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.
Highlights
We found that transient protonation changes of glutamic acid residue E141 and, most notably, arginine R148 facilitate bidirectional proton transfer in [FeFe]-hydrogenases
The active-ready geometry of Hox is characterized by a square-pyramidal configuration of both metal ions, a μCO ligand, and an open coordination site at Fed.[15−17] While this geometry is conserved in Hred′ and Hhyd,[27,30] the structural changes upon reduction of the diiron site are under debate.[36]
We demonstrate how in situ infrared spectroscopy was applied to analyze the hydrogen-bonding network of the catalytic proton transfer pathway in [FeFe]-hydrogenases
Summary
Hydrogenases are gas-processing iron−sulfur enzymes that catalyze the reversible reduction of protons to molecular hydrogen in all kingdoms of life.[1,2] Most hydrogenases are biased energy toward H2 oxidation, metabolism and H2 for example, in sensing.[3−5] The the context of [FeFe]-hydrogenases from bacteria and algae, in contrast, are truly bidirectional with similar eaffincdienccayta.6ly−z8eCHom[2] boinxiidnagtihoinghatnudrnoHv2erefvroelquutieonncies theH(1+0/H0020reHdo[2] xs−c1o)uapnled,9a−1c1attahleytaicctmiviedspioteinctopfaoctteonrtioafl close to [FeFe]-hydrogenases (“H-cluster”) inspired the design of numerous biomimetic complexes for H2 production.[12−14]The H-cluster comprises a conventional [4Fe-4S] center linked to a bimetallic iron−sulfur complex (Figure 1a).[15−17]The diiron site carries two terminal carbonyl and cyanide ligands (CO, CN−) as well as a single carbonyl ligand in Fe− Fe bridging position (μCO).[18−20] An aminodithiolate (ADT)group connects the proximal and distal iron ion (Fep and Fed, relative to the [4Fe-4S] center)[21] and functions as proton relay between active site cofactor and protein environment.[22]. The oxidized resting state (Hox)[23−25] can be distinguished from intermediates with a reduced [4Fe-4S] center (Hred′, Hhyd)[26−32] or a reduced diiron site (Hred, Hsred).[33−35] These states are formed upon concerted proton and electron transfer. The active-ready geometry of Hox is characterized by a square-pyramidal configuration of both metal ions, a μCO ligand, and an open coordination site at Fed.[15−17] While this geometry is conserved in Hred′ and Hhyd,[27,30] the structural changes upon reduction of the diiron site are under debate.[36] The H-cluster may undergo rigorous ligand rearrangement forming a μH geometry, which would exclude both Hred and Hsred from catalytic turnover.[35] Alternatively, diiron site geometries with a bridging[37,38] or “semi-bridging” CO ligand[20] have been
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