Proton Coupled Electronic Rearrangement within the H-Cluster as an Essential Step in the Catalytic Cycle of [FeFe] Hydrogenases

Abstract

The active site of [FeFe] hydrogenases, the H-cluster, consists of a [4Fe-4S] cluster connected via a bridging cysteine to a [2Fe] complex carrying CO and CN ligands as well as a bridging aza-dithiolate ligand (ADT) of which the amine moiety serves as a proton shuttle between the protein and the H-cluster. During the catalytic cycle, the two subclusters change oxidation states: [4Fe-4S] ⇔ [4Fe-4S] and [Fe(I)Fe(II)] ⇔ [Fe(I)Fe(I)] thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H + 2e ⇄ H. Using FTIR spectro-electrochemistry on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) at different pH values, we resolve the redox and protonation events in the catalytic cycle and determine their intrinsic thermodynamic parameters. We show that the singly reduced state H of the H-cluster actually consists of two species: H = [4Fe-4S] - [Fe(I)Fe(II)] and HH = [4Fe-4S] - [Fe(I)Fe(I)] (H) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe-4S] subcluster at similar midpoint-potentials (-375 vs -418 mV); the protonation event (H/HH) has a pK ≈ 7.2.

Direct Observation of an Iron-Bound Terminal Hydride in [FeFe]-Hydrogenase by Nuclear Resonance Vibrational Spectroscopy

“…2,9-11 The active oxidized state H ox is represented as [4Fe–4S] H 2+ -Fe(I)Fe(II). Two singly reduced states have recently been identified: H red =[4Fe–4S] H + -Fe(I)Fe(II) and H red H + =[4Fe–4S] H 2+ Fe(I)Fe(I)(NH 2 + ) where protonation of the ADT amine is coupled to electronic rearrangement within the H-cluster.…”

“…Two singly reduced states have recently been identified: H red =[4Fe–4S] H + -Fe(I)Fe(II) and H red H + =[4Fe–4S] H 2+ Fe(I)Fe(I)(NH 2 + ) where protonation of the ADT amine is coupled to electronic rearrangement within the H-cluster. 9 The doubly reduced state is characterized as H sred H + =[4Fe–4S] H + -Fe(I)Fe(I)(NH 2 + ). According to widely accepted models of the catalytic cycle, 4-7 H ox interacts with molecular hydrogen which is deprotonated by the pendant ADT amine in concert with electron transfer from the [2Fe] H subsite to the [4Fe–4S] H 2+ cluster yielding an Fe(II)Fe(II)-bound hydride.…”

Electrolyte Engineering as a Key Strategy Towards a Sustainable Energy Scenario?

“…In the literature, the underlying elementary processes are discussed controversially, particularly for the case of [FeFe] hydrogenases . The main differences are related to the binding of hydrogen in the active site and the corresponding protonation sites (precursor structures) in the protein environment or at the active center . Hitherto, the influence of the electrolyte composition on the activity and reaction mechanism of hydrogenases has been practically ignored.…”

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

“…Hred und Hred′ sind zwei unterschiedliche, einfach‐reduzierte Zustände. Während der H‐Cluster in Hred das Elektron am [FeFe]‐Kofaktor trägt, liegt in Hred′ ein reduziertes [4Fe‐4S]‐Zentrum vor . Weder Hred noch Hred′ sind ESR‐aktiv .…”