Proton‐Coupled Reduction of the Catalytic [4Fe‐4S] Cluster in [FeFe]‐Hydrogenases

In nature, [FeFe]‐hydrogenases catalyze the uptake and release of molecular hydrogen (H₂) at a unique iron‐sulfur cofactor. The absence of an electrochemical overpotential in the H₂ release reaction makes [FeFe]‐hydrogenases a prime example of efficient biocatalysis. However, the molecular details of hydrogen turnover are not yet fully understood. Herein, we characterize the initial one‐electron reduction of [FeFe]‐hydrogenases by infrared spectroscopy and electrochemistry and present evidence for proton‐coupled electron transport during the formation of the reduced state Hred′. Charge compensation stabilizes the excess electron at the [4Fe‐4S] cluster and maintains a conservative configuration of the diiron site. The role of Hred′ in hydrogen turnover and possible implications on the catalytic mechanism are discussed. We propose that regulation of the electronic properties in the periphery of metal cofactors is key to orchestrating multielectron processes.     

A [RuRu] Analogue of an [FeFe]‐Hydrogenase Traps the Key Hydride Intermediate of the Catalytic Cycle

The active site of the [FeFe]‐hydrogenases features a binuclear [2Fe]_H sub‐cluster that contains a unique bridging amine moiety close to an exposed iron center. Heterolytic splitting of H₂ results in the formation of a transient terminal hydride at this iron site, which, however is difficult to stabilize. We show that the hydride intermediate forms immediately when [2Fe]_H is replaced with [2Ru]_H analogues through artificial maturation. Outside the protein, the [2Ru]_H analogues form bridging hydrides, which rearrange to terminal hydrides after insertion into the apo‐protein. H/D exchange of the hydride only occurs for [2Ru]_H analogues containing the bridging amine moiety.     

Caught in the Hinact: Crystal Structure and Spectroscopy Reveal a Sulfur Bound to the Active Site of an O2‐stable State of [FeFe] Hydrogenase

[FeFe] hydrogenases are the most active H₂ converting catalysts in nature, but their extreme oxygen sensitivity limits their use in technological applications. The [FeFe] hydrogenases from sulfate reducing bacteria can be purified in an O₂‐stable state called H_inact. To date, the structure and mechanism of formation of H_inact remain unknown. Our 1.65 Å crystal structure of this state reveals a sulfur ligand bound to the open coordination site. Furthermore, in‐depth spectroscopic characterization by X‐ray absorption spectroscopy (XAS), nuclear resonance vibrational spectroscopy (NRVS), resonance Raman (RR) spectroscopy and infrared (IR) spectroscopy, together with hybrid quantum mechanical and molecular mechanical (QM/MM) calculations, provide detailed chemical insight into the H_inact state and its mechanism of formation. This may facilitate the design of O₂‐stable hydrogenases and molecular catalysts.     

Aerobic Damage to [FeFe]‐Hydrogenases: Activation Barriers for the Chemical Attachment of O2

[FeFe]‐hydrogenases are the best natural hydrogen‐producing enzymes but their biotechnological exploitation is hampered by their extreme oxygen sensitivity. The free energy profile for the chemical attachment of O₂ to the enzyme active site was investigated by using a range‐separated density functional re‐parametrized to reproduce high‐level ab initio data. An activation free‐energy barrier of 13 kcal mol^?1 was obtained for chemical bond formation between the di‐iron active site and O₂, a value in good agreement with experimental inactivation rates. The oxygen binding can be viewed as an inner‐sphere electron‐transfer process that is strongly influenced by Coulombic interactions with the proximal cubane cluster and the protein environment. The implications of these results for future mutation studies with the aim of increasing the oxygen tolerance of this enzyme are discussed.     

An S‐Oxygenated [NiFe] Complex Modelling Sulfenate Intermediates of an O2‐Tolerant Hydrogenase

To understand the molecular details of O₂‐tolerant hydrogen cycling by a soluble NAD⁺‐reducing [NiFe] hydrogenase, we herein present the first bioinspired heterobimetallic S‐oxygenated [NiFe] complex as a structural and vibrational spectroscopic model for the oxygen‐inhibited [NiFe] active site. This compound and its non‐S‐oxygenated congener were fully characterized, and their electronic structures were elucidated in a combined experimental and theoretical study with emphasis on the bridging sulfenato moiety. Based on the vibrational spectroscopic properties of these complexes, we also propose novel strategies for exploring S‐oxygenated intermediates in hydrogenases and similar enzymes.     

[FeFe]‐Hydrogenase with Chalcogenide Substitutions at the H‐Cluster Maintains Full H2 Evolution Activity

The [FeFe]‐hydrogenase HYDA1 from Chlamydomonas reinhardtii is particularly amenable to biochemical and biophysical characterization because the H‐cluster in the active site is the only inorganic cofactor present. Herein, we present the complete chemical incorporation of the H‐cluster into the HYDA1‐apoprotein scaffold and, furthermore, the successful replacement of sulfur in the native [4Fe_H] cluster with selenium. The crystal structure of the reconstituted pre‐mature HYDA1[4Fe4Se]_H protein was determined, and a catalytically intact artificial H‐cluster variant was generated upon in vitro maturation. Full hydrogen evolution activity as well as native‐like composition and behavior of the redesigned enzyme were verified through kinetic assays, FTIR spectroscopy, and X‐ray structure analysis. These findings reveal that even a bioinorganic active site with exceptional complexity can exhibit a surprising level of compositional plasticity.     

High‐Frequency Fe–H Vibrations in a Bridging Hydride Complex Characterized by NRVS and DFT

High‐spin iron species with bridging hydrides have been detected in species trapped during nitrogenase catalysis, but there are few general methods of evaluating Fe?H bonds in high‐spin multinuclear iron systems. An ⁵⁷Fe nuclear resonance vibrational spectroscopy (NRVS) study on an Fe(μ‐H)₂Fe model complex reveals Fe?H stretching vibrations for bridging hydrides at frequencies greater than 1200 cm^?1. These isotope‐sensitive vibrational bands are not evident in infrared (IR) spectra, showing the power of NRVS for identifying hydrides in this high‐spin iron system. Complementary density functional theory (DFT) calculations elucidate the normal modes of the rhomboidal iron hydride core.     

Influence of the [2Fe]H Subcluster Environment on the Properties of Key Intermediates in the Catalytic Cycle of [FeFe] Hydrogenases: Hints for the Rational Design of Synthetic Catalysts

Nature's recipe : A theoretical study analyzes how the environment of the [FeFe] hydrogenase's catalytic cofactor affects its chemical properties, particularly the relative stability of complexes with bridging and terminal hydride ligands (see picture; Fe teal, S yellow, C green, N blue, O red, H gray). The results help to elucidate key rules for the design of bioinspired synthetic catalysts for H₂ production.

     

Isoprenoid Biosynthesis in Pathogenic Bacteria: Nuclear Resonance Vibrational Spectroscopy Provides Insight into the Unusual [4Fe‐4S] Cluster of the E. coli LytB/IspH Protein

The LytB/IspH protein catalyzes the last step of the methylerythritol phosphate (MEP) pathway which is used for the biosynthesis of essential terpenoids in most pathogenic bacteria. Therefore, the MEP pathway is a target for the development of new antimicrobial agents as it is essential for microorganisms, yet absent in humans. Substrate‐free LytB has a special [4Fe‐4S]²⁺ cluster with a yet unsolved structure. This motivated us to use synchrotron‐based nuclear resonance vibrational spectroscopy (NRVS) in combination with quantum chemical‐molecular mechanical (QM/MM) calculations to gain more insight into the structure of substrate‐free LytB. The apical iron atom of the [4Fe‐4S]²⁺ is clearly linked to three water molecules. We additionally present NRVS data of LytB bound to its natural substrate, (E)‐4‐hydroxy‐3‐methylbut‐2‐en‐1‐yl diphosphate (HMBPP) and to the inhibitors (E)‐4‐amino‐3‐methylbut‐2‐en‐1‐yl diphosphate and (E)‐4‐mercapto‐3‐methylbut‐2‐en‐1‐yl diphosphate.     

Nitrosylation of Nitric‐Oxide‐Sensing Regulatory Proteins Containing [4Fe‐4S] Clusters Gives Rise to Multiple Iron–Nitrosyl Complexes

The reaction of protein‐bound iron–sulfur (Fe‐S) clusters with nitric oxide (NO) plays key roles in NO‐mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe‐S clusters has been hampered by a lack of information about the nature of the iron‐nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe‐4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe₂(NO)₄(Cys)₂]) and Roussin's Black Salt (RBS, [Fe₄(NO)₇S₃]. In the latter case, the absence of ³²S/³⁴S shifts in the Fe?S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.     

On‐Line Determination of Arsenic Species in Sea Water by Selective Hydride Generation Coupled with Inductively Coupled Plasma — Mass Spectrometry

A method for direct de termination of total in organic arsenic (III+V), arsenic (III) and dimethylarsinate (DMA) in sea water was developed by combining continuous‐flow selective hydride generation and inductively coupled plasma mass spectrometry (ICP‐MS) is presented. The principle underlying selective hydride generation is based on proper control of the reaction conditions for achieving separation of the respective arsenic species. The effects of pH and composition of reaction media on mutual interference between the arsenic species were investigated in detail. The results indicate that the appropriate media for the selective determination of total in organic arsenic, DMA and As(III) are 6 M HNO₃, acetate buffer at pH = 4.63 and citrate buffer at pH = 6.54, respectively. The concentrations of total inorganic arsenic species, As(III+V), and As(III) were respectively deter mined and that of As(V) was obtained by the difference between them. As to the concentration of DMA, it was obtained after correction from the interference caused by As(III) and As(V). By following the established procedure, the detection lim its (as based on 3‐sigma criterion) for As(III+V), As(III) and DMA were 0.050, 0.009, and 0.002 ng/mL, respectively. There liability of the pro posed method was evaluated in terms of precision and spike addition. The results indicated that the precision of better than 3% and spike recovery of 95 to 105% for all the arsenic species tested in the natural sea water samples can be obtained.     

[FeFe] Hydrogenase: Protonation of {2Fe3S} Systems and Formation of Super‐reduced Hydride States

The synthesis and crystallographic characterization of a complex possessing a well‐defined {2Fe3S(μ‐H)} core gives access to a paramagnetic bridging hydride with retention of the core geometry. Chemistry of this 35‐electron species within the confines of a thin‐layer FTIR spectro‐electrochemistry cell provides evidence for a unprecedented super‐reduced Fe^I(μ‐H)Fe^I intermediate.