Temperature-dependent iron motion in extremophile rubredoxins - no need for 'corresponding states'.

Extremophile organisms are known that can metabolize at temperatures down to - 25 °C (psychrophiles) and up to 122 °C (hyperthermophiles). Understanding viability under extreme conditions is relevant for human health, biotechnological applications, and our search for life elsewhere in the universe. Information about the stability and dynamics of proteins under environmental extremes is an important factor in this regard. Here we compare the dynamics of small Fe-S proteins - rubredoxins - from psychrophilic and hyperthermophilic microorganisms, using three different nuclear techniques as well as molecular dynamics calculations to quantify motion at the Fe site. The theory of 'corresponding states' posits that homologous proteins from different extremophiles have comparable flexibilities at the optimum growth temperatures of their respective organisms. Although 'corresponding states' would predict greater flexibility for rubredoxins that operate at low temperatures, we find that from 4 to 300 K, the dynamics of the Fe sites in these homologous proteins are essentially equivalent.

Facile electrocatalytic proton reduction by a [Fe-Fe]-hydrogenase bio-inspired synthetic model bearing a terminal CN- ligand.

An azadithiolate bridged CN- bound pentacarbonyl bis-iron complex, mimicking the active site of [Fe-Fe] H2ase is synthesized. The geometric and electronic structure of this complex is elucidated using a combination of EXAFS analysis, infrared and Mössbauer spectroscopy and DFT calculations. The electrochemical investigations show that complex 1 effectively reduces H+ to H2 between pH 0-3 at diffusion-controlled rates (1011 M-1 s-1) i.e. 108 s-1 at pH 3 with an overpotential of 140 mV. Electrochemical analysis and DFT calculations suggests that a CN- ligand increases the pKa of the cluster enabling hydrogen production from its Fe(i)-Fe(0) state at pHs much higher and overpotential much lower than its precursor bis-iron hexacarbonyl model which is active in its Fe(0)-Fe(0) state. The formation of a terminal Fe-H species, evidenced by spectroelectrochemistry in organic solvent, via a rate determining proton coupled electron transfer step and protonation of the adjacent azadithiolate, lowers the kinetic barrier leading to diffusion controlled rates of H2 evolution. The stereo-electronic factors enhance its catalytic rate by 3 order of magnitude relative to a bis-iron hexacarbonyl precursor at the same pH and potential.

Phonon-assisted electron-proton transfer in [FeFe] hydrogenases: Topological role of clusters.

[FeFe] hydrogenases are enzymes that have acquired a unique capacity to synthesize or consume molecular hydrogen (H2). This function relies on a complex catalytic mechanism involving the active site and two distinct electron and proton transfer networks working in concert. By an analysis based on terahertz vibrations of [FeFe] hydrogenase structure, we are able to predict and identify the existence of rate-promoting vibrations at the catalytic site and the coupling with functional residues involved in reported electron and proton transfer networks. Our findings suggest that the positioning of the cluster is influenced by the response of the scaffold to thermal fluctuations, which in turn drives the formation of networks for electron transfer through phonon-assisted mechanisms. Thus, we address the problem of linking the molecular structure to the catalytic function through picosecond dynamics, while raising the functional gain brought by the cofactors or clusters, using the concept of fold-encoded localized vibrations.

Nitrogenase Chemistry at 10 Kelvin─Phototautomerization and Recombination of CO-Inhibited α-H195Q Enzyme.

CO-bound forms of nitrogenase are N2-reduction inhibited and likely intermediates in Fischer-Tropsch chemistry. Visible-light photolysis at 7 K was used to interrogate all three known CO-related EPR-active forms as exhibited by the α-H195Q variant of Azotobacter vinelandii nitrogenase MoFe protein. The hi(5)-CO EPR signal converted to the hi-CO EPR signal, which reverted at 10 K. FT-IR monitoring revealed an exquisitely light-sensitive "Hi-2" species with bands at 1932 and 1866 cm-1 that yielded "Hi-1" with bands at 1969 and 1692 cm-1, which reverted to "Hi-2". The similarities of photochemical behavior and recombination kinetics showed, for the first time, that hi-CO EPR and "Hi-1" IR signals arise from one chemical species. hi(5)-CO EPR and "Hi-2" IR signals are from a second species, and lo-CO EPR and "Lo-2" IR signals, formed after prolonged illumination, are from a third species. Comparing FT-IR data with CO-inhibited MoFe-protein crystal structures allowed assignment of CO-bonding geometries in these species.

From inert gas to fertilizer, fuel and fine chemicals: N2 reduction and fixation.

The 100th anniversary of a leading nitrogen fixation technology developer like CASALE SA is a reason to reflect over the 20th century successful solution of the problem of world food supply, and to look out for solutions for sustainable developments with respect to ammonia production. We review the role of nitrogen as essential chemical constituent in photosynthesis and biology, and component of ammonia as it is used as fertilizer for primary production by photosynthesis for farming and food supply and its future role as energy carrier. While novel synthesis methods and very advanced synchrotron based x-ray analytical techniques are being developed, we feel it is important to refer to the historical and economical context of nitrogen. The breaking of the N≡N triple bond remains a fundamental chemical and energetic problem in this context. We review the electrochemical ammonia synthesis as an energetically and environmentally benign method. Two relatively novel X-ray spectroscopy methods, which are relevant for the molecular understanding of the catalysts and biocatalysts, i.e. soft X-ray absorption spectroscopy and nuclear resonant vibration spectroscopy are presented. We illustrate the perceived reality in fertilizer usage on the field, and fertilizer production in the factory complex with photos and thus provide a contrast to the academic view of the molecular process of ammonia function and production.

Carbon monoxide binding to α-R277H Mo-nitrogenase - Evidence for multiple pH-dependent species from IR-monitored photolysis.

The nitrogenase (N2ase) enzyme family is responsible for the conversion of dinitrogen into biologically accessible ammonia, a critical step in the global nitrogen cycle. Carbon monoxide (CO) has long been known as an inhibitor of dinitrogen reduction, but it can also be reduced to hydrocarbons catalyzed by all three N2ases, namely the wild-type Mo enzyme and select variants and the V and Fe nitrogenases, both of which are orders of magnitude more effective. CO interactions with N2ases are thus relevant to both dinitrogen fixation and Fischer-Tropsch-like chemistry. Here, we investigated the interaction of CO with the α-R277H variant of the Azotobacter vinelandii N2ase MoFe protein, in which the α-subunit 277Arg residue is replaced by His and results in production of only the S = 3/2 EPR signal (denoted as hi(5)-CO). Fourier-transform infrared (FT-IR) spectroscopy was used to follow the photolysis of CO bound to the α-R277H variant under cryogenic conditions. Multiple EPR-silent species were observed with FT-IR spectroscopic signatures previously assigned to CO-inhibited forms of the α-H195Q and α-H195N N2ase variants. The distribution of these CO-inhibited forms varied dramatically with pH over the range of pH 6.5 to pH 8.5, indicating protonation/deprotonation involvement.

NRVS and DFT of MitoNEET: Understanding the Special Vibrational Structure of a [2Fe-2S] Cluster with (Cys)3(His)1 Ligation.

The human mitochondrial protein, mitoNEET (mNT), belongs to the family of small [2Fe-2S] NEET proteins that bind their iron-sulfur clusters with a novel and characteristic 3Cys:1His coordination motif. mNT has been implicated in the regulation of lipid and glucose metabolisms, iron/reactive oxygen species homeostasis, cancer, and possibly Parkinson's disease. The geometric structure of mNT as a function of redox state and pH is critical for its function. In this study, we combine 57Fe nuclear resonance vibrational spectroscopy with density functional theory calculations to understand the novel properties of this important protein.

Vibrational Perturbation of the [FeFe] Hydrogenase H-Cluster Revealed by 13C2H-ADT Labeling.

[FeFe] hydrogenases are highly active catalysts for the interconversion of molecular hydrogen with protons and electrons. Here, we use a combination of isotopic labeling, 57Fe nuclear resonance vibrational spectroscopy (NRVS), and density functional theory (DFT) calculations to observe and characterize the vibrational modes involving motion of the 2-azapropane-1,3-dithiolate (ADT) ligand bridging the two iron sites in the [2Fe]H subcluster. A -13C2H2- ADT labeling in the synthetic diiron precursor of [2Fe]H produced isotope effects observed throughout the NRVS spectrum. The two precursor isotopologues were then used to reconstitute the H-cluster of [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1), and NRVS was measured on samples poised in the catalytically crucial Hhyd state containing a terminal hydride at the distal Fe site. The 13C2H isotope effects were observed also in the Hhyd spectrum. DFT simulations of the spectra allowed identification of the 57Fe normal modes coupled to the ADT ligand motions. Particularly, a variety of normal modes involve shortening of the distance between the distal Fe-H hydride and ADT N-H bridgehead hydrogen, which may be relevant to the formation of a transition state on the way to H2 formation.

Exploring Structure and Function of Redox Intermediates in [NiFe]-Hydrogenases by an Advanced Experimental Approach for Solvated, Lyophilized and Crystallized Metalloenzymes.

To study metalloenzymes in detail, we developed a new experimental setup allowing the controlled preparation of catalytic intermediates for characterization by various spectroscopic techniques. The in situ monitoring of redox transitions by infrared spectroscopy in enzyme lyophilizate, crystals, and solution during gas exchange in a wide temperature range can be accomplished as well. Two O2 -tolerant [NiFe]-hydrogenases were investigated as model systems. First, we utilized our platform to prepare highly concentrated hydrogenase lyophilizate in a paramagnetic state harboring a bridging hydride. This procedure proved beneficial for 57 Fe nuclear resonance vibrational spectroscopy and revealed, in combination with density functional theory calculations, the vibrational fingerprint of this catalytic intermediate. The same in situ IR setup, combined with resonance Raman spectroscopy, provided detailed insights into the redox chemistry of enzyme crystals, underlining the general necessity to complement X-ray crystallographic data with spectroscopic analyses.

Nuclear Resonance Vibrational Spectroscopy: A Modern Tool to Pinpoint Site-Specific Cooperative Processes.

Nuclear resonant vibrational spectroscopy (NRVS) is a synchrotron radiation (SR)-based nuclear inelastic scattering spectroscopy that measures the phonons (i.e., vibrational modes) associated with the nuclear transition. It has distinct advantages over traditional vibration spectroscopy and has wide applications in physics, chemistry, bioinorganic chemistry, materials sciences, and geology, as well as many other research areas. In this article, we present a scientific and figurative description of this yet modern tool for the potential users in various research fields in the future. In addition to short discussions on its development history, principles, and other theoretical issues, the focus of this article is on the experimental aspects, such as the instruments, the practical measurement issues, the data process, and a few examples of its applications. The article concludes with introduction to non-57Fe NRVS and an outlook on the impact from the future upgrade of SR rings.

High-Frequency Fe-H and Fe-H2 Modes in a trans-Fe(η2-H2)(H) Complex: A Speed Record for Nuclear Resonance Vibrational Spectroscopy.

Nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) are complementary tools for studying the vibrational and geometric structures of specific isotopically labeled molecular systems. Here we apply NRVS and DFT to characterize the trans-[57Fe(η2-H2)(H)(dppe)2][BPh4] [dppe = 1,2-bis(diphenylphosphino)ethane] complex. Heretofore, most NRVS observations have centered on the spectral region below 1000 cm-1, where the 57Fe signal is strongest. In this work, we show that state-of-the-art synchrotron facilities can extend the observable region to 2000 cm-1 and likely beyond, in measurements that require less than 1 day. The 57Fe-H stretch was revealed at 1915 cm-1, along with the asymmetric 57Fe-H2 stretch at 1774 cm-1. For a small fraction of the H2-dissociated product, the 57Fe-H stretch was detected at 1956 cm-1. The unique sensitivity to 57Fe motion and the isolated nature of the Fe-H/H2 stretching modes enabled NRVS to quantitatively analyze the sample composition.

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 Hinact . To date, the structure and mechanism of formation of Hinact remain unknown. Our 1.65 Šcrystal 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 Hinact state and its mechanism of formation. This may facilitate the design of O2 -stable hydrogenases and molecular catalysts.

The large subunit of the regulatory [NiFe]-hydrogenase from Ralstonia eutropha - a minimal hydrogenase?

Chemically synthesized compounds that are capable of facilitating the reversible splitting of dihydrogen into protons and electrons are rare in chemists' portfolio. The corresponding biocatalysts - hydrogenases - are, however, abundant in the microbial world. [NiFe]-hydrogenases represent a major subclass and display a bipartite architecture, composed of a large subunit, hosting the catalytic NiFe(CO)(CN)2 cofactor, and a small subunit whose iron-sulfur clusters are responsible for electron transfer. To analyze in detail the catalytic competence of the large subunit without its smaller counterpart, we purified the large subunit HoxC of the regulatory [NiFe]-hydrogenase of the model H2 oxidizer Ralstonia eutropha to homogeneity. Metal determination and infrared spectroscopy revealed a stoichiometric loading of the metal cofactor. This enabled for the first time the determination of the UV-visible extinction coefficient of the NiFe(CO)(CN)2 cofactor. Moreover, the absence of disturbing iron-sulfur clusters allowed an unbiased look into the low-spin Fe2+ of the active site by Mössbauer spectroscopy. Isolated HoxC was active in catalytic hydrogen-deuterium exchange, demonstrating its capacity to activate H2. Its catalytic activity was drastically lower than that of the bipartite holoenzyme. This was consistent with infrared and electron paramagnetic resonance spectroscopic observations, suggesting that the bridging position between the active site nickel and iron ions is predominantly occupied by water-derived ligands, even under reducing conditions. In fact, the presence of water-derived ligands bound to low-spin Ni2+ was reflected by the absorption bands occurring in the corresponding UV-vis spectra, as revealed by time-dependent density functional theory calculations conducted on appropriate in silico models. Thus, the isolated large subunits indeed represent simple [NiFe]-hydrogenase models, which could serve as blueprints for chemically synthesized mimics. Furthermore, our data point to a fundamental role of the small subunit in preventing water access to the catalytic center, which significantly increases the H2 splitting capacity of the enzyme.

Vibrational characterization of a diiron bridging hydride complex - a model for hydrogen catalysis.

A diiron complex containing a bridging hydride and a protonated terminal thiolate of the form [(µ,κ2-bdtH)(µ-PPh2)(µ-H)Fe2(CO)5]+ has been investigated through 57Fe nuclear resonance vibrational spectroscopy (NRVS) and interpreted using density functional theory (DFT) calculations. We report the Fe-µH-Fe wagging mode, and indications for Fe-µD stretching vibrations in the D-isotopologue, observed by 57Fe-NRVS. Our combined approach demonstrates an asymmetric sharing of the hydride between the two iron sites that yields two nondegenerate Fe-µH/D stretching vibrations. The studied complex provides an important model relevant to biological hydrogen catalysis intermediates. The complex mimics proposals for the binuclear metal sites in [FeFe] and [NiFe] hydrogenases. It is also an appealing prototype for the 'Janus intermediate' of nitrogenase, which has been proposed to contain two bridging Fe-H-Fe hydrides and two protonated sulfurs at the FeMo-cofactor. The significance of observing indirect effects of the bridging hydride, as well as obstacles in its direct observation, is discussed in the context of biological hydrogen intermediates.

Hydroxy-bridged resting states of a [NiFe]-hydrogenase unraveled by cryogenic vibrational spectroscopy and DFT computations.

The catalytic mechanism of [NiFe]-hydrogenases is a subject of extensive research. Apart from at least four reaction intermediates of H2/H+ cycling, there are also a number of resting states, which are formed under oxidizing conditions. Although not directly involved in the catalytic cycle, the knowledge of their molecular structures and reactivity is important, because these states usually accumulate in the course of hydrogenase purification and may also play a role in vivo during hydrogenase maturation. Here, we applied low-temperature infrared (cryo-IR) and nuclear resonance vibrational spectroscopy (NRVS) to the isolated catalytic subunit (HoxC) of the heterodimeric regulatory [NiFe]-hydrogenase (RH) from Ralstonia eutropha. Cryo-IR spectroscopy revealed that the HoxC protein can be enriched in almost pure resting redox states suitable for NRVS investigation. NRVS analysis of the hydrogenase catalytic center is usually hampered by strong spectral contributions of the FeS clusters of the small, electron-transferring subunit. Therefore, our approach to investigate the FeS cluster-free, 57Fe-labeled HoxC provided an unprecedented insight into the [NiFe] site modes, revealing their contributions in a spectral range otherwise superimposed by FeS cluster-derived bands. Rationalized by density functional theory (DFT) calculations, our data provide structural descriptions of the previously uncharacterized hydroxy- and water-containing resting states. Our work highlights the relevance of cryogenic vibrational spectroscopy and DFT to elucidate the structure of barely defined redox states of the [NiFe]-hydrogenase active site.

In Vitro Assembly as a Tool to Investigate Catalytic Intermediates of [NiFe]-Hydrogenase.

[NiFe]-hydrogenases catalyze the reversible reaction H2 ⇄ 2H+ + 2e-. Their basic module consists of a large subunit, coordinating the NiFe(CO)(CN)2 center, and a small subunit that carries electron-transferring iron-sulfur clusters. Here, we report the in vitro assembly of fully functional [NiFe]-hydrogenase starting from the isolated large and small subunits. Activity assays complemented by spectroscopic measurements revealed a native-like hydrogenase. This approach was used to label exclusively the NiFe(CO)(CN)2 center with 57Fe, enabling a clear view of the catalytic site by means of nuclear resonance vibrational spectroscopy. This strategy paves the way for in-depth studies of [NiFe]-hydrogenase catalytic intermediates.

Assignment of protonated R-homocitrate in extracted FeMo-cofactor of nitrogenase via vibrational circular dichroism spectroscopies.

Protonation of FeMo-cofactor is important for the process of substrate hydrogenation. Its structure has been clarified as Δ-Mo*Fe7S9C(R-homocit*)(cys)(Hhis) for the efforts of nearly 30 years, while it remains controversial whether FeMo-cofactor is protonated or deprotonated with chelated ≡C-O(H) homocitrate. We have used protonated molybdenum(V) lactates 1 and its enantiomer as model compounds for R-homocitrate in FeMo-cofactor of nitrogenase. Vibrational circular dichroism (VCD) spectrum of 1 at 1051 cm-1 is attributed to ≡C-OH vibration, and molybdenum(VI) R-lactate at 1086 cm-1 is assigned as ≡C-O α-alkoxy vibration. These vibrations set up labels for the protonation state of coordinated α-hydroxycarboxylates. The characteristic VCD band of NMF-extracted FeMo-cofactor is assigned to ν(C-OH), which is based on the comparison of molybdenum(VI) R-homocitrate. Density Functional Theory calculations are in consistent with these assignments. To the best of our knowledge, this is the first time that protonated R-homocitrate in FeMo-cofactor is confirmed by VCD spectra.

Spectroscopic and Computational Evidence that [FeFe] Hydrogenases Operate Exclusively with CO-Bridged Intermediates.

[FeFe] hydrogenases are extremely active H2-converting enzymes. Their mechanism remains highly controversial, in particular, the nature of the one-electron and two-electron reduced intermediates called HredH+ and HsredH+. In one model, the HredH+ and HsredH+ states contain a semibridging CO, while in the other model, the bridging CO is replaced by a bridging hydride. Using low-temperature IR spectroscopy and nuclear resonance vibrational spectroscopy, together with density functional theory calculations, we show that the bridging CO is retained in the HsredH+ and HredH+ states in the [FeFe] hydrogenases from Chlamydomonas reinhardtii and Desulfovibrio desulfuricans, respectively. Furthermore, there is no evidence for a bridging hydride in either state. These results agree with a model of the catalytic cycle in which the HredH+ and HsredH+ states are integral, catalytically competent components. We conclude that proton-coupled electron transfer between the two subclusters is crucial to catalysis and allows these enzymes to operate in a highly efficient and reversible manner.

Asymmetry in the Ligand Coordination Sphere of the [FeFe] Hydrogenase Active Site Is Reflected in the Magnetic Spin Interactions of the Aza-propanedithiolate Ligand.

[FeFe] hydrogenases are very active enzymes that catalyze the reversible conversion of molecular hydrogen into protons and electrons. Their active site, the H-cluster, contains a unique binuclear iron complex, [2Fe]H, with CN- and CO ligands as well as an aza-propane-dithiolate (ADT) moiety featuring a central amine functionality that mediates proton transfer during catalysis. We present a pulsed 13C-ENDOR investigation of the H-cluster in which the two methylene carbons of ADT are isotope labeled with 13C. We observed that the corresponding two 13C hyperfine interactions are of opposite sign and corroborated this finding using density functional theory calculations. The spin polarization in the ADT ligand is shown to be linked to the asymmetric coordination of the distal iron site with its terminal CN- and CO ligands. We propose that this asymmetry is relevant for the enzyme reactivity and is related to the (optimal) stabilization of the iron-hydride intermediate in the catalytic cycle.

Insights from 125Te and 57Fe nuclear resonance vibrational spectroscopy: a [4Fe-4Te] cluster from two points of view.

Iron-sulfur clusters are common building blocks for electron transport and active sites of metalloproteins. Their comprehensive investigation is crucial for understanding these enzymes, which play important roles in modern biomimetic catalysis and biotechnology applications. We address this issue by utilizing (Et4N)3[Fe4Te4(SPh)4], a tellurium modified version of a conventional reduced [4Fe-4S]+ cluster, and performed both 57Fe- and 125Te-NRVS to reveal its characteristic vibrational features. Our analysis exposed major differences in the resulting 57Fe spectrum profile as compared to that of the respective [4Fe-4S] cluster, and between the 57Fe and 125Te profiles. DFT calculations are applied to rationalize structural, electronic, vibrational, and redox-dependent properties of the [4Fe-4Te]+ core. We herein highlight the potential of sulfur/tellurium exchange as a method to isolate the iron-only motion in enzymatic systems.