Accumulating the hydride state in the catalytic cycle of [FeFe]-hydrogenases

Abstract

H2 turnover at the [FeFe]-hydrogenase cofactor (H-cluster) is assumed to follow a reversible heterolytic mechanism, first yielding a proton and a hydrido-species which again is double-oxidized to release another proton. Three of the four presumed catalytic intermediates (Hox, Hred/Hred and Hsred) were characterized, using various spectroscopic techniques. However, in catalytically active enzyme, the state containing the hydrido-species, which is eponymous for the proposed heterolytic mechanism, has yet only been speculated about. We use different strategies to trap and spectroscopically characterize this transient hydride state (Hhyd) for three wild-type [FeFe]-hydrogenases. Applying a novel set-up for real-time attenuated total-reflection Fourier-transform infrared spectroscopy, we monitor compositional changes in the state-specific infrared signatures of [FeFe]-hydrogenases, varying buffer pH and gas composition. We selectively enrich the equilibrium concentration of Hhyd, applying Le Chatelier’s principle by simultaneously increasing substrate and product concentrations (H2/H+). Site-directed manipulation, targeting either the proton-transfer pathway or the adt ligand, significantly enhances Hhyd accumulation independent of pH.

Reaction Coordinate Leading to H2 Production in [FeFe]-Hydrogenase Identified by Nuclear Resonance Vibrational Spectroscopy and Density Functional Theory

“…29 These turn-over conditions give high steady state levels of H hyd , which we rationalize as follows. With such a high concentration of reductant, the rate of electron transfer to the hydrogenase is very high.…”

From Enzymes to Functional Materials—Towards Activation of Small Molecules

“…Structuralsimilaritiesbetweens elected natural enzymesa nd natural minerals. [23,26,[34][35][36][37][38][39][40][41][42][43]…”

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

“…Dieser sogenannte “H‐Cluster” besteht aus einem [4Fe‐4S]‐Zentrum, welches kovalent an einen [FeFe]‐Kofaktor gebunden ist (Abbildung ). Der Wasserstoffumsatz findet an dem distal zum [4Fe‐4S]‐Zentrum lokalisierten Eisenion des bimetallischen Kofaktors statt (Fe d ) . Eine Aminodithiolatgruppe (adt) am [FeFe]‐Kofaktor fungiert als Protonrelais in der heterolytischen Oxidation von H 2 .…”

“…Auch der hydridische Zustand Hhyd zeigt ein reduziertes [4Fe‐4S]‐Zentrum. Trotzdem erlaubt der “superoxidierte” [FeFe]‐Kofaktor mit einem terminalen Hydrid am Fe d eine eindeutige Charakterisierung von Hhyd . Im Gegensatz dazu konnte per Dichtefunktionaltheorie (DFT) gezeigt werden, dass Hred und Hsred ein verbrückendes Hydrid am [FeFe]‐Kofaktor tragen .…”

“…Auch Hhyd ist durch einen μ CO‐Liganden charakterisiert . Es ist eine reizvolle Aufgabe, nun die Infrarotsignatur von Hhyd per DFT‐Rechnungen auszuwerten.…”