How [FeFe]-hydrogenase facilitates bidirectional proton transfer

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

Infrared Characterization of the Bidirectional Oxygen-Sensitive [NiFe]-Hydrogenase from E. coli

[NiFe]-hydrogenases are gas-processing metalloenzymes that catalyze the conversion of dihydrogen (H2) to protons and electrons in a broad range of microorganisms. Within the framework of green chemistry, the molecular proceedings of biological hydrogen turnover inspired the design of novel catalytic compounds for H2 generation. The bidirectional “O2-sensitive” [NiFe]-hydrogenase from Escherichia coli HYD-2 has recently been crystallized; however, a systematic infrared characterization in the presence of natural reactants is not available yet. In this study, we analyze HYD-2 from E. coli by in situ attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR) under quantitative gas control. We provide an experimental assignment of all catalytically relevant redox intermediates alongside the O2- and CO-inhibited cofactor species. Furthermore, the reactivity and mutual competition between H2, O2, and CO was probed in real time, which lays the foundation for a comparison with other enzymes, e.g., “O2-tolerant” [NiFe]-hydrogenases. Surprisingly, only Ni-B was observed in the presence of O2 with no indications for the “unready” Ni-A state. The presented work proves the capabilities of in situ ATR FTIR spectroscopy as an efficient and powerful technique for the analysis of biological macromolecules and enzymatic small molecule catalysis

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

[FeFe]-hydrogenases are efficient metalloenzymes that catalyze the oxidation and evolution of molecular hydrogen, H2. They serve as a blueprint for the design of synthetic H2-forming catalysts. [FeFe]-hydrogenases harbor a six-iron cofactor that comprises a [4Fe-4S] cluster and a unique diiron site with cyanide, carbonyl, and hydride ligands. To address the ligand dynamics in catalytic turnover and upon carbon monoxide (CO) inhibition, we replaced the native aminodithiolate group of the diiron site by synthetic dithiolates, inserted into wild-type and amino acid variants of the [FeFe]-hydrogenase HYDA1 from Chlamydomonas reinhardtii. The reactivity with H2 and CO was characterized using in situ and transient infrared spectroscopy, protein crystallography, quantum chemical calculations, and kinetic simulations. All cofactor variants adopted characteristic populations of reduced species in the presence of H2 and showed significant changes in CO inhibition and reactivation kinetics. Differences were attributed to varying interactions between polar ligands and the dithiolate head group and/or the environment of the cofactor (i.e., amino acid residues and water molecules). The presented results show how catalytically relevant intermediates are stabilized by inner-sphere hydrogen bonding suggesting that the role of the aminodithiolate group must not be restricted to proton transfer. These concepts may inspire the design of improved enzymes and biomimetic H2-forming catalysts

Spectroscopical Investigations on the Redox Chemistry of [FeFe]-Hydrogenases in the Presence of Carbon Monoxide

[FeFe]-hydrogenases efficiently catalyzes hydrogen conversion at a unique [4Fe–4S]-[FeFe] cofactor, the so-called H-cluster. The catalytic reaction occurs at the diiron site, while the [4Fe–4S] cluster functions as a redox shuttle. In the oxidized resting state (Hox), the iron ions of the diiron site bind one cyanide (CN−) and carbon monoxide (CO) ligand each and a third carbonyl can be found in the Fe–Fe bridging position (µCO). In the presence of exogenous CO, A fourth CO ligand binds at the diiron site to form the oxidized, CO-inhibited H-cluster (Hox-CO). We investigated the reduced, CO-inhibited H-cluster (Hred´-CO) in this work. The stretching vibrations of the diatomic ligands were monitored by attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR). Density functional theory (DFT) at the TPSSh/TZVP level was employed to analyze the cofactor geometry, as well as the redox and protonation state of the H-cluster. Selective 13CO isotope editing, spectro-electrochemistry, and correlation analysis of IR data identified a one-electron reduced, protonated [4Fe–4S] cluster and an apical CN− ligand at the diiron site in Hred´-CO. The reduced, CO-inhibited H-cluster forms independently of the sequence of CO binding and cofactor reduction, which implies that the ligand rearrangement at the diiron site upon CO inhibition is independent of the redox and protonation state of the [4Fe–4S] cluster. The relation of coordination dynamics to cofactor redox and protonation changes in hydrogen conversion catalysis and inhibition is discussed

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

In der Natur katalysieren [FeFe]-Hydrogenasen die Abgabe und Aufnahme von molekularem Wasserstoff (H2) an einem einzigartigen Eisen-Schwefel-Kofaktor. Das geringe elektrochemische Überpotential in der Wasserstoffabgabe-Reaktion macht die [FeFe]-Hydrogenasen zu einem hervorragenden Beispiel für effiziente Biokatalyse. Gegenwärtig sind die molekularen Details des Wasserstoffumsatzes jedoch noch nicht vollständig verstanden. Daher adressieren wir in dieser Untersuchung die initiale Reduktion des katalytischen Zentrums der [FeFe]-Hydrogenasen mittels Infrarotspektroskopie und Elektrochemie und zeigen, dass der reduzierte Zustand Hred′ durch protonengekoppelten Elektronentransport gebildet wird. Ladungskompensation bindet das überschüssige Elektron am [4Fe-4S]-Zentrum und führt zu einer Stabilisierung der konservativen Konfiguration des [FeFe]-Kofaktors. Die Rolle von Hred′ beim Wasserstoffumsatz und mögliche Auswirkungen auf den katalytischen Mechanismus werden diskutiert. Es liegt nahe, dass die Regulation elektronischer Eigenschaften in der Umgebung von metallischen Kofaktoren die Grundlage für Multielektronenprozesse bildet

In‐Liquid Plasma Modified Nickel Foam: NiOOH/NiFeOOH Active Site Multiplication for Electrocatalytic Alcohol, Aldehyde, and Water Oxidation

The oxygen evolution reaction (OER) and the value-added oxidation of renewable organic substrates are critical to supply electrons and protons for the synthesis of sustainable fuels. To meet industrial requirements, new methods for a simple, fast, environmental-friendly and cheap synthesis of robust, self-supported and high surface area electrodes are required. Herein, a novel in-liquid plasma (plasma electrolysis) approach for the growth of hierarchical nanostructures on nickel foam is reported on. Under morphology retention, iron can be doped into this high surface area electrode. For the oxidation of 5-(hydroxymethyl)furfural and benzyl alcohol, the iron-free, plasma-treated electrode is more suitable reaching current densities up to 800 mA cm$^{-2}$ with Faradaic efficiencies above 95%. For the OER, the iron-doped nickel foam electrode reaches the industrially relevant current density of 500 mA cm$^{-2}$ at 1.473 ± 0.013$_{VRHE}$ (60 °C) and shows no activity decrease over 140 h. The different effects of iron doping are rationalized using methanol probing and in situ Raman spectroscopy. Furthermore, the intrinsic activity is separated from the number of active sites, and, for the organic oxidation reactions, diffusion limitations are revealed. The authors anticipate that the plasma modified nickel foam will be suitable for various (electro)catalytic processes

Protonation/reduction dynamics at the [4Fe–4S] cluster of the hydrogen-forming cofactor in [FeFe]-hydrogenases

The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen conversion catalysts in nature. Their active-site cofactor (H-cluster) comprises a [4Fe–4S] cluster linked to a unique diiron site that binds three carbon monoxide (CO) and two cyanide (CN−) ligands. Understanding microbial hydrogen conversion requires elucidation of the interplay of proton and electron transfer events at the H-cluster. We performed real-time spectroscopy on [FeFe]-hydrogenase protein films under controlled variation of atmospheric gas composition, sample pH, and reductant concentration. Attenuated total reflection Fourier-transform infrared spectroscopy was used to monitor shifts of the CO/CN− vibrational bands in response to redox and protonation changes. Three different [FeFe]-hydrogenases and several protein and cofactor variants were compared, including element and isotopic exchange studies. A protonated equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially accumulated at acidic pH and under reducing conditions. We show that the one- electron reduced state Hred′ represents an intrinsically protonated species. Interestingly, the formation of HoxH and Hred′ was independent of the established proton pathway to the diiron site. Quantum chemical calculations of the respective CO/CN− infrared band patterns favored a cysteine ligand of the [4Fe–4S] cluster as the protonation site in HoxH and Hred′. We propose that proton-coupled electron transfer facilitates reduction of the [4Fe–4S] cluster and prevents premature formation of a hydride at the catalytic diiron site. Our findings imply that protonation events both at the [4Fe–4S] cluster and at the diiron site of the H-cluster are important in the hydrogen conversion reaction of [FeFe]-hydrogenases

Infrared Characterization of the Bidirectional Oxygen-Sensitive [NiFe]-Hydrogenase from <i>E. coli</i>

[NiFe]-hydrogenases are gas-processing metalloenzymes that catalyze the conversion of dihydrogen (H2) to protons and electrons in a broad range of microorganisms. Within the framework of green chemistry, the molecular proceedings of biological hydrogen turnover inspired the design of novel catalytic compounds for H2 generation. The bidirectional &#8220;O2-sensitive&#8222; [NiFe]-hydrogenase from Escherichia coli HYD-2 has recently been crystallized; however, a systematic infrared characterization in the presence of natural reactants is not available yet. In this study, we analyze HYD-2 from E. coli by in situ attenuated total reflection Fourier-transform infrared spectroscopy (ATR FTIR) under quantitative gas control. We provide an experimental assignment of all catalytically relevant redox intermediates alongside the O2- and CO-inhibited cofactor species. Furthermore, the reactivity and mutual competition between H2, O2, and CO was probed in real time, which lays the foundation for a comparison with other enzymes, e.g., &#8220;O2-tolerant&#8222; [NiFe]-hydrogenases. Surprisingly, only Ni-B was observed in the presence of O2 with no indications for the &#8220;unready&#8222; Ni-A state. The presented work proves the capabilities of in situ ATR FTIR spectroscopy as an efficient and powerful technique for the analysis of biological macromolecules and enzymatic small molecule catalysis

Site-selective protonation of the one-electron reduced cofactor in [FeFe]-hydrogenase

Hydrogenases are bidirectional redox enzymes that catalyze hydrogen turnover in archaea, bacteria, and algae. While all types of hydrogenase show H-2 oxidation activity, [FeFe]-hydrogenases are excellent H-2 evolution catalysts as well. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site equipped with carbon monoxide and cyanide ligands. The active site niche is connected with the solvent by two distinct proton transfer pathways. To analyze the catalytic mechanism of [FeFe]-hydrogenase, we employ operando infrared spectroscopy and infrared spectro-electrochemistry. Titrating the pH under H-2 oxidation or H-2 evolution conditions reveals the influence of site-selective protonation on the equilibrium of reduced cofactor states. Governed by pK(a) differences across the active site niche and proton transfer pathways, we find that individual electrons are stabilized either at the [4Fe-4S] cluster (alkaline pH values) or at the diiron site (acidic pH values). This observation is discussed in the context of the complex interdependence of hydrogen turnover and bulk pH

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

Hydrogenases are microbial redox enzymes that catalyze H2 oxidation and proton reduction (H2 evolution). While all hydrogenases show high oxidation activities, the majority of [FeFe]-hydrogenases are excellent H2 evolution catalysts as well. Their active site cofactor comprises a [4Fe-4S] cluster covalently linked to a diiron site equipped with carbon monoxide and cyanide ligands that facilitate catalysis at low overpotential. Distinct proton transfer pathways connect the active site niche with the solvent, resulting in a non-trivial dependence of hydrogen turnover and bulk pH. To analyze the catalytic mechanism of [FeFe]-hydrogenase, we employ in situ infrared spectroscopy and infrared spectro-electrochemistry. Titrating the pH under H2 oxidation or H2 evolution conditions reveals the influence of site-selective protonation on the equilibrium of reduced cofactor states. Governed by pKa differences across the active site niche and proton transfer pathways, we find that individual electrons are stabilized either at the [4Fe-4S] cluster (alkaline pH values) or at the diiron site (acidic pH values). This observation is discussed in the context of the natural pH dependence of hydrogen turnover as catalyzed by [FeFe]-hydrogenase.<br /