Synthesis of a miniaturized [FeFe] hydrogenase model system

Abstract

The reaction occurring during artificial maturation of [FeFe] hydrogenase has been recreated using molecular systems. The formation of a miniaturized [FeFe] hydrogenase model system, generated through the combination of a [4Fe4S] cluster binding oligopeptide and an organometallic Fe complex, has been monitored by a range of spectroscopic techniques. A structure of the final assembly is suggested based on EPR and FTIR spectroscopy in combination with DFT calculations. The capacity of this novel H-cluster model to catalyze H2 production in aqueous media at mild potentials is verified in chemical assays.

Fig. 1. Schematic representation of the inorganic cofactor constituting the active site of [FeFe]-hydrogenase and the model complexes employed in this study. (A): The complete H-cluster; (B): the [2Fe] subsite model [Fe₂(adt)(CO)₄(CN)₂]^2–, 1; (C): a truncated structure of P. aerogenes ferredoxin I, the -CIACGAC- core motif on which the FdM maquette is based is highlighted in yellow. Heteroatom colour coding: Fe = orange; S = yellow; N = blue and O = red. A and C generated from Protein Data Bank entries ; 3C8Y (selected amino acids removed to provide an unobstructed view of the cofactor) and ; 1DUR respectively.

Fig. 2. Spectroscopic properties of the as-prepared and reduced forms of [4Fe4S]–FdM; (Top): UV/Vis spectra, [4Fe4S]²⁺–FdM (20 μM black line) and [4Fe4S]⁺–FdM (20 μM, red line), (inset): apo-FdM (128 μM); (Bottom) X-band EPR spectra of [4Fe4S]²⁺–FdM (120 μM, black solid line), [4Fe4S]⁺–FdM (120 μM, red solid line) and a reduced solution of iron and sodium sulfide in the presence of β-mercaptoethanol (dashed line). All samples prepared in HEPES buffer (50 mM, pH 8.0). EPR spectra recorded at 10 K; microwave frequency 9.28 GHz; modulation amplitude: 10 G; microwave power: 1 mW.

Scheme 1. Schematic representation of the formation of 2 proceeding via the binding of the reduced [4Fe4S] cluster to complex 1, resulting in the formation of a mixed valence complex with concomitant formation of H₂ and release of a CO ligand.

Fig. 3. FTIR spectra of 1 treated with [4Fe4S]–FdM under reducing or non-reducing conditions. Complex 1 in aqueous buffer (spectrum C); 1 + 2 eq. of [4Fe4S]²⁺–FdM (300 μM) (spectrum B); 1 + 2 eq. of [4Fe4S]⁺–FdM (300 μM) (spectrum A). Arrows indicate the new peaks assigned to 2 (red) and the consumption of 1 (black); peaks not assigned to 1 or 2 indicated with asterisk (see main text). All samples prepared in HEPES buffer (50 mM, pH 8) and spectra recorded after 30 minutes incubation time with 1. Calculated spectra are obtained using an [Fe₂(adt)(CO)₄(CN)₂]^2– model of 1 and an [4Fe-4S]-S(Cys)-[2Fe] model of 2 (see Fig. S9 and S13 for details). Results for alternative isomers and protonation states are given in the ESI.