holoenzyme. This was consistent with infrared and electron paramagnetic resonance spectroscopic observations, suggesting that the bridging position between the active site nickel and iron ions is predominantly occupied by water-derived ligands, even under reducing conditions. In fact, the presence of water-derived ligands bound to low-spin Ni²⁺was reflected by the absorption bands occurring in the corresponding UV-vis spectra, as revealed by time-dependent density functional theory calculations conducted on appropriatein silicomodels. Thus, the isolated large subunits indeed represent simple [NiFe]-hydrogenase models, which could serve as blueprints for chemically synthesized mimics. Furthermore, our data point to a fundamental role of the small subunit in preventing water access to the catalytic center, which significantly increases the H₂splitting capacity of the enzyme.

Full text of DOI:10.1039/d0sc01369b

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and O. Lenz, Chem. Sci., 2020, DOI: 10.1039/D0SC01369B.
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The large subunit of the regulatory [NiFe]-hydrogenase from Ralstonia eutrophaDO–I: 1A0.10m39i/nD0iSmC0a1l369B
hydrogenase?†
In memory of Maurizio Bruschi.
Giorgio Caserta,a* Christian Lorent,a Alexandre Ciaccafava,a Matthias Keck,b Raffaella Breglia,c
Claudio Greco,c Christian Limberg,b Peter Hildebrandt,a Stephen P. Cramer,d Ingo Zebger,a* and
Oliver Lenza*
a Institut für Chemie, Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany
b Department of Chemistry, Humboldt-Universität zu Berlin, Brook-Taylor-Straße 2, 12489 Berlin,
Germany
c Department of Earth and Environmental Sciences, Milano-Bicocca University, Piazza della Scienza
1, 20126 Milan, Italy
d SETI Institute, 189 Bernardo Ave., Mountain View, CA 94043, United States
*corresponding authors
Keywords: Hydrogenase, H/D exchange reaction, O2-tolerance, infrared (IR), electron paramagnetic
resonance (EPR), Mössbauer.
Abstract
Chemically synthesized compounds that are capable in the reversible splitting of dihydrogen into
protons and electrons are rare in the chemists’ portfolio. The corresponding biocatalysts –
hydrogenases– are, however, abundant in the microbial world. [NiFe]-hydrogenases represent a major
subclass and display a bipartite architecture, composed of a large subunit, hosting the catalytic
NiFe(CO)(CN)2 cofactor, and a small subunit whose iron-sulfur clusters are responsible for electron
transfer. To analyze in detail the catalytic competence of the large subunit without its smaller
counterpart, we purified the large subunit HoxC of the regulatory [NiFe]-hydrogenase of the model
H2 oxidizer Ralstonia eutropha to homogeneity. Metal determination and infrared spectroscopy
revealed a stoichiometric loading with the metal cofactor. This enabled for the first time to determine
the UV-visible extinction coefficient of the NiFe(CO)(CN)2 cofactor. Moreover, the absence of
disturbing iron-sulfur clusters allowed an unbiased look onto the low-spin Fe2+ of the active site by
Mössbauer spectroscopy. Isolated HoxC was active in catalytic hydrogen-deuterium exchange,
demonstrating its capacity to activate H2. The catalytic activity was drastically lower than that of the
bipartite holoenzyme. This was consistent with infrared and electron paramagnetic resonance
spectroscopic observations, suggesting that the bridging position between the active site nickel and
iron ions is predominantly occupied by water-derived ligands, even under reducing conditions. In
fact, the presence of water-derived ligands bound to low-spin Ni2+ was reflected by the absorption
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bands occurring in the corresponding UV-vis spectra, as revealed by time-depDeOnId: 1e0n.1t039d/De0nSsCi0t1y369B
functional theory calculations conducted on appropriate in silico models. Thus, isolated large subunits
indeed represent simple [NiFe]-hydrogenase models, which could serve as blueprints for chemically
synthesized mimics. Furthermore, our data point to a fundamental role of the small subunit in
preventing water access to the catalytic center, which significantly increases the H2 splitting capacity
of the enzyme.
INTRODUCTION
[NiFe]-hydrogenases are abundant enzymes in prokaryotes1,2and catalyze the reversible
conversion of molecular hydrogen into proton and electrons.3,4 These metalloproteins display a
bipartite architecture that provides the necessary infrastructure for the controlled interchange of
substrates and products between the protein surface and the deeply buried catalytic center (Fig. 1).
The large subunit of [NiFe]-hydrogenases harbors the catalytic nickel-iron site in addition to a gas
channel network and a proton translocation pathway. The small subunit contains one to three iron-
sulfur clusters, which can have different compositions, but always serve as electron relay connecting
the active site with the physiological electron acceptors/donors. In the absence of the physiological
electron acceptors/donors, hydrogenase activity can be assayed by using artificial redox partners, such
as methyl viologen or methylene blue. The site where these compounds interact with the electron
transport chain, however, remained unclear.
To elucidate whether small subunits serve merely as electronic wires or provide additional
functions that support hydrogenase activity, it appears appropriate to investigate [NiFe]-hydrogenase
large subunits independently from their counterpart. In fact, we have previously analyzed by
biochemical and spectroscopic means the isolated precursor of the large subunit, preHoxG, of the O2-
tolerant membrane-bound [NiFe]-hydrogenase (MBH) of Ralstonia eutropha.5 The protein was
capable of H2 activation – even in the presence of O2 –, but only a restricted number of the active site
redox states known to contribute to the catalytic cycle have been observed. While this study clearly
provided first evidence for H2 conversion without the small subunit, the results could not be
generalized because of two constraints. First, the active site contained a sub-stoichiometric amount
of nickel, and, second, the large subunit was still equipped with the typical C-terminal extension,
which is usually cleaved off upon insertion of the catalytic center.5 In fact, the crystal structure of the
premature hydrogenase large subunit, HyhL, from Thermococcus kodakarensis suggests the
displacement (relative to the situation in the holoprotein) of one of the four cysteines coordinating
the catalytic nickel-iron site because of the presence of the C-terminal extension.6 Recent experiments
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conducted to unravel the chaperone function of the C-terminal extension of the EDsOcIh: 1e0r.1i0c3h9i/Da0ScCo0l1i369B
[NiFe]-hydrogenase 2 large subunit precursor support this assumption.7
A
B
C
Cys
NiIII
Cys
Cys
S
S
S
CO
FeII
CN
Cys
O
H
S
Nir-B
e- -e-
Cys
NiII
Cys
NiII
Cys
Cys
Cys
S
Cys
S
S
S
S
S
H+
- H+
CO
CN
CO
CN
FeII
FeII
Cys
Cys
O
H
O
S
S
CN
CN
H
H
HoxC
MBH
Nir-SI
Nir-SII
O
H2
Cys
NiII
Cys
Cys
S
S
S
CO
CN
FeII
Cys
S
e- H+
CN
H2
Nia-S
Cys
S
Cys
Cys
Cys
Cys
Cys
S
S
S
H-
S
S
CO
CN
CO
NiII
NiI
FeII
FeII
CN
Cys
S+
Cys
S+
CN
CN
H
H
Ni-L
Nia-SR
Cys
S
Cys
Cys
S
e- H+
CO
CN
NiIII
S
H-
FeII
Cys
S
CN
Nia-C
Figure 1. Structure of [NiFe]-hydrogenase and proposed active site states. (A) Schematic
representation of the ready (subscript “r”) and active (subscript “a”) states of the active site. The grey
box comprises the interconvertible states observed for HoxCai. Dashed boxes indicate states identified
in native RH and its isolated large subunit HoxC. (B) Homology model of HoxC generated by using
the
PHYRE2
Protein
Fold
Recognition
Server
(http://www.sbg.bio.ic.ac.uk/~phyre2/html/page.cgi?id=index). The MBH large subunit HoxG from
R. eutropha, which shares 34 % amino acid identity with HoxC, served as the template (3RGW) (C)
Overall structure of the membrane-bound [NiFe]-hydrogenase of R. eutropha. The [NiFe] cofactor
and [FeS] clusters are depicted as spheres. The large and small subunit is depicted in cyan and green,
respectively. Element color code of the cofactors: Fe, brown; Ni, green; S, yellow; C, grey; N, blue;
O, red.
In the present study, we focused our attention on the large subunit of the regulatory hydrogenase
(RH) from R. eutropha, which belongs to the small subgroup of [NiFe]-hydrogenases whose large
subunits do not possess a C-terminal extension.8 The RH contains an otherwise standard-like catalytic
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center (Fig. 1), in which a nickel and an iron ion are bridged by two cysteines. Two further cysteines
DOI: 10.1039/D0SC01369B
act as terminal ligands to the Ni, while one CO and two CN− ligands complete the coordination sphere
of the Fe. This results in a NiFeII(CO)(CN)2 configuration of the active site, in which only the nickel
changes its redox state in response to treatment of the enzymes with natural and artificial
oxidants/reductants. The RH has already served as a “model” for a number of mostly spectroscopic
studies including electron paramagnetic resonance (EPR), infrared (IR), and resonance Raman (RR)
spectroscopy.9–13 This is because of the apparent simplicity of the RH which can be selectively
trapped in certain redox states of the active site. In its as-isolated form, the catalytic center resides in
a NiII-(–)-FeII(CO)(CN)2 configuration (with (–) indicating a vacant bridging position between the
two metals) known as the Nia-S state, whose structure has been recently confirmed via X-ray
crystallography.14 Upon treatment with H2, the active site adopts predominantly the Nia-C state
characterized by a bridging hydride (NiIII-(H–)-FeII(CO)(CN)2).9 Illumination at cryogenic
temperatures, converts the Nia-C state into the Ni-L state,15 which is discussed to be an intermediate
of the catalytic cycle (Fig. 1).16 The first known intermediate upon H2 incubation, Nia-SR (NiII-(H–)-
FeII(CO)(CN)2), represents a state commonly found in “standard” [NiFe]-hydrogenases, but it is not
accumulated to large amounts in RH.12,13 The same is true for the Nir-S and Nir-B states carrying an
OH– ligand in the bridging position, which is supposed to derive from either H2O or O2 (Fig. 1). The
apparent lack of these oxidized states has been considered to be a consequence of the O2 tolerance of
the RH.17
Apart from the catalytically active large subunit HoxC, the RH also includes the small subunit
HoxB, which carries three [4Fe-4S] clusters. Previous experiments already showed that the HoxC
subunit can be isolated without HoxB. However, the purified protein was only substoichiometrically
(~ 50%) loaded with nickel and thought to be redox-inactive.18,19
Here, we resumed our previous investigations on isolated HoxC, thereby aiming for a protein
equipped stoichiometrically with the NiFeII(CO)(CN)2 cofactor. By adapting a new cultivation
protocol for the HoxC-overproducing strain we succeeded in purifying homogenous protein, which
turned out to be capable in H2 activation. Moreover, the optimized protein preparation allowed for a
unique spectroscopic view onto the catalytic center of a [NiFe]-hydrogenase, which in the native
enzyme is blurred by the spectral contributions of the iron-sulfur clusters present in the small subunit
in case of UV–visible, Mössbauer, RR, and nuclear resonance vibrational spectroscopy. On the basis
of the unusual catalytic and spectroscopic properties of the isolated large subunit, we were able to
derive important information on the role of the small subunit for the function of [NiFe]-hydrogenase.
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DOI: 10.1039/D0SC01369B
EXPERIMENTAL
Bacterial strains and cultivation
Strains R. eutropha HF574(pGE537)18 and R. eutropha HF574 (pGE567)17 were used for
overproduction of RH (composed of single copies of HoxC and C-terminally truncated HoxB carrying
a Strep-tag II at the C-terminus) and isolated HoxC (carrying a Strep-tag II at the N-terminus). Basic
mineral medium contained H16 buffer (25 mM Na2HPO4, 11 mM KH2PO4 pH 7.0) supplemented
with mineral salts (37.5 mM NH4Cl, 1 µM NiCl2, 18 µM FeCl3, 68 µM CaCl2 and 810 µM MgSO4).
R. eutropha pre-cultures were grown for 48 h at 30 °C and 120 rpm in basic mineral medium
containing 0.4 % fructose and 22.5 µM tetracycline. In contrast to a previous, less time-consuming,
but more aerobic cultivation strategy,18 main cultures were grown in baffled Erlenmeyer flasks filled
to 80% of their capacity with basic mineral medium containing with 0.05% fructose, 0.4 % glycerol
and 22.5 µM tetracycline. Upon inoculation with pre-culture, the cultivation was performed at 30 °C
and 120 rpm until the optical density at 436 nm reached a value of 11-13. Cells were harvested by
centrifugation (11 500 x g, 4 °C, 15 min), and the cell pellet was flash frozen in liquid nitrogen and
stored at –80 °C until further use. Mineral medium for production of [57Fe]-labelled HoxC contained
57FeCl3 instead of 56FeCl3 with natural isotope distribution.
Protein purification
The cell pellets were resuspended in lysis buffer (3-5 mL of buffer per g of cell paste) consisting of
50 mM Tris-HCl, pH 8.0, 150 mM NaCl, protease inhibitor cocktail (complete EDTA-free, Roche)
and DNase I (Roche). Cells in the resulting suspension were disrupted in a French pressure cell (G.
Heinemann Ultraschall and Labortechnik, Schwäbisch Gmünd, Germany) at 1.24∙108 Pa (18,000 psi).
Crude extracts were ultracentrifuged (100,000 x g) for 40 min at 4 °C, and the soluble fraction was
loaded onto a Strep-Tactin high-capacity column (IBA, Göttingen, Germany). The column was
washed with ten bed volumes of washing buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl), and the
proteins were eluted with 4 bed volumes of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl and 3 mM D-
desthiobiotin. The eluted proteins were concentrated by ultrafiltration (4,000 x g, 4 °C) using Amicon
Ultra Ultracel 30 kDa cut-off concentrators (Millipore). The resulting protein solution was filled up
with 20 volumes of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl and concentrated again by ultrafiltration.
The final concentrate was flash-frozen in liquid nitrogen and stored at –80 °C. Size-exclusion
chromatography analysis of HoxC was performed on Superdex S 200 Increase 10/300 GL column
using a buffer composed of 50 mM Tris-HCl pH 8.0, 150 mM NaCl. Protein standards were run under
the same conditions to establish a calibration curve. The protein concentration was determined using
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a Pierce BCA Protein Assay kit (Thermo Scientific). A solution of bovine serum albDuOmI: i1n0.1s0e39r/vDe0SdC0a1s369B
the standard. The protein purity was assessed by SDS-PAGE and subsequent Coomassie Brilliant
Blue G-250 staining. Immunological detection of HoxC was carried out as described before.18
Polyclonal antibodies raised against RH were used for HoxC and RH detection. Alkaline
phosphatase-labelled goat-anti-rabbit IgG (Dianova, Hamburg, Germany) was used as secondary
antibody.
Metal content analysis
The nickel and iron content of HoxC was determined by inductively coupled plasma optical emission
spectrometer (ICP-OES Optima 2100, Perkin Elmer), taking Ni and Fe traces in the purification
buffer into account. Three samples each containing 500 µL of HoxC solution 10 µM were used for
the metal content analysis.
Activity assays
H2 oxidation. H2-mediated reduction of methylene blue was measured spectrophotometrically using
a Cary50 UV−vis spectrophotometer (Varian, Agilent, Santa Clara, California) as previously
described.20 The reaction was performed in a glass cuvette (1 cm path length) at 30 °C. The 2.0-mL
reaction contained a buffer mixture of 50 mM K2HPO4 and 100 mM citric acid (pH 5.0 – 8.0) and 0.2
mM methylene blue. Upon saturation of the mixture by bubbling with H2 gas, the protein sample was
injected using a gas-tight Hamilton syringe. H2-dependent reduction of the dye was followed at
570 nm using a molar extinction coefficient of 13.1 mM1 cm1. Measurements were repeated in
triplicate using two different protein samples.
Catalytic H/D exchange reaction. D2 and HD production rates were calculated upon uptake of H2 in
D2O-based buffer. The amount of D2 and HD gases was measured in a modified O2 electrode chamber
(DW2/2, Hansatech, King’s Lynn, U.K.) connected by a homemade brass adaptor to a mass
spectrometer (OmniStar G3D 301 T3 Quadrupole, Pfeiffer Vacuum, Berlin, Germany). The measured
mass-to-charge ratios (m/z) for H2, HD and for D2 were 2, 3 and 4 respectively, and mass spectrometer
settings have been described previously.21 The reaction chamber was separated from the vacuum side
of the mass spectrometer by a gas-permeable Teflon membrane (12.5 µm, Hansatech). The reaction
volume was 1.9 mL, and the measurement was performed at room temperature under continuous
stirring at 1250 rpm. The reaction buffer contained 50 mM K2HPO4/100 mM citric acid in D2O (pH
3.0 - 9.0) and was bubbled with H2 gas until saturation. Then, protein samples were injected using a
gas-tight Hamilton syringe. D2 and HD production rates were monitored, and data analysis was
performed as described previously.21
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DOI: 10.1039/D0SC01369B
Spectroscopy
UV-Visible spectroscopy. UV-Visible absorption spectra have been measured using a Cary300
UV−vis spectrophotometer (Varian, Agilent, Santa Clara, California). Spectra were accumulated
between 250-800 nm using washing buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl) as a reference
for the baseline. HoxC spectra were acquired using 200 µM and 15 µM protein solution.
Infrared (IR) spectroscopy. HoxC and RH proteins solutions were transferred into a gas-tight,
homemade, temperature controlled (10 °C), transmission cell equipped with two sandwiched CaF2
windows separated by a Teflon spacer (optical pathlength of 50 µm). Spectra with 2 cm1 resolution
were recorded on a Tensor 27 Fourier-Transform spectrometer from Bruker equipped with a liquid
nitrogen cooled mercury cadmium telluride (MCT) detector. Thereby, 200 scans were averaged for a
single spectrum. The sample compartment was purged with dried air. A buffer spectrum (50 mM
Tris-HCl 8.0, 150 mM NaCl) was chosen as reference to calculate the corresponding absorbance
spectra. Bruker OPUS software 7.5 was used for data evaluation and Origin 2018b for preparation of
the figures. HoxC samples were analyzed in a concentration range of 0.5-1.0 mM. RH was analyzed
in a concentration range of 0.2-0.4 mM. HoxC samples used for pH studies have been equilibrated at
the desired pH using a buffered solution of 100 mM K2HPO4 and 50 mM citric acid. K3Fe(CN)6 (E0´=
0.420 V vs SHE at pH 8.0)22, Na2S2O4, K2IrCl6 (E0´= +0.96 V vs SHE at pH 5.0)23 were freshly
prepared in 50 mM Tris-HCl pH 8.0, 150 mM NaCl. K3Fe(CN)6 and K4Fe(CN)6 spectra were
independently acquired to assign the individual ν(C≡N) stretching modes (Fig. S8).
Electron Paramagnetic Resonance spectroscopy. HoxC samples were analyzed in a concentration
range of c.a. 0.5-1.0 mM and RH at 0.1-0.3 mM (70-100 µL). HoxC and RH protein solutions were
transferred into 4 mm quartz EPR tubes, quenched in cold ethanol (193 K) and frozen in liquid
nitrogen for further analysis. A Bruker EMXplus spectrometer equipped with an ER 4122 SHQE
resonator, an Oxford EPR 900 helium flow cryostat and an Oxford ITC4 temperature controller was
used for EPR measurements. The baseline correction of the experimental spectra was done by
subtracting a spectrum of buffer solution measured with the same parameters. If necessary, the spectra
were additionally corrected by using a polynom or spline function. Experimental parameters: 1 mW
microwave power, microwave frequency 9.29 GHz, modulation amplitude 10 G and 100 kHz
modulation frequency. Spectra simulation was performed with the Matlab toolbox Easyspin 5.2.25.
Mössbauer. Zero-field Mössbauer spectra were recorded on a SEECO MS6 spectrometer that
comprises the following instruments: a JANIS CCS-850 cryostat, including a CTI-CRYOGENICS
closed cycle 10 K refrigerator, and a CTI-CRYOGENICS 8200 helium compressor. The cold head
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and sample mount are equipped with calibrated DT-670-Cu-1.4L silicon diode temperature probes
DOI: 10.1039/D0SC01369B
and heaters. Temperature is controlled by a LAKESHORE 335 temperature controller. Spectra are
recorded using a LND-45431 Kr gas proportional counter with beryllium window connected to the
SEECO W204 γ-ray spectrometer that includes a high voltage supply, a 10 bit and 5 μs ADC and two
single channel analyzers. Motor control and recording of spectra is taken care of by the W304
resonant γ-ray spectrometer. For the reported spectra a RIVERTEC MCO7.114 source (57Co in Rh
matrix) with an activity of about 1 GBq was used. All spectra were recorded on frozen solutions at
13 K and data were accumulated for about 24 hours. Mössbauer data were processed and simulated
using the WMOSS4 program (www.wmoss.org). Isomeric shifts are referenced to α-iron at room
temperature. [57Fe]-HoxC and [57Fe]-HoxCox were analyzed in a concentration range of ca. 1-1.5 mM.
Time-dependent density functional theory (TDDFT) calculations. The active site models used for the
TDDFT calculations include Ni and Fe atoms and their first coordination spheres, and their initial
geometries were based on the [NiFe]-hydrogenase active site of Desulfovibrio fructosovorans (pdb
code: 1YRQ).24 The four metal-coordinating cysteine residues were truncated at the level of their C
atoms, whose coordination sphere was subsequently saturated by adding an H atom. Calculations on
the resulting models was carried out using the CAM-B3LYP exchange-correlation functional.25 A
def2-TZVP basis set26 was performed for all atoms to carry out geometry optimization, which was
followed by the UV-vis spectra prediction, as detailed below. All calculations were carried out with
the GAUSSIAN16 software,27 upon constraining the cysteine C atoms at their crystallographic
positions. If not otherwise stated, the cysteine C atoms were the only geometric constraints imposed
to the models. The models were soaked in the continuum polarizable model PCM28,29 implemented
in GAUSSIAN16. We chose a dielectric constant of  = 4, which was shown to be appropriate for
metalloprotein modeling,30–32 and the remaining PCM parameters were kept at their default values.27
For our models, we considered both water-ligated and hydroxo-ligated species, which had an overall
charge of -2 and -3 e, respectively. Self-consistent field computations were carried out within the
closed-shell framework. Indeed, efforts to localize broken-symmetry open-shell singlets33 led to
convergence on non-spin-polarized solutions. As far as the computation of UV-vis theoretical spectra
is concerned, TDDFT calculations were performed using the optimized geometries and the
corresponding electronic structures. Altogether 20 states were considered for TDDFT computations
for each of the optimized structures.
RESULTS
HoxC contains an integer NiFe(CO)(CN)2 cofactor
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As-isolated HoxC protein (HoxCai) was overproduced in R. eutropha and subsequently purified by
DOI: 10.1039/D0SC01369B
affinity chromatography as described in the Experimental and the SI (Fig. S1, S2). ICP-OES
measurements of purified HoxCai samples revealed the presence of (1.10 ± 0.05) Fe and (1.06 ± 0.03)
Ni (Fe/Ni = 0.96), indicating full occupancy with the [NiFe] cofactor. The pale brownish color of the
protein samples tempted us to perform UV-vis absorption spectroscopy. Indeed, a diluted HoxCai
sample (15 µM) displayed a weak absorption in the region between 360 nm and 400 nm, while a more
concentrated sample (200 µM) showed even two maxima at 370 nm and 390 nm (Fig. 2A). Based on
the protein concentration, we calculated an apparent molar extinction coefficient of ε = (3800 ± 400)
M1cm1. We assign this particular UV-vis absorption to the NiFe(CO)(CN)2 cofactor, which was
thus far undetectable due to the superimposed absorption of the FeS clusters present in the
hydrogenase small subunit.
Further evidence for the correct incorporation of the [NiFe] cofactor into HoxCai was obtained by IR
spectroscopy, which probes the ν(C≡O) (1870-2020 cm1) and ν(C≡N) (2030-2150 cm1) stretching
modes of the CO and CN– ligands of the [NiFe] catalytic center.4,34 After aerobic purification, the IR
spectrum of HoxCai exhibited two CO bands at 1941 cm1 and 1952 cm1 and two broad CN-related
absorptions at 2066 cm1 and 2084 cm1. The IR spectrum of as-isolated RH, for comparison, shows
frequencies at 1943 cm1, 2070 cm1, and 2081 cm1, attributed to the Nia-S state of the active site
(Fig. 1, Fig. 2B, Table S1).13,35
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DOI: 10.1039/D0SC01369B
A
B
C
Figure 2. Spectroscopic characterization of HoxCai. (A) UV-vis absorption spectra of diluted (solid
line) and concentrated (inset, dash-dotted line) HoxCai showing two bands at 370 and 390 nm. (B)
Comparison of IR spectra of HoxCai (top) and as-isolated RH (bottom) at a concentration of 0.2 mM
and pH 8.0. (C) IR spectra of HoxCai recorded at pH 8.0 (trace a) and pH 6.0 (trace b) normalized to
the absorption intensity at 1941 cm1. Trace c (magnified by a factor 2.5 to match intensity in a)
displays the difference spectrum of “a” minus “b” to isolate the active site state prevailing at high pH.
In the difference spectrum d, trace b is subtracted from trace c to isolate the species dominating at
low pH. Trace e represents the spectrum of HoxCai, first incubated at pH 6.0 and subsequently
buffered at pH 8.0, which demonstrates interconvertibility of the active site states.
Thus, metal determination, UV-vis, and IR spectroscopy confirmed that HoxC is completely
loaded with an intact NiFe(CO)(CN)2 cofactor. The IR band broadening, however, indicated a certain
structural heterogeneity of the active site in HoxCai when compared to RH. In fact, the presence of
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two CO vibrations suggests different conformations or/and redox states of the [NiFe] cofactor. To get
DOI: 10.1039/D0SC01369B
a clue about the origin of these different conformations, we checked first the integrity of the isolated
HoxCai by size-exclusion chromatography (Fig. S2). Indeed, HoxCai occurred in a monomeric and a
dimeric version. Both versions were subjected to IR spectroscopy which revealed identical spectra
(Fig. S3). Thus, the two different CO vibrations, were not a result of the mixture of monomeric and
dimeric HoxCai, and all further experiments were therefore performed with HoxCai protein obtained
after affinity chromatography. Remarkably, extended exposure of HoxCai to carbon monoxide,
hydrogen gas or even sodium dithionite (4-fold molar excess) revealed no significant changes of the
original IR spectrum (Fig. S4). Thus, HoxCai does apparently not bind exogenous CO and withstands
reduction at physiological redox potentials.
To investigate whether the two HoxCai active site states related to the CO stretching modes at
1941 cm1 and 1952 cm1 represent different protonation states of the “same” species, we carried out
pH dependent IR spectroscopic measurements (Fig. 2C). HoxCai, which was initially purified at pH
8.0, was equilibrated with buffer at pH 6.0. This pH shift resulted in an increase of the stretching band
at 1941 cm1, while the absorption at 1952 cm1 dropped in intensity (Fig. 2C, traces a and b). The
process turned out to be reversible, as the return to pH 8.0 revealed the initial ratio of the bands at
1952 cm1 and 1941 cm1 (Fig. 2C, trace e). As the HoxCai protein was instable at pH values lower
than 5.5 and higher than 9.0, it was impossible to isolate the [NiFe] site in only one of the two states.
However, by calculating difference spectra (pH 8.0 – pH 6.0), we were able to deconvolute the
CO/CN bands and to delineate the two individual states in almost pure forms (Fig. 2C, traces c and
d). Based on the results of the pH-dependent IR analysis of HoxCai and the apparent lack of reactivity
of the protein towards exogenous H2 and CO gases (Fig. S4), we attributed the two CO modes to the
two isoelectronic Nir-SI and Nir-SII states, which just differ in their degree of protonation. Taking into
account previous observations for standard [NiFe]-hydrogenases,36–38 the main CO band at 1952 cm1
was assigned to Nir-SI, which supposedly contains a metal-bridging hydroxo ligand resulting in a NiII-
(OH–)-FeII(CO)(CN)2 configuration of the active site. By lowering the pH, the bridging hydroxo
ligand becomes protonated, thereby yielding a bound H2O molecule,37,39 which results in the Nir-SII
state with a NiII-(HOH)-FeII(CO)(CN)2 structure and a ν(C≡O) stretching vibration at 1941 cm1.
The presence of two interconvertible water-based active site species in the HoxCai preparation
prompted us to investigate whether the UV-vis spectrum absorptions shown in Fig. 2A can be
reproduced by time-dependent density functional theory (TDDFT) calculations based on geometry-
optimized [NiFe] cofactor models. Indeed, the characteristic absorption maxima below 300 nm as
well as 370 and 390 nm were mimicked only by assuming the co-presence of NiII-(HOH)-FeII and
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NiII-(OH)-FeII species, both of them carrying low-spin metal centers (see SI, Figs. S5-S8. Tables S2-
DOI: 10.1039/D0SC01369B
S4).
In contrast to HoxCai, as-isolated RH resided predominantly in the Nia-S state at pH values ranging
between 5.5 and 9.0 (Fig. S6A). Interestingly, the IR spectrum contained also a minor ν(C≡O)
stretching mode at 1963 cm1, which was most dominant at pH 5.5 (Fig. S6A). Upon further exposure
to O2 (at pH 5.5), which was accomplished by slow air diffusion into the transmission cell, the band
at 1963 cm1 increased further, and ν(C≡N) stretching vibrations at 2089 and 2097 cm1 became
clearly visible (Fig. S6B). The CO-related band at 1963 cm1 has already been assigned to the Nir-B
state of RH, while the corresponding ν(C≡N) stretching modes were not resolved in the previous
study.12
The [NiFe] cofactor of HoxC responds to chemical oxidation
The IR spectrum of HoxCai did not show any detectable changes when the protein was incubated with
either H2 or CO (Fig. S4). Treatment with two different chemical oxidants, K3Fe(CN)6 and K2Ir(Cl)6,
however, resulted in distinct frequency upshifts of the CO- and CN-related bands (Fig. 3A, Fig. S7A
and S7B), indicating an oxidation of the [NiFe] cofactor. The IR spectrum of K3Fe(CN)6-oxidized
HoxC (HoxCox) revealed a broadened CO absorption at 1978 cm1 with a shoulder at 1969 cm1. CN
ligand-related signals were observed at 2081 and 2090 cm1 (Fig. 3A, Table S1). The prominent band
3
at 2116 cm1 corresponds to the ν(C≡N) stretching of Fe(CN)6 , while the peak at 2036 cm1 belongs
to the ν(C≡N) stretching of the by 1e electron reduced Fe(CN)62 (Fig. S8). A very similar spectrum
was obtained upon incubation of HoxCai with the stronger oxidant K2Ir(Cl)6 (Fig. 3A). While 2-3
equivalents of K3Fe(CN)6 were sufficient to oxidize HoxC completely, 25 equivalents of K2Ir(Cl)6
were required to obtain quantitatively oxidized [NiFe] cofactor. The higher demand for the iridium
compound can be explained by its redox potential of +0.96 V, which is large enough to extract
electrons from H2O.40 Nonetheless, since the treatments with K2Ir(Cl)6 and K3Fe(CN)6 resulted in
exactly the same HoxC-derived IR bands, we can exclude that further ν(C≡O) or ν(C≡N) stretching
2
modes were covered by the contribution of Fe(CN)6 /Fe(CN)63 modes (Fig. 3A, Fig. S7A, S7B and
Fig. S8). As for HoxCai, treatment of RH enzyme with K3Fe(CN)6 led to the formation of Nir-B, but
only in minuscule amounts (Fig. S9A).
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DOI: 10.1039/D0SC01369B
A
B
Figure 3. IR and EPR spectroscopic characterization of HoxC preparations treated with chemical
oxidants and reductants. (A) IR spectra (normalized to the protein concentration) of redox-treated
HoxC preparations. From bottom to top: HoxCai, HoxCox1 (HoxCai oxidized with 28 equivalents of
K2IrCl6) HoxCox2 (HoxCai oxidized with 4 equivalents of K3Fe(CN)6), and HoxC´ (prepared from
K3Fe(CN)6-oxidized HoxCox, subsequently reduced with Na2S2O4). Color-coded (redox) states: red,
Nir-SI; black, Nir-SII; orange, Ni-L; magenta, Nir-B´; and blue, Nir-B´´. Absorptions of [Fe(CN)6]3
(2116 cm1) and [Fe(CN)6]2 (2036 cm1) were partially cut to emphasize the vibrational modes of
the active site. (B) EPR spectra of redox-treated HoxC preparations measured at 20 K. From bottom
to top: HoxC (HoxCai treated with sodium dithionite), HoxCai, HoxCox (HoxCai oxidized with 2
equivalents of K3Fe(CN)6, and HoxC´ (prepared from K3Fe(CN)6-oxidized HoxCox, subsequently
reduced with Na2S2O4). The asterisk in the HoxC´ spectrum denotes the signal of a so far unknown
paramagnetic Ni species. The simulated spectrum of HoxCox including the Nir-B´ and Nir-B´´ species
is shown as dotted line. The simulations of the single EPR spectra are shown in Fig. S7D.
The blue shift of the ν(C≡O) stretching modes upon oxidation (Fig. 3A) indicated a change in the
redox state of the catalytic center of HoxCai. In case of [NiFe]-hydrogenases, nickel has been
identified to play the key role as it can adopt the diamagnetic 2+ state as well as the paramagnetic 1+
and 3+ states (Fig. 1).4,41 Therefore, we investigated the different redox states of HoxC by EPR
spectroscopy (Fig. 3B). HoxCai appeared to be EPR silent, which is in accordance with a low-spin
NiII-FeII configuration of the active site as present in the diamagnetic Nir-SI and Nir-SII states. The
EPR spectrum of HoxCox displayed a rather rhombic signal (Fig. 3B blue trace, Fig. S7C). By using
numeric simulation, the complexity of the spectrum was deconvoluted and interpreted with at least
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two different overlapping rhombic signals of paramagnetic (S=1/2) NiIII species (FigD. OSI:71D0.1)0.39F/Du0rStCh0e1r369B
EPR measurements at varying microwave power and different temperatures revealed a very similar
spin relaxation behavior and, hence, indicated a structural and electronic similarity of the two species
(Fig. S10). Based on the observed characteristic blue shift in the IR spectra upon oxidation, the
presence of paramagnetic NiIII, and the reducibility of the state (see below), we infer a NiIII-(OH–)-
FeII(CO)(CN)2 configuration in HoxCox. In classical heterodimeric [NiFe]-hydrogenases this active
site configuration is designated Nir-B (Fig. 1).42,43 Based on the relative intensities of the CO
vibrations at 1969 and 1978 cm1 (Fig. S7B) and the corresponding intensities of the simulated EPR
spectra (Fig. S7D), we assign these frequencies to Nir-B´ and Nir-B´´, respectively (Table S5).
Notably, accurate simulation of the Nir-B´´-related EPR spectrum required the implementation of an
unusually large g-strain. This indicates structural heterogeneity, which is probably caused by an
enhanced flexibility of the [NiFe] cofactor. Such flexibility would also explain the large band
broadening observed for the ν(C≡O) stretching mode at 1978 cm1 (Fig. 3A, Fig. S7A and S7B). It is
worth to note that a small paramagnetic signal indicative for Nir-B was also observed for RH protein
(Fig. S9B), which correlates with the IR-based ν(C≡O) stretching mode at 1963 cm1 (Fig. S6).12
A
B
57
Figure 4. Mössbauer spectra of Fe-labelled HoxC in the as-isolated form (A) and chemically
oxidized with K3Fe(CN)6 (B). Black dots represent the experimental data. Red lines indicate the
simulations, and the corresponding isomer shifts (δ) and quadrupole splittings (ΔEQ) are shown. Low-
intensity signals of high-spin FeII were also detected (δ = 1.43, ΔEQ = 3.02 mm s1 for HoxCai and δ
= 1.43, ΔEQ = 3.13 mm s1 for HoxCox) representing 8-10 % of the total iron content.
To identify the redox and spin state(s) of the active site iron, we performed Mössbauer
spectroscopy on 57Fe-labelled HoxC protein. In fact, the isolated HoxC subunit allowed for the first
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time an unadulterated view on the single iron in the catalytic center, which is usuallyDmOI:a1s0k.1e03d9/bD0ySCth01e369B
numerous iron atoms of the FeS clusters in standard hydrogenase preparations containing the small
subunit. Mössbauer spectroscopy showed that the active site iron ion in HoxCai is characterized by a
low isomer shift (δ = 0.14 mm s1), which hardly differs from HoxCox (δ = 0.15 mm s1) (Fig. 4 and
Fig. S11). The additional low-intensity resonances are derived from a high-spin FeII contamination
with a 8-10 % partitioning. Also the quadrupole splitting parameters of the iron ions in HoxC (ΔEQ
= 0.65 mm s1) and HoxCox (ΔEQ = 0.59 mm s1) are comparable (Table S6), reflecting a similar
electric field and, thus, ligand environment in both states. These observations are in agreement with
an active site iron(II) ion persisting in the low-spin state, which is consistent with the current view on
the catalytic properties of the [NiFe] cofactor.4
The oxidized [NiFe] cofactor of HoxC responds to chemical reduction
As described above, HoxCai protein turned out to be inert for reductants (Fig. S4). Therefore, we
tested whether K3Fe(CN)6-oxidized HoxCox can be reduced. Indeed, upon addition of sodium
dithionite, the CO- and CN-related bands shifted back to their original positions as previously
observed for HoxCai (Fig. 3A). Only a minor change in the ratio of the Nir-SI and Nir-SII states was
observed. Because of the almost identical IR pattern compared to HoxCai, we designated the
dithionite-treated HoxCox to as HoxC´. Notably, a minor broad band appeared at 1889 cm1 upon
reductive treatment (Fig. 3A). CO stretching vibrations in this spectral region are commonly related
to the Ni-L state, characterized by a NiI-(–)-FeII(CO)(CN)2 configuration of the active site.4,44 EPR
spectroscopy confirmed the presence of a paramagnetic species in HoxC´ resembling the spectral
signature of Ni-L (Fig. 3B, Table S5). In accordance with the IR data (Fig. 3A), however, the Ni-L
state occurs only in traces (Fig. 3A).
H2 splitting capacity of HoxC
[NiFe]-hydrogenases are known to perform the catalytic hydrogen/deuterium (H/D) exchange
reaction,5,45,46 which is independent of long-range electron transfer mediated by the iron-sulfur
clusters in the small subunit. Therefore, it is particularly suited for probing the H2 binding and
splitting capacity of isolated large subunits.5 The catalytic H/D exchange reaction occurs according
to the reactions:
1) H2 + D2O ⇌ HD + HDO
2) HD + D2O ⇌ D2 + HDO
Thus, the reversible H2 splitting capacity of a given catalyst can be followed by measuring H2
consumption and the concomitant production of HD and D2. HoxC samples were placed in D2O-
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based buffer and the reaction was started by addition of H2-saturated buffer. The release of gaseous
DOI: 10.1039/D0SC01369B
products was measured with a mass spectrometer by monitoring the mass-to-charge ratios (m/z) 3 and
4, indicative of HD and D2, respectively (Fig. 5A). Purified HoxC showed highest activities at pH
4.5, with HD and D2 production rates of 0.89 ∙ 103 s1 and 0.19 ∙ 103 s1, respectively (HD/D2 = 4.5,
Table S7). To verify the significance of these low activities, we conducted control experiments with
bovine serum albumin and a hydrogenase maturase complex, consisting of the HypCD proteins
carrying the Fe(CO)(CN)2 unit of the [NiFe] cofactor.47 Both samples did not show any catalytic H/D
exchange activity (Fig. S12). Furthermore, the HD and D2 evolution rates of purified HoxC increased
linearly dependent on both the protein (Fig. S13) and H2 concentration (Fig. S14). Notably, no
differences in activity and the HD/D2 ratios were detected for the monomeric and dimeric forms of
HoxC (Fig. S15). The HoxC-mediated H/D exchange activity decreased at pH values < 4.0, which
was likely caused by protein instability. At pH values above 7.0, HD and D2 production were not
detected anymore. A comparative investigation was performed with the RH protein, revealing highest
HD and D2 production rates of 460 ∙ 10-3 s1 and 1802 ∙ 103 s1, respectively, at pH 7.0 (Fig. S16,
Table S7). At pH 5.0 the HD production of RH was 235 ∙ 103 s1, i.e. more than 200-fold higher than
that of HoxC (Fig. S16, Fig. 5A). RH-mediated activity was observed at pH 3 up to pH 9, which
correlates with the prevailing Nia-S species in as-isolated RH within a broad pH interval (Fig. S6A).
A
B
Figure 5. HoxC-mediated H2 activation. (A) HD and D2 production rates of HoxCai as a function of
the pH. 100-200 µg of HoxCai were incubated with H2 gas in a D2O-based buffer at different pH
values. (B) HD and D2 production rates of HoxCai (pH 4.5) in the presence of different O2
concentrations. The values provided at the top of the columns denote the rates in relation to the rate
determined in the absence of O2. Error bars indicate the standard deviation calculated from three
independent measurements.
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Next, we investigated whether HoxC is able to split H2 reversibly also in the preseDnOcIe: 10o.1f03O9/2D.0SICn01a369B
previous study, we showed that the catalytic H/D exchange performed by the purified premature
MBH large subunit, preHoxG, is only barely affected by O2, even if added at high concentration.5
Native RH has also been shown to sustain catalytic activity in the presence of O2.48 We measured the
catalytic H/D exchange activity of HoxC at pH 4.5 in the presence of 5, 10, and 20% (v/v) O2-
saturated buffer while keeping the initial H2 concentration (Fig. 5B) constant. When compared to O2-
free reaction conditions, HoxC maintained 84.5% of HD and 58% of D2 production rates at 20% O2.
Notably, the HD production activity was less affected by O2 than the D2 production (Fig. 5B). This
can be easily explained by the longer exposure of the catalytic center to O2 in the case of the complete
conversion of H2 into D2 via HD.
We also tested the capability of HoxC to transfer electrons derived from H2 oxidation to an
artificial electron acceptor. Indeed, HoxC catalyzed the H2-mediated reduction of methylene blue, a
two-electron acceptor with E0’ = + 40 mV, with a specific activity of (0.010 ± 0.001) U mg1 at pH
5.2 (Fig. S17). It is, however, unclear whether the electrons are transferred to the dye in a step-wise
mechanism or even as a hydride. No activity was determined in the absence H2 (replaced by N2) or
without protein. Moreover, the activity significantly decreased at pH values lower than 4.0 and higher
than 5.5, which is consistent with the pH dependency of the catalytic H/D exchange reaction (Fig.
5A). Purified RH protein, for comparison, catalyzes H2 oxidation with a specific activity of (3.8 ± 0.5)
U mg1 at pH 7.0. At pH 5.2, RH activity decreases to 2.0 ± 0.1 U mg1, which is still more than 200-
fold higher than that of HoxC (Fig. S17).
DISCUSSION
In this study we investigated an isolated, fully assembled large subunit of a [NiFe]-hydrogenase by
means of biochemical and spectroscopic methods. Isolated [NiFe]-hydrogenase large subunits have
been analyzed before, but they were either sub-stoichiometrically loaded with nickel5,18 or even did
not contain at all metal ions.6 The stoichiometric presence of both nickel and iron in HoxC allowed
for the first time determination of the UV-visible molar extinction coefficient of the [NiFe] cofactor
with ε = 3800 M1cm1. This value is much lower than that of [4Fe-4S] clusters49 (13,000-16,000
M1cm1) which generally dominate the electronic absorbance properties of [NiFe]-hydrogenases.
Due to the lack of more appropriate in vitro/in silico model systems, we compared the HoxC
spectroscopic signatures to those of bioinspired [NiFe] model compounds carrying low-spin (S=0)
NiII and FeII ions.50-52 Moreover, the UV-vis spectrum of HoxCai could be well-reproduced by TDDFT
calculations on active site models carrying low-spin NiII and FeII centers that are occupied by water
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derived ligands, which perfectly agrees with the corresponding biochemical and speDcOtrI:o1s0c.1o03p9i/Dc0dSCa0t1a369B
(see below).
The presence of an intact [NiFe(CO)(CN)2] cofactor was verified by IR spectroscopy. However, in
contrast to RH (HoxBC), which occurs almost completely in the catalytically competent Nia-S state
(NiII-(–)-FeII(CO)(CN)2)10 (Fig. 2), HoxCai resides predominantly in the Nir-S conformation (NiII-
(OH–)-FeII(CO)(CN)2). Absence of EPR-detectable signals for HoxCai further supports a diamagnetic
low-spin NiII-FeII arrangement of the active site, as suggested to be present as well in standard [NiFe]-
hydrogenases.10,14,53 Thus, despite the aerobic purification, the active site nickel remained in the 2+
state, while the iron center retained a low-spin FeII configuration. The latter was verified by
Mössbauer spectroscopy of 57Fe-labelled HoxC protein (Fig. 4). So far, Mössbauer studies on [NiFe]-
hydrogenases (13,54) including RH have revealed spectra that were dominated by iron-sulfur cluster
signals from three [4Fe-4S] clusters, which required sophisticated simulation procedures to
deconvolute the contributions of single Fe species. In case of RH, for instance, the signal of the active
site iron contributed only with 7 % to the total spectrum (Table S6).13 Nevertheless, the isomer shift
and quadrupole splitting of the iron atom of the RH active site in the Nia-S state have been calculated
with 0.1 mm s1 and 1.60 mm s1, respectively. In reduced RH, which resides predominantly in the
in Nia-C state, however, the isomer shift and quadrupole splitting shifted to 0.07 mm s1 and 0.69 mm
s1, respectively, suggesting significant changes in the ligand environment of the FeII ion upon
reduction. In fact, the observed decrease in the quadrupole splitting has been correlated with the
change from a 5-fold coordinated FeII in the Nia-S state [NiII-(–)-FeII(CO)(CN)2] to a 6-fold
coordinated FeII in Nia-C [NiIII-(H–)-FeII(CO)(CN)2].13 Our Mössbauer investigations on 57Fe-labelled
HoxC allowed an exclusive view on the signal of the low-spin FeII in the active site (Fig. 4 and Fig.
S11). Interestingly, the quadrupole splitting parameters for HoxCai and HoxCox are similar (0.65 vs.
0.59 mm s1) to each other and both are close to that of the Nia-C state of RH (0.69 mm s1).13 This
suggests that the number of iron ligands did not vary between the three species, but only the oxidation
state of the nickel. This observation is supportive for a 6-fold coordination of the iron atoms of both
HoxCai and HoxCox, in line with the proposed presence of bridging H2O or OH– ligands of the active
site in the Nir-S and Nir-B states.
According to our EPR measurements, the [NiFe] site changed from a diamagnetic state in HoxCai
into a paramagnetic state in HoxCox (Fig. 3B). The corresponding spectrum, however, contained two
different species whose spectral signatures deviate from the standard Nir-B state (Fig. S7D). Hence,
we designate these so far unknown states as Nir-B´ and Nir-B´´ (see Table S5 for g-values). The
occurrence of two states instead of a pure Nir-B spectrum supports different arrangements of the
[NiFe] core, as a result of an increased conformational flexibility of HoxC lacking the small subunit
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HoxB. The increased conformational flexibility compared to the native enzyme might also explain
DOI: 10.1039/D0SC01369B
the relatively high frequency of the CO modes of the Nir-B species (Fig. 3A). Chemical reduction of
HoxCox with dithionite resulted in a minor appearance of Ni-L species (NiI-(–)-FeII(CO)(CN)2), which
is nowadays discussed to be an intermediate of the catalytic cycle.16,55 In the present case, Ni-L seems
to be derived from the paramagnetic Nir-B`-Nir-B´´ species under 2e- reduction provided by the
dithionite treatment (reductant) involving water and/or OH- release and remains kinetically trapped.
However, most of the dithionite-reduced HoxCox population remained diamagnetic and was
dominated by Nir-SI and Nir-SII species (Fig. 3).
Our data show that HoxC is redox-active and occurs in active site states common to [NiFe]-
hydrogenases. However, apart from minor amounts of Ni-L, no states related to the catalytic cycle
(Fig. 1A) were enriched. In fact, some of the intermediates observed for HoxC, including all Nir
states, are essentially absent in fully assembled RH. Although the isolated HoxC and the HoxC
subunit in RH share the same amino acids composition, the absence of the small subunit obviously
changes the environment of the active site in a way that prevents canonical H2/H+ cycling.
Nevertheless, HoxC is capable of activating molecular hydrogen, as has been previously observed for
the isolated large subunit, preHoxG, of the membrane bound hydrogenase of R. eutropha.5 The
catalytic H/D exchange activity of the two proteins in D2O-based buffer is based on their capacity to
split H2 into H+ and H-, resulting in the detectable products HD and D2. The HD/D2 production rates
were 0.89 ∙ 103/0.193 ∙ 103 s1 for HoxC and 0.74 ∙ 103 /2.04 ∙ 103 s1 for preHoxG.5 The RH and
MBH of R. eutropha, by contrast, showed HD and D2 production rates of 460 ∙ 103 s1/1802 ∙ 103
s1 and 11.21 ∙ 103 s1/12.55 ∙ 103 s1, respectively (Table S7).5 Thus, HoxC has a considerably
lower activity than RH but an only 12 times lower HD production rate compared to that of MBH.
Besides, HoxC activity was linearly dependent on the amount of enzyme and substrate (Fig. S13 and
S14). We conclude that the absence of the confining and hydrophobic environment created by the
small subunit decreased the affinity for H2. Consistently, only small variations in the HD/D2 ratios
were observed for different HoxC concentrations (Table S8), which indicates the absence of a
prominent cage effect. By contrast, the corresponding HD/D2 ratios of native [NiFe]-hydrogenases
were shown to be highly dependent on the enzyme concentration.46,56
Notably, the highest HoxC activity was observed at rather acidic conditions, and only negligible
activity was measured at physiological pH. This is consistent with our IR data that show that the
Nir-SI state (NiII-(OH–)-FeII(CO)(CN)2) dominates at high pH. Upon lowering the pH, the bridging
hydroxo ligand becomes protonated resulting in the formation of the Nir-SII species (NiII-(HOH)-
FeII(CO)(CN)2). An acid-base equilibrium has also been described for Ni-S states of standard [NiFe]-
hydrogenases39,57–59, where accumulation of the Nia-S state was observed as a result of protonation of
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the bridging OH– ligand of the Nir-S state. The corresponding H2O molecule might be located in
DOI: 10.1039/D0SC01369B
proximity of the active site. In case of HoxCai, with a rather solvent-exposed catalytic cofactor,
however, water may remain in the bridging position of the active site. Only a small HoxC fraction in
the Nir-SII state lost the bridging water ligand, resulting in the formation of the catalytically active
Nia-S species. This hypothesis is in line with the broadening of the CO and CN stretching modes of
HoxCai, which became more pronounced at low pH (Fig. 2C). This can be interpreted with Nia-S-
related absorption bands underneath those of the prevailing Nir-SI and Nir-SII species.
Apart from explicit deviations in activity, HoxC, RH and preHoxG, and MBH exhibit also
different HD/D2 ratios (Table S7). While the HD production rate of HoxC is much higher than that
of D2, RH, preHoxG, and MBH show the opposite trend. This observation supports the assumption
that the active site of HoxC lies in proximity to the protein surface (Fig. 1), resulting in a facilitated
release of HD. In case of preHoxG, the C-terminal extension may shield the catalytic center,5 and in
RH/MBH the active site is deeply buried within the holoenzyme35,60. This results in the retention of
HD gas close to the active site, facilitating a second catalytic cycle, leading to the predominant
formation of D2.
It has been outlined before that at metal centers H2 can compete both thermodynamically and
kinetically with H2O as a ligand, and the electrophilicity of a metal center must be just right to favor
binding of H2 over water.61 Consequently, binding and processing of H2 should be particularly easy
at dedicated metal centers where the effective H2O concentration is low. Thus, based on the peculiar
biochemical and spectroscopic properties of HoxC, one important role of the small hydrogenase
subunit seems to guarantee a hydrophobic environment of the catalytic center. This becomes evident
when regarding the catalytic H/D exchange activity, which is independent of long-range electron
transfer and, hence, should be independent of the electron-transfer capacity of the small subunit.
Nevertheless, HoxC showed a 200-fold lower H/D exchange activity than native RH, suggesting a
significantly increased competition between H2 and H2O for binding at the active site. Preventing
water access to the active site and the resulting formation of oxidized states such as Nir-B is a
prerequisite particularly for the H2-sensing function of the RH, which needs to react immediately if
H2 is available.17 The potential inhibitory role of water in catalysis should also be taken into account
for chemical synthesis of [NiFe] mimics that are functional in aqueous solutions.62
Apart from active site protection, the small subunit is likely responsible for activating the catalytic
center in a redox-dependent manner upon complex formation with the large subunit. We showed that
HoxCai is “locked” primarily in the Nir-S state(s) protecting the enzyme from redox inactivation. The
presence of an OH–/H2O ligand in the bridging position between iron and nickel prevents interaction
with H2 and potentially harmful molecules such as CO and O2 at physiological potential. This is
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particularly important for hydrogenase maturation under aerobic conditions as iDtOoI: c10c.1u0r3s9/Di0nSC0R1.369B
eutropha.63
CONCLUSIONS
Our biochemical and spectroscopic investigations of the isolated large subunit HoxC of the regulatory
[NiFe]-hydrogenase reveal that the catalytic center is equipped with stoichiometric amounts of nickel
and iron as well as diatomic CN– and CO ligands coordinated to the latter. Moreover, the protein is
capable of H2 activation as shown by catalytic H/D exchange and methylene blue reduction
measurements. The fact, that the HoxC activities were drastically lower compared to native RH may
increase the possibility to trap early (short-lived) intermediates that so far escaped from spectroscopic
detection. For instance, by exchanging presumably proton-accepting amino acid residues near the
catalytic center, e.g. invariant glutamate and arginine residues,64–66 could enable the identification of
the proposed dihydrogen-metal sigma complex of the active site. Based on its distorted coordination
within the active site, nickel is currently assumed to be initial target of dihydrogen.67 Corresponding
experimental evidence, however, is not yet available.
Remarkably, HoxC retains the ability to split H2 in the presence of molecular O2, making it a
companion of the isolated large subunit, preHoxG, of the membrane-bound [NiFe]-hydrogenase of
R. eutropha.5 To unravel whether this is a general feature, it is of high interest to isolate catalytically
competent large subunits of O2-sensitive [NiFe]-hydrogenases and to investigate their catalytic H/D
exchange properties in the absence and presence of O2. In fact, the premature large subunit, preHyhL,
of the O2-sensitive [NiFe]-hydrogenase of Thermooccus kodakarensis has already been isolated and
its crystal structure was determined.6 Intriguingly, the active site pocket revealed to be metal cofactor-
free. Therefore, the RH large subunit, HoxC, represents thus far the best characterized miniaturized
model of a [NiFe]-hydrogenase, and the biochemical and biophysical data presented in this study
might be used as benchmark to inspire chemists to synthetize well-performing biomimetic complexes
able split and produce H2 under ambient conditions.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors are grateful to Janna Schoknecht for excellent technical assistance in RH preparation,
Anne-Christine Schulz for providing HypCD samples, Dr. Stefan Rieske and Prof. Dr. Silke
Leimkühler (Universität Potsdam) for ICP-OES measurements, and Dr. Lars Lauterbach (TU Berlin)
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for the design of a versatile sample holder for Mössbauer spectroscopic investigations of biological
DOI: 10.1039/D0SC01369B
samples. Quantum chemical calculations were performed on the HPC Marconi cluster at CINECA
(CINECA-Bicocca agreement). G.C., O.L., I.Z., P.H. and S.P.C are grateful to the Einstein
Foundation Berlin for funding (grant number EVF-2016-277). This work was also funded by the
Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany´s
Excellence Strategy – EXC 2008 – 390540038 – UniSysCat. The authors are indebted for EU
financial support (Article 38.1.2, GA) within the European Union’s Horizon 2020 research and
innovation program under grant agreement No 810856. S.P.C. acknowledges funding for his work
through NIH GM-65440.
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Footnote
† Electronic supplementary information (ESI) available: Control experiments.
25