Dihydrogen activation by sulfido-bridged dinuclear Ru/Ge complexes: insight into the [NiFe] hydrogenase unready state

A S/SH bridged hetero-dinuclear Ru/Ge complex cation reacted with H(2) to afford the μ-S/μ-H complex. The reaction was considerably slower compared to that of the μ-S/μ-OH complex. Thus, the μ-S/μ-SH and μ-S/μ-OH complexes might provide models for the unready and ready states, respectively, of [NiFe] hydrogenase.     

High-spin Ni(II), a surprisingly good structural model for [NiFe] hydrogenase.

The first density functional calculations on high-spin (HS) Ni(II) models for the active site of the [NiFe] hydrogenases predict a ligand arrangement about Ni that is in better agreement with the crystal structures than previous predictions for low-spin (LS) Ni(II) models. With the crystal structures' geometry, the HS form is approximately 20 kcal/mol lower in energy than the LS one.     

Electronic structure of a binuclear nickel complex of relevance to [NiFe] hydrogenase

The binuclear complex [Ni(2)(L)(MeCN)(2)](3+) (L(2-) = compartmental macrocycle incorporating imine N and thiolate S donors) has a Ni(III) center bridged via two thiolate S-donors to a diamagnetic Ni(II) center. The ground-state has dominant 3d(z)(1)(2) character similar to that observed for [NiFe] hydrogenases in which Ni(III) is bridged via two thiolate donors to a diamagnetic center (Fe(II)). The system has been studied by X-ray crystallography and pulse EPR, ESEEM, and ENDOR spectroscopy in order to determine the extent of spin-delocalization onto the macrocycle L(2-). The hyperfine coupling constants of six nitrogen atoms have been identified and divided into three sets of two equivalent nitrogens. The most strongly coupled nitrogen atoms (a(iso) approximately 53 MHz) stem from axially bound solvent acetonitrile molecules. The two macrocycle nitrogens on the Ni(III) side have a coupling of a(iso) approximately 11 MHz, and those on the Ni(II) side have a coupling of a(iso) approximately 1-2 MHz. Density functional theory (DFT) calculations confirm this assignment, while comparison of the calculated and experimental (14)N hyperfine coupling constants yields a complete picture of the electron-spin density distribution. In total, 91% spin density is found at the Ni(III) of which 72% is in the 3d(z)(2) orbital and 16% in the 3d(xy) orbital. The Ni(II) contains -3.5% spin density, and 7.5% spin density is found at the axial MeCN ligands. In analogy to hydrogenases, it becomes apparent that binding of a substrate to Ni at the axial positions causes a redistribution of the electron charge and spin density, and this redistribution polarizes the chemical bonds of the axial ligand. For [NiFe] hydrogenases this implies that the H(2) bond becomes polarized upon binding of the substrate, which may facilitate its heterolytic splitting.     

Resorcin[6]arene as a building block for tubular crystalline state architectures

A resorcin[6]arene with an r-trans-cis-trans-cis-trans configuration of the pendant ethyl groups forms tubular crystal structures.     

Photoswitchable electrochemical behaviour of a [FeFe] hydrogenase model with a dithienylethene derivative

A diiron dithiolate complex 1o with a dithienylethene (DTE) phosphine ligand has been elaborately designed and fully investigated by spectroscopic and DFT computational studies. Upon irradiation with UV light, the DTE moiety in complex 1o undergoes an excellent photocyclization reaction to attain ring-closed state 1c in high yield (>95%), accompanied by a colour change from orange to deep blue. On the other hand, upon irradiation with visible light (>460 nm), ring-closed form 1c in CH(3)CN solution reverts perfectly into ring-open form 1o. Both 1o and 1c were characterised by IR, (1)H, (31)P, (19)F NMR and electrochemical spectroscopy. The electrochemical behaviours of both the open and closed forms were investigated by cyclic voltammetry. Upon photocyclization reaction, a 290 mV (from -2.29 V to -2.00 V) positive shift is induced in the potential of electrochemical catalytic proton reduction, due to the electron-withdrawing effect of the ring-closed DTE moiety. Consequently, complex 1 can reversibly photoswitch the potential of proton reduction on the [FeFe] moiety.     

Original design of an oxygen-tolerant [NiFe] hydrogenase: major effect of a valine-to-cysteine mutation near the active site

Hydrogenases are efficient biological catalysts of H(2) oxidation and production. Most of them are inhibited by O(2), and a prerequisite for their use in biotechnological applications under air is to improve their oxygen tolerance. We have previously shown that exchanging the residue at position 74 in the large subunit of the oxygen-sensitive [NiFe] hydrogenase from Desulfovibrio fructosovorans could impact the reaction of the enzyme with O(2) (Dementin, S.; J. Am. Chem. Soc. 2009, 131, 10156-10164; Liebgott, P. P.; Nat. Chem. Biol. 2010, 6, 63-70). This residue, a valine in the wild-type enzyme, located at the bottleneck of the gas channel near the active site, has here been exchanged with a cysteine. A thorough characterization using a combination of kinetic, spectroscopic (EPR, FTIR), and electrochemical studies demonstrates that the V74C mutant has features of the naturally occurring oxygen-tolerant membrane-bound hydrogenases (MBH). The mutant is functional during several minutes under O(2), has impaired H(2)-production activity, and has a weaker affinity for CO than the WT. Upon exposure to O(2), it is converted into the more easily reactivatable inactive form, Ni-B, and this inactive state reactivates about 20 times faster than in the WT enzyme. Control experiments carried out with the V74S and V74N mutants indicate that protonation of the position 74 residue is not the reason the mutants reactivate faster than the WT enzyme. The electrochemical behavior of the V74C mutant toward O(2) is intermediate between that of the WT enzyme from D. fructosovorans and the oxygen-tolerant MBH from Aquifex aeolicus.     

A triad [FeFe] hydrogenase system for light-driven hydrogen evolution

A novel molecular triad [FeFe]-H(2)ase 1, and its model complexes 2 and 3 have been successfully constructed. The multistep PET and long-lived Fe(i)Fe(0) species were found to be responsible for the better performance of triad 1 than that of 2 with 3 for light-driven H(2) evolution.     

Ni(xbsms)Ru(CO)2Cl2]: a bioinspired nickel-ruthenium functional model of [NiFe] hydrogenase

As a model of the active site of [NiFe] hydrogenases, a dinuclear nickel-ruthenium complex [Ni(xbsms)Ru(CO)2Cl2] was synthesized and fully characterized. The three-dimensional structure reveals a nickel center in a square-planar dithioether-dithiolate environment connected to a ruthenium moiety via a Ni(mu-SR)2Ru bridge. This complex catalyzes hydrogen evolution by electroreduction of the weakly acidic Et3NH+ ions in N,N-dimethylformamide and is therefore the first functional bioinspired model of [NiFe] hydrogenases.     

The electrochemical polymerisation of a [2]rotaxane

We report the synthesis of a dithienylpyrrole-stoppered rotaxane and its subsequent electrochemical polymerisation onto a platinum working electrode surface. We have shown that the tetracationic cyclophane moiety of the rotaxane does not impair electropolymerisation of this derivative. Indeed, functionalised films can be conveniently prepared by oxidative polymerisation of the dithienylpyrrole stopper units, to yield a network of rotaxane units interconnected by a conducting polymer backbone.     

Structural studies of the carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F: suggestion for the initial activation site for dihydrogen

The carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F has been characterized by X-ray crystallography and absorption and resonance Raman spectroscopy. Nine crystal structures of the [NiFe]hydrogenase in the CO-bound and CO-liberated forms were determined at 1.2-1.4 A resolution. The exogenously added CO was assigned to be bound to the Ni atom at the Ni-Fe active site. The CO was not replaced with H(2) in the dark at 100 K, but was liberated by illumination with a strong white light. The Ni-C distances and Ni-C-O angles were about 1.77 A and 160 degrees, respectively, except for one case (1.72 A and 135 degrees ), in which an additional electron density peak between the CO and Sgamma(Cys546) was recognized. Distinct changes were observed in the electron density distribution of the Ni and Sgamma(Cys546) atoms between the CO-bound and CO-liberated structures for all the crystals tested. The novel structural features found near the Ni and Sgamma(Cys546) atoms suggest that these two atoms at the Ni-Fe active site play a role during the initial H(2)-binding process. Anaerobic addition of CO to dithionite-reduced [NiFe]hydrogenase led to a new absorption band at about 470 nm ( approximately 3000 M(-1)cm(-1)). Resonance Raman spectra (excitation at 476.5 nm) of the CO complex revealed CO-isotope-sensitive bands at 375/393 and 430 cm(-1) (368 and 413 cm(-1) for (13)C(18)O). The frequencies and relative intensities of the CO-related Raman bands indicated that the exogenous CO is bound to the Ni atom with a bent Ni-C-O structure in solution, in agreement with the refined structure determined by X-ray crystallography.     

Catalysts for hydrogen evolution from the [NiFe] hydrogenase to the Ni2P(001) surface: the importance of ensemble effect

Density functional theory (DFT) was employed to investigate the behavior of a series of catalysts used in the hydrogen evolution reaction (HER, 2H(+) + 2e(-) --> H(2)). The kinetics of the HER was studied on the [NiFe] hydrogenase, the [Ni(PS3*)(CO)](1)(-) and [Ni(PNP)(2)](2+) complexes, and surfaces such as Ni(111), Pt(111), or Ni(2)P(001). Our results show that the [NiFe] hydrogenase exhibits the highest activity toward the HER, followed by [Ni(PNP)(2)](2+) > Ni(2)P > [Ni(PS3*)(CO)](1)(-) > Pt > Ni in a decreasing sequence. The slow kinetics of the HER on the surfaces is due to the fact that the metal hollow sites bond hydrogen too strongly to allow the facile removal of H(2). In fact, the strong H-Ni interaction on Ni(2)P(001) can lead to poisoning of the highly active sites of the surface, which enhances the rate of the HER and makes it comparable to that of the [NiFe] hydrogenase. In contrast, the promotional effect of H-poisoning on the HER on Pt and Ni surfaces is relatively small. Our calculations suggest that among all of the systems investigated, Ni(2)P should be the best practical catalyst for the HER, combining the high thermostability of the surfaces and high catalytic activity of the [NiFe] hydrogenase. The good behavior of Ni(2)P(001) toward the HER is found to be associated with an ensemble effect, where the number of active Ni sites is decreased due to presence of P, which leads to moderate bonding of the intermediates and products with the surface. In addition, the P sites are not simple spectators and directly participate in the HER.     

A microscopic model for gas diffusion dynamics in a [NiFe]-hydrogenase

We describe and apply a microscopic model for the calculation of gas diffusion rates in a [NiFe]-hydrogenase. This enzyme has attracted much interest for use as a H(2) oxidising catalyst in biofuel cells, but a major problem is their inhibition by CO and O(2). In our model, the diffusive hopping of gas molecules in the protein interior is coarse grained using a master equation approach with transition rates estimated from equilibrium and non-equilibrium pulling simulations. Propagating the rate matrix in time, we find that the probability for a gas molecule to reach the enzyme active site follows a mono-exponential increase. Fits to a phenomenological rate law give an effective diffusion rate constant for CO that is in very good agreement with experimental measurements. We find that CO prefers to move along the canonical 'hydrophobic' main channel towards the active site, in contrast to O(2) and H(2), which were previously shown to explore larger fractions of the protein. Differences in the diffusion of the three gases are discussed in light of recent efforts to engineer a gas selectivity filter in the enzyme.     

Hydrogen bonding affects the [NiFe] active site of Desulfovibrio vulgaris Miyazaki F hydrogenase: a hyperfine sublevel correlation spectroscopy and density functional theory study

Pulse electron paramagnetic resonance and hyperfine sublevel correlation spectroscopy have been used to investigate nitrogen coordination of the active site of [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in its oxidized "ready" state. The obtained (14)N hyperfine (A = [+1.32, +1.32, +2.07] MHz) and nuclear quadrupole (e(2)qQ/h = -1.9 MHz, eta = 0.37) coupling constants were assigned to the N(epsilon) of a highly conserved histidine (His88) by studying a hydrogenase preparation in which the histidines were (15)N labeled. The histidine is hydrogen-bonded via its N(epsilon)-H to the nickel-coordinating sulfur of a cysteine (Cys549) that carries an appreciable amount of spin density. Through the hydrogen bond a small fraction of the spin density ( approximately 1%) is delocalized onto the histidine ring giving rise to an isotropic (14)N hyperfine coupling constant of about 1.6 MHz. These conclusions are supported by density functional calculations. The measured (14)N quadrupole coupling constants are related to the polarization of the N(epsilon)-H bond, and the respective hydrogen bond can be classified as being weak.     

Direct detection of a hydrogen ligand in the [NiFe] center of the regulatory H2-sensing hydrogenase from Ralstonia eutropha in its reduced state by HYSCORE and ENDOR spectroscopy

The regulatory H2-sensing [NiFe] hydrogenase of the beta-proteobacterium Ralstonia eutropha displays an Ni-C "active" state after reduction with H2 that is very similar to the reduced Ni-C state of standard [NiFe] hydrogenases. Pulse electron nuclear double resonance (ENDOR) and four-pulse ESEEM (hyperfine sublevel correlation, HYSCORE) spectroscopy are applied to obtain structural information on this state via detection of the electron-nuclear hyperfine coupling constants. Two proton hyperfine couplings are determined by analysis of ENDOR spectra recorded over the full magnetic field range of the EPR spectrum. These are associated with nonexchangeable protons and belong to the beta-CH(2) protons of a bridging cysteine of the NiFe center. The signals of a third proton exhibit a large anisotropic coupling (Ax = 18.4 MHz, Ay = -10.8 MHz, Az = -18 MHz). They disappear from the 1H region of the ENDOR spectra after exchange of H2O with 2H2O and activation with 2H2 instead of H2 gas. They reappear in the 2H region of the ENDOR and HYSCORE spectra. Based on a comparison with the spectroscopically similar [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F, for which the g-tensor orientation of the Ni-C state with respect to the crystal structure is known (Foerster et al. J. Am. Chem. Soc. 2003, 125, 83-93), an assignment of the 1H hyperfine couplings is proposed. The exchangeable proton resides in a bridging position between the Ni and Fe and is assigned to a formal hydride ion. After illumination at low temperature (T = 10 K), the Ni-L state is formed. For the Ni-L state, the strong hyperfine coupling observed for the exchangeable hydrogen in Ni-C is lost, indicating a cleavage of the metal-hydride bond(s). These experiments give first direct information on the position of hydrogen binding in the active NiFe center of the regulatory hydrogenase. It is proposed that such a binding situation is also present in the active Ni-C state of standard hydrogenases.     

Reversible heterolysis of H2 mediated by an M-S(thiolate) bond (M = Ir, Rh): a mechanistic implication for [NiFe] hydrogenase

Facile H2 heterolysis was found to be mediated by coordinatively unsaturated Cp*Ir and Cp*Rh thiolate complexes. The reaction of iridium complex is reversible, and the formation of an intermediary Ir-H/thiol complex was detected. The reversible conversion between thiolate complex+H2 and hydride complex+thiol provides an intriguing functional model of [NiFe] hydrogenase.     

Construction of Heterometallic Clusters in a Small Peptide Scaffold as [NiFe]-Hydrogenase Models: Development of a Synthetic Methodology

[NiFe]-hydrogenases are enzymes that catalyze the reversible interconversion of protons and hydrogen at a heterobimetallic site containing Ni and Fe. This organometallic site has served as an inspiration for the synthesis of a number of biomimetic complexes, but, unfortunately, most close structural mimics have shown little to no reactivity with either of the substrates for hydrogenases. This suggests that interactions between the metallo-active site and the protein scaffold are crucial in tuning reactivity. As a first step toward development of peptide-based models, in this paper we demonstrate a synthetic strategy for construction of peptide coordinated, cysteinyl thiolate bridged Ni-M complexes in which M is a hetero-organometallic fragment. We utilize the seven amino acid peptide ACDLPCG as a scaffold for construction of these peptide-coordinated metallocenters. This peptide binds Ni in an N(2)S(2) environment consisting of the amino terminus, an amide nitrogen, and the two cysteinyl thiolates. We show that these thiolates serve as reactive sites for formation of heterometallic complexes in which they serve as bridging ligands. The method is general, and a number of heterometallic fragments including Ru(η(6)-arene)(2+), M(CO)(4)(piperidine) for M = Mo and W, and Fe(2)(CO)(6) were successfully incorporated, and the resulting metallopeptides characterized via a range of spectroscopic techniques. This methodology serves as the first step to construction of hydrogenase peptidomimetics that incorporate defined outer coordination sphere interactions intended to tune reactivity.