Vibrational spectroscopy reveals the initial steps of biological hydrogen evolution - PubMed (original) (raw)

. 2016 Nov 1;7(11):6746-6752.

doi: 10.1039/c6sc01098a. Epub 2016 Jul 11.

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Vibrational spectroscopy reveals the initial steps of biological hydrogen evolution

S Katz et al. Chem Sci. 2016.

Abstract

[FeFe] hydrogenases are biocatalytic model systems for the exploitation and investigation of catalytic hydrogen evolution. Here, we used vibrational spectroscopic techniques to characterize, in detail, redox transformations of the [FeFe] and [4Fe4S] sub-sites of the catalytic centre (H-cluster) in a monomeric [FeFe] hydrogenase. Through the application of low-temperature resonance Raman spectroscopy, we discovered a novel metastable intermediate that is characterized by an oxidized [FeIFeII] centre and a reduced [4Fe4S]1+ cluster. Based on this unusual configuration, this species is assigned to the first, deprotonated H-cluster intermediate of the [FeFe] hydrogenase catalytic cycle. Providing insights into the sequence of initial reaction steps, the identification of this species represents a key finding towards the mechanistic understanding of biological hydrogen evolution.

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Figures

Fig. 1

Fig. 1. (Top) Skeletal formula representation of the H-cluster. Fed (Fep) refers to the distal (proximal) iron ion of the [FeFe] sub-site, and the vacant coordination site at Fed is marked by an asterisk. Apart from the thiolate bridging the two cofactor moieties, cysteine residues are omitted for the sake of clarity. (Bottom) Schematic representations of three experimentally detected catalytic intermediates of the H-cluster (highlighted in red) are shown together with the corresponding CO-inhibited states.– In the Hred state, a proton is thought to be bound to the bridging aza-dithiolate ligand, while in the Hsred state it is probably transferred to an amino acid side chain. Arabic and roman numbers indicate the charge of the inorganic [4Fe4S] core and the formal oxidation state of the [FeFe] moiety, respectively. For mixed valence species, the formal mono- and divalent state is arbitrarily assigned to the proximal and distal Fe atom of the [FeFe] moiety, respectively.

Fig. 2

Fig. 2. Low-temperature RR spectra (80 K) of the reduced synthetic [FeFe]-adt complex (red, 25 mM, 514 nm excitation), thionine-oxidized apo-HydA1 (blue, 1.4 mM, 458 nm excitation), and _in vitro_-matured holo-HydA1(adt) (black, 2 mM, 488 nm excitation). Color-coded schematic representations depict the chemical (cofactor) species reflected by the individual RR spectra. Apart from a thiolate bridging the two H-cluster moieties, cysteine residues are omitted for the sake of clarity. Spectral regions reflecting normal modes with major contributions from Fe–S, Fe–CN, and Fe–CO coordinates are indicated.–,– Spectra of holo- and apo-HydA1 were normalized with respect to the band intensity of a (non-resonantly excited) phenylalanine sidechain mode of the protein matrix at ca. 1005 cm–1 (not shown here, see ESI 1†). The spectrum of the synthetic [FeFe]-adt complex was scaled to match the spectrum of holo-HydA1(adt) in terms of maximum band intensities in the region of Fe–CO centred normal modes.

Fig. 3

Fig. 3. Baseline-corrected vibrational spectra of (A) thionine-oxidized, (B) CO-treated, (C) as-isolated (dithionite-reduced), and (D) H2-reduced _in vitro_-matured holo-HydA1(adt). Low-temperature RR spectra (80 K, 488 nm excitation) are presented in two parts for the sake of clarity. The middle panel displays the spectral region reflecting Fe–CO/CN vibrations of the [FeFe] moiety, while the left panel is dominated by normal modes of the [4Fe4S] cluster.–,–, The difference spectra in the middle panel (traces B–D, black lines) were calculated by subtracting the spectrum of thionine-oxidized holo-HydA1 (trace A, shown in grey) from coloured traces B–D prior to baseline-correction. RR spectra are normalized as described in the Experimental details section. Low-temperature IR spectra (80 K) of the CO and CN stretching modes of the [FeFe] moiety are depicted in the right panel (see Table 1 for band assignments). Lines in light and saturated colours represent spectra recorded in the dark and during blue light illumination (460 nm), respectively. Interestingly, IR spectra of H2-treated holo-HydA1(adt) exhibit a significant photo-induced decrease of the band at 1881 cm–1 and a concomitant absorbance increase at 1954 cm–1 (trace D). Previously, both bands were assigned to a single Hsred state, which appears unlikely according to this observation.

Fig. 4

Fig. 4. New proposal for the catalytic cycle of [FeFe] hydrogenase, expanded from ref. 5. Intermediates probed by RR spectroscopy are labelled in blue, and the novel H′red species is additionally highlighted by a light blue shade. Arabic and roman numbers indicate the charge of the inorganic [4Fe4S] core and the formal oxidation state of the [FeFe] moiety, respectively. For mixed valence species, the formal mono- and divalent state is arbitrarily assigned to the proximal and distal Fe atom of the [FeFe] moiety, respectively.

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