Stepwise isotope editing of [FeFe]-hydrogenases exposes cofactor dynamics

Abstract

The six-iron cofactor of [FeFe]-hydrogenases (H-cluster) is the most efficient H 2 -forming catalyst in nature. It comprises a diiron active site with three carbon monoxide (CO) and two cyanide (CN − ) ligands in the active oxidized state (H ox ) and one additional CO ligand in the inhibited state (H ox -CO). The diatomic ligands are sensitive reporter groups for structural changes of the cofactor. Their vibrational dynamics were monitored by real-time attenuated total reflection Fouriertransform infrared spectroscopy. Combination of 13 CO gas exposure, blue or red light irradiation, and controlled hydration of three different [FeFe]-hydrogenase proteins produced 8 H ox and 16 H ox -CO species with all possible isotopic exchange patterns. Extensive density functional theory calculations revealed the vibrational mode couplings of the carbonyl ligands and uniquely assigned each infrared spectrum to a specific labeling pattern. For H ox -CO, agreement between experimental and calculated infrared frequencies improved by up to one order of magnitude for an apical CN − at the distal iron ion of the cofactor as opposed to an apical CO. For H ox , two equally probable isomers with partially rotated ligands were suggested. Interconversion between these structures implies dynamic ligand reorientation at the H-cluster. Our experimental protocol for site-selective 13 CO isotope editing combined with computational species assignment opens new perspectives for characterization of functional intermediates in the catalytic cycle.[FeFe]-hydrogenase | isotope editing | infrared spectroscopy | density functional theory | cofactor dynamics T he reduction of protons to form molecular hydrogen (H 2 ) is catalyzed by [FeFe]-hydrogenases (1, 2). With a turnover rate of up to 10,000 H 2 molecules per second in a thermodynamically reversible reaction (3-5), [FeFe]-hydrogenases inspired synthetic hydrogen catalysts (6-8) and renewable fuel technology applications (9, 10). The mechanism of catalysis at the active site cofactor (H-cluster) needs to be elucidated. Further information on functional intermediates is required (11-16) and expected to emerge from spectroscopic studies on H-cluster constructs carrying siteselective isotopic reporter groups (17)(18)(19)(20) Formation of H ox -CO does not affect the formal redox state of the H-cluster, but leads to increased spin delocalization over the diiron site (28). CO binding inhibits H 2 turnover and protects the enzyme against O 2 and light-induced degradation (24,29,30).The vibrational modes of the CO and CN − ligands at the diiron site are well accessible by infrared (IR) spectroscopy because they are separated from protein backbone and liquid water bands.Infrared spectroscopy therefore has pioneered elucidation of the molecular structure of the H-cluster and identification of several redox states (24, 31). In particular, the CO stretching frequencies are highly sensitive to structural isomerism, redox transitions, ligand binding, and isotope exchange (11,12,15,18,24,31,32). 13 CO editing ...

Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis

“…Active Strep HybC-His HybO heterocomplex was isolated by histidine affinity chromatography (Co 2ϩ -charged resin). Protein samples were concentrated and analyzed by ATR FTIR as reported earlier (47).…”

“…All FTIR spectroscopy was conducted on a rapid-scan Tensor 27 spectrometer (Bruker Optik, Ettlingen, Germany) equipped with a three-reflection ZnSe/silicon crystal ATR cell (Smiths Detection, Wiesbaden Germany) as described earlier (27,28,47). The spectrometer was situated in an anaerobic gas chamber (Coy Laboratories, Grass Lake, MI) in a water-free atmosphere of typically 99% N 2 and 1% H 2 .…”

The Geometry of the Catalytic Active Site in [FeFe]-Hydrogenases is Determined by Hydrogen Bonding and Proton Transfer

“…While the overall IR signature of Hox-CO was conserved in all cofactor variants, ODT and EDT showed pronounced up-shifts, in particular of the highest-frequency CO band (25 cm -1 or 12 cm -1 , respectively). It is important to note that this band results from a coupled vibrational mode involving all CO ligands and must not be assigned to exogenous CO. 19 The mean differences in CNstretching frequencies for Hox-CO ODT and EDT are the same as in Hox (Fig. S4).…”

“…8). We propose an intrinsically flexible diiron site geometry19,40 that facilitates stabilization of polar ligands at the distal iron ion in both catalytic intermediates (Hhyd) and inhibited species (Hox-CO). Comparing H 2 oxidation and CO inhibition allows dissecting the influence of proton transfer and hydrogen bonding on the geometry of the H-cluster.…”

Protonengekoppelte Reduktion des katalytischen [4Fe‐4S]‐Zentrums in [FeFe]‐Hydrogenasen

“…Die Struktur des H‐Clusters lässt sich anhand der charakteristischen Infrarotabsorption der CO‐ und CN‐Liganden untersuchen . Abbildung zeigt das Vibrationsprofil der eingestellten Spezies in der Kofaktorvariante HYDA1 pdt bei verschiedenen pH‐Werten und ansteigend reduzierenden Potentialen.…”