How [FeFe]-Hydrogenase Facilitates Bidirectional Proton Transfer

Abstract

Hydrogenases are metalloenzymes that catalyze the conversion of protons and molecular hydrogen, H2. [FeFe]-hydrogenases show particularly high rates of hydrogen turnover and have inspired numerous compounds for biomimetic H2 production. Two decades of research on the active site cofactor of [FeFe]-hydrogenases have put forward multiple models of the catalytic proceedings. In comparison, our understanding of proton transfer is poor. Previously, residues were identified forming a hydrogen-bonding network between active site cofactor and bulk solvent; however, the exact mechanism of catalytic proton transfer remained inconclusive. Here, we employ in situ infrared difference spectroscopy on the [FeFe]-hydrogenase from Chlamydomonas reinhardtii evaluating dynamic changes in the hydrogen-bonding network upon photoreduction. While proton transfer appears to be impaired in the oxidized state (Hox), the presented data support continuous proton transfer in the reduced state (Hred). Our analysis allows for a direct, molecular unique assignment to individual amino acid residues. We found that transient protonation changes of glutamic acid residue E141 and, most notably, arginine R148 facilitate bidirectional proton transfer in [FeFe]-hydrogenases.

Characterization of a putative sensory [FeFe]-hydrogenase provides new insight into the role of the active site architecture

“…1). [42][43][44] Moreover, the genes encoding M2e and M2f enzymes are oen located on the same operon as group A hydrogenases. 5 In light of these similarities, we have now denoted the enzyme as Tam HydS (previously Tam HydA 40 ).…”

Operando Infrared Spectroscopy for the Analysis of Gas-processing Metalloenzymes

“…Moreover, the Δ75 cm -1 downshift from 1680 to 1605 cm -1 in D2O can be assigned to a deprotonation of arginine R148. 71 In the SH frequency regime around 2500 cm -1 (compare Figure 4) any difference signals are missing. This makes protonation or hydrogen-bonding changes involving the cysteine residue C169 in the proton transfer pathway rather unlikely.…”

“…Therefore, investigating 'Hred -Hox' difference spectra conveyed a dynamic understanding of the hydrogen-bonding changes that facilitate proton transfer ( Figure 14A). 71 The respective changes in the frequency regime below 1800 cm -1 for [FeFe]-hydrogenase CrHydA1 are shown in Figure 14B. Negative bands belong to Hox (note the large µCO band at 1802 cm -1 in the main panel), positive bands belong to Hred.…”

“…Whether this effect stems from direct heating (thermal radiation) or light-heat conversion in the protein film is currently unclear; however, facilitating measurements with a minimum of unspecific changes, thorough temperature control, high power densities (i.e., laser irradiation), or efficient absorbers are recommended (Section 5). 71 Combining ATR FTIR spectroscopy with UV/visible or Resonance Raman spectroscopy (as discussed in the Outlook), both unspecific changes and the electronic excitation of sample may influence the observables. Here, ATR FTIR spectroscopy can serve as a 'dark reference'.…”

Site-selective Protonation of the One-electron Reduced Cofactor in [FeFe]-Hydrogenase

“…[13][14][15] The secondary amine of the H-cluster's aminodithiolate ligand (ADT) has been suggested to serve as proton relay between the diiron site and the amino acid residues of the catalytic PT pathway. [16][17][18][19][20][21] Moreover, it acts as an inner-sphere hydrogenbonding donor to a number of apical ligands at the distal iron ion (Fed). 22 Figure 1C depicts a schematic representation of the H-cluster with potential binding sites for hydrogen species.…”