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Electron paramagnetic resonance spectroscopy was used in studying the effect of copper and zinc ions on manganese-depleted photosystem II (PS II). Copper and zinc ions were shown to have similar effects on PS II systems although a higher concentration of zinc ions was needed to elicit the same effect as the copper ions. The differing redox and binding behavior of the copper ions in the iron site in PS II compared to its behavior in the bacterial reaction center is seen as further proof of the varying iron coordination in the two systems.

Shifts of the special pair redox potential of the photosynthetic reaction center of Rhodobacter sphaeroides were studied for various point mutations in the region adjacent to the special pair. The computed shifts of the midpoint potential of double and triple mutants can approximately be obtained from the corresponding shifts of the single point mutations. The computed shifts agree with the measured values for all single and double mutants.

The loss or addition of hydrogen bonds changes the energy of the internal charge transfer state in the bacteriochlorophyll (BChl) -bacteriopheophytin (BPhe) dimer in the reaction center of Rhodobacter sphaeroides. The addition of a bond increases the primary donor's (P) oxidation-reduction midpoint potential in BChl and BPhe. This is accompanied by an increased rate of charge recombination from the primary quinone. The hydrogen bonds increase the transfer state's energy in BChl and stabilize the state in BPhe.

Mutants of Rhodobacter sphaeroides, in which hydrogen bonds were added or removed in the reaction center (RC), produced an increase or decrease in the oxidation midpoint potential. The RC is a membrane protein, which brings about electron transfer in photosynthesis and has three protein subunits that bind the cofactors: four bacteriochlorophyll, a (BChl a) and other units. Electro nuclear double resonance and triple resonance reactions for mutants with various combinations of the four carbonyl oxygens of the BChl dimer were studied.

A comparative study of the reaction centers (RCs) of the four species of purple bacteria, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum and Rhodospirillum centenum, using electron paramagnetic resonance, electron nuclear double resonance and electron nuclear triple resonance spectroscopies, reveals differences in the spin density distribution of the dimer across the various species. The asymmetries are due to different orbital energies of the two bacteriochlorophylls halves of the dimer. The reaction center environment influences the electronic structure of the cofactors and the electron transfer rates.

Study is presented to demonstrate that the combination of pulsed high-field EPR spectroscopy at the W band with its extension to pulsed electron-electron double resonance (PELDOR) and relaxation -induced dipolar modulation enhancement (RIDME) offers a powerful tool for obtaining three-dimensional structure of radical-pair systems with large interspin distances.

The temperature dependence of the electron spin-lattice relaxation time T1 was measured for the S(sub 0) state of the oxygen-evolving complex in photosystem II and for two dinuclear manganese model complexes by pulse EPR. For the latter complex, an energy separation between the ground and the first excited electronic state was determined.

The superoxidized Mn(super III)Mn(super IV) state of dimanganase catalase from Thermus thermophilus and a series of structurally similar Mn(super III)Mn(super IV) complexes were investigated using continuous wave (CW) and pulsed EPR spectroscopy at X- (9 GHz), Q- (34 GHz) frequencies. An important experimental aspect is the elimination of Mn(super II) impurities, which give rise to very intense lines superimposed to the spectrum of the Mn(super III)Mn(super IV) complex.

The photosynthesis reaction center is an integral membrane protein that carries out the initial charge-separation reactions of photosynthesis. Upon light excitation, a pair (P) of bacteriochlorophylls (Bchls) donates an electron to a bacteriopheophytin (H(sub L), generating an ion-pair (P(sub +)H(sub L)(super -)).

The anisotropic transverse relaxation times T(sub 2) of the primary and secondary acceptor quinone radical anions Q(sub A)(super -.) and Q(sub B)(super -.) in Zn-substituted photosynthetic bacterial reaction of Rhodobacter sohaeroides R26 by means of 2D high-field-frequency electron spin-echo spectroscopy are investigated. The anisotropy of the relaxation times is related to anisotropic stochastic fluctuations of the quinones in their protein binding pockets, which are temperature-dependant.

Research was conducted to examine the replacement of the Fe2+ by Zn2+ in the bacterial reaction center (RC) of Rhodopseudomonas viridis by a chemical method. The procedure for the iron substitution is described. The obtained ZnRCs were characterized and the primary quinone radical anion Q(sub A)-* and the radical pair P960+*Q(sub A)-* were investigated through ENDOR, ESEEM, electron paramagnetic resonance and transient/pulsed EPR methods.

Davies-type pulsed (super 1)H electron nuclear double resonance measurements were carried out at a magnetic field of 3.4 T and a microwave frequency of 95 GHz (W-band). The small g-anisotropy of the semiquinone anion radicals could be resolved in frozen solutions by taking advantage of the increased electron Zeeman interaction at high field. The measurements were performed on a variety of randomly oriented semiquinone anion radicals in frozen alcoholic solution and on the primary ubiquinone anion radical, Q(sub A)(super -*), in frozen photosynthetic bacterial reaction centers of Rhodobacter sphaeroides.

The temperature dependence of the electron spin—lattice relaxation time T1 was measured for the S0 state of the oxygen-evolving complex (OEC) in photosystem II and for two dinuclear manganese model complexes by pulse EPR using the inversion—recovery method. For [Mn(III)Mn(IV)(μ-O)2bipy4]- ClO4, the Raman relaxation process dominates at temperatures below 50 K. In contrast, Orbach type relaxation was found for [Mn(II)Mn(III)(μ-OH)(μ-piv)2(Me3tacn)2](ClO4)2 between 4.3 and 9 K. For the latter complex, an energy separation of 24.7-28.0 cm-1 between the ground and the first excited electronic state was determined. In the So state of photosystem II, the T1 relaxation times were measured in the range of 4.3-6.5 K. A comparison with the relaxation data (rate and pre-exponential factor) of the two model complexes and of the S2 state of photosystem II indicates that the Orbach relaxation process is dominant for the So state and that its first excited state lies 21.7 ± 0.4 cm-1 above its ground state. The results are discussed with respect to the structure of the OEC in photosystem II. [ABSTRACT FROM AUTHOR]

[FeFe] hydrogenases are highly active catalysts for hydrogen conversion. Their active site has two components: a [4Fe-4S] electron relay covalently attached to the H 2 binding site and a diiron cluster ligated by CO, CN - , and 2-azapropane-1,3-dithiolate (ADT) ligands. Reduction of the [4Fe-4S] site was proposed to be coupled with protonation of one of its cysteine ligands. Here, we used time-resolved infrared (TRIR) spectroscopy on the [FeFe] hydrogenase from Chlamydomonas reinhardtii ( Cr HydA1) containing a propane-1,3-dithiolate (PDT) ligand instead of the native ADT ligand. The PDT modification does not affect the electron transfer step to [4Fe-4S] H but prevents the enzyme from proceeding further through the catalytic cycle. We show that the rate of the first electron transfer step is independent of the pH, supporting a simple electron transfer rather than a proton-coupled event. These results have important implications for our understanding of the catalytic mechanism of [FeFe] hydrogenases and highlight the utility of TRIR.

[FeFe] hydrogenases are highly active catalysts for the interconversion of molecular hydrogen with protons and electrons. Here, we use a combination of isotopic labeling, 57 Fe 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 - 13 C 2 H 2 - 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 ( Cr HydA1), and NRVS was measured on samples poised in the catalytically crucial H hyd state containing a terminal hydride at the distal Fe site. The 13 C 2 H isotope effects were observed also in the H hyd spectrum. DFT simulations of the spectra allowed identification of the 57 Fe 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 H 2 formation.

In vivo expression of metalloproteins requires specific metal trafficking and incorporation machinery inside the cell. Synthetic designed metalloproteins are typically purified without the target metal, which is subsequently introduced through in vitro reconstitution. The extra step complicates protein optimization by high-throughput library screening or laboratory evolution. We demonstrate that a designed coiled-coil iron-sulfur protein (CCIS) assembles robustly with [4Fe-4S] clusters in vivo . While in vitro reconstitution produces a mixture of oligomers that depends on solution conditions, in vivo production generates a stable homotrimer coordinating a single, diamagnetic [4Fe-4S] 2+ cluster. The multinuclear cluster of in vivo assembled CCIS is more resistant to degradation by molecular oxygen. Only one of the two metal coordinating half-sites is required in vivo, indicating specificity of molecular recognition in recruitment of the metal cluster. CCIS, unbiased by evolution, is a unique platform to examine iron-sulfur protein biogenesis and develop synthetic multinuclear oxidoreductases.

Oxidoreductase enzymes often perform technologically useful chemical transformations using abundant metal cofactors with high efficiency under ambient conditions. The understanding of the catalytic mechanism of these enzymes is, however, highly dependent on the availability of well-characterized and optimized time-resolved analytical techniques. We have developed an approach for rapidly injecting electrons into a catalytic system using a photoactivated nanomaterial in combination with a range of redox mediators to produce a potential jump in solution, which then initiates turnover via electron transfer (ET) to the catalyst. The ET events at the nanomaterial-mediator-catalyst interfaces are, however, highly sensitive to the experimental conditions such as photon flux, relative concentrations of system components, and pH. Here, we present a systematic optimization of these experimental parameters for a specific catalytic system, namely, [FeFe] hydrogenase from Chlamydomonas reinhardtii ( Cr HydA1). The developed strategies can, however, be applied in the study of a wide variety of oxidoreductase enzymes. Our potential jump system consists of CdSe/CdS core-shell nanorods as a photosensitizer and a series of substituted bipyridinium salts as mediators with redox potentials in the range from -550 to -670 mV (vs SHE). With these components, we screened the effect of pH, mediator concentration, protein concentration, photosensitizer concentration, and photon flux on steady-state photoreduction and hydrogen production as well as ET and potential jump efficiency. By manipulating these experimental conditions, we show the potential of simple modifications to improve the tunability of the potential jump for application to study oxidoreductases.

The active site of [FeFe] hydrogenase features a binuclear iron cofactor Fe 2 ADT(CO) 3 (CN) 2 , where ADT represents the bridging ligand aza-propane-dithiolate. The terminal diatomic ligands all coordinate in a basal configuration, and one CO bridges the two irons leaving an open coordination site at which the hydrogen species and the competitive inhibitor CO bind. Externally supplied CO is expected to coordinate in an apical configuration. However, an alternative configuration has been proposed in which, due to ligand rotation, the CN - bound to the distal Fe becomes apical. Using selective 13 C isotope labeling of the CN - and CO ext ligands in combination with pulsed 13 C electron-nuclear-nuclear triple resonance spectroscopy, spin polarization effects are revealed that, according to density functional theory calculations, are consistent with only the "unrotated" apical CO ext configuration.

[FeFe] hydrogenases are extremely active H 2 -converting enzymes. Their mechanism remains highly controversial, in particular, the nature of the one-electron and two-electron reduced intermediates called H red H + and H sred H + . In one model, the H red H + and H sred H + 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 H sred H + and H red H + 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 H red H + and H sred H + 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.

[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 13 C-ENDOR investigation of the H-cluster in which the two methylene carbons of ADT are isotope labeled with 13 C. We observed that the corresponding two 13 C 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.

Hydrogenases are metalloenzymes that catalyze the reversible oxidation of H 2 . The [FeFe] hydrogenases are generally biased toward proton reduction and have high activities. Several different catalytic mechanisms have been proposed for the [FeFe] enzymes based on the identification of intermediate states in equilibrium and steady state experiments. Here, we examine the kinetic competency of these intermediate states in the [FeFe] hydrogenase from Chlamydomonas reinhardtii ( Cr HydA1), using a laser-induced potential jump and time-resolved IR (TRIR) spectroscopy. A CdSe/CdS dot-in-rod (DIR) nanocrystalline semiconductor is employed as the photosensitizer and a redox mediator efficiently transfers electrons to the enzyme. A pulsed laser induces a potential jump, and TRIR spectroscopy is used to follow the population flux through each intermediate state. The results clearly establish the kinetic competency of all intermediate populations examined: H ox , H red , H red H + , H sred H + , and H hyd . Additionally, a new short-lived intermediate species with a CO peak at 1896 cm -1 was identified. These results establish a kinetics framework for understanding the catalytic mechanism of [FeFe] hydrogenases.

[FeFe] hydrogenases interconvert H 2 into protons and electrons reversibly and efficiently. The active site H-cluster is composed of two sites: a unique [2Fe] subcluster ([2Fe] H ) covalently linked via cysteine to a canonical [4Fe-4S] cluster ([4Fe-4S] H ). Both sites are redox active and electron transfer is proton-coupled, such that the potential of the H-cluster lies very close to the H 2 thermodynamic potential, which confers the enzyme with the ability to operate quickly in both directions without energy losses. Here, one of the cysteines coordinating [4Fe-4S] H (Cys362) in the [FeFe] hydrogenase from the green algae Chlamydomonas reinhardtii ( CrHydA1) was exchanged with histidine and the resulting C362H variant was shown to contain a [4Fe-4S] cluster with a more positive redox potential than the wild-type. The change in the [4Fe-4S] cluster potential resulted in a shift of the catalytic bias, diminishing the H 2 production activity but giving significantly higher H 2 oxidation activity, albeit with a 200 mV overpotential requirement. These results highlight the importance of the [4Fe-4S] cluster as an electron injection site, modulating the redox potential and the catalytic properties of the H-cluster.

By a combination of electron paramagnetic resonance spectroscopy, finite-temperature ab initio simulations, and electronic structure analyses, the activation of molecular dioxygen at the interface of gold nanoparticles and titania in Au/TiO 2 catalysts is explained at the atomic scale by tracing processes down to the molecular orbital picture. Direct evidence is provided that excess electrons in TiO 2 , for example created by photoexcitation of the semiconductor, migrate to the gold particles and from there to oxygen molecules adsorbed at gold/titania perimeter sites. Superoxide species are formed more efficiently in this way than on the bare TiO 2 surface. This catalytic effect of the gold nanoparticles is attributed to a weakening of the internal O-O bond, leading to a preferential splitting of the molecule at shorter bond lengths together with a 70% decrease of the dissociation free energy barrier compared to the non-catalyzed case on bare TiO 2 . The findings are an important step forward in the clarification of the role of gold in (photo)catalytic processes.

[FeFe] hydrogenases catalyze proton reduction and hydrogen oxidation with high rates and efficiency under physiological conditions, but are highly oxygen sensitive. The [FeFe] hydrogenase from Desulfovibrio desulfuricans ( DdHydAB) can be purified under air in an oxygen stable inactive state H ox air . The formation of the H ox air state in vitro allows the handling of hydrogenases in air, making their implementation in biotechnological applications more feasible. Here, we report a simple and robust protocol for the formation of the H ox air state in DdHydAB and the [FeFe] hydrogenase from Chlamydomonas reinhardtii, which is based on high potential inactivation in the presence of sulfide.

[FeFe]-hydrogenases, HydAs, are unique biocatalysts for proton reduction to H 2 . However, they suffer from a number of drawbacks for biotechnological applications: size, number and diversity of metal cofactors, oxygen sensitivity. Here we show that HydA from Megasphaera elsdenii (MeHydA) displays significant resistance to O 2 . Furthermore, we produced a shorter version of this enzyme (MeH-HydA), lacking the N-terminal domain harboring the accessory FeS clusters. As shown by detailed spectroscopic and biochemical characterization, MeH-HydA displays the following interesting properties. First, a functional active site can be assembled in MeH-HydA in vitro, providing the enzyme with excellent hydrogenase activity. Second, the resistance of MeHydA to O 2 is conserved in MeH-HydA. Third, MeH-HydA is more biased toward proton reduction than MeHydA, as the result of the truncation changing the rate limiting steps in catalysis. This work shows that it is possible to engineer HydA to generate an active hydrogenase that combines the resistance of the most resistant HydAs and the simplicity of algal HydAs, containing only the H-cluster.

Hydride state intermediates are known to occur in various hydrogen conversion enzymes, including the highly efficient [FeFe] hydrogenases. The intermediate state involving a terminal iron-bound hydride has been recognized as crucial for the catalytic mechanism, but its occurrence has up to now eluded unequivocal proof under (near) physiological conditions. Here we show that the terminal hydride in the [FeFe] hydrogenase from Chlamydomonas reinhardtii can be directly detected using solution 1 H NMR spectroscopy at room temperature, opening new avenues for detailed in situ investigations under catalytic conditions.

Sensory type [FeFe] hydrogenases are predicted to play a role in transcriptional regulation by detecting the H 2 level of the cellular environment. These hydrogenases contain the hydrogenase domain with distinct modifications in the active site pocket, followed by a Per-Arnt-Sim (PAS) domain. As yet, neither the physiological function nor the biochemical or spectroscopic properties of these enzymes have been explored. Here, we present the characterization of an artificially maturated, putative sensory [FeFe] hydrogenase from Thermotoga maritima (HydS). This enzyme shows lower hydrogen conversion activity than prototypical [FeFe] hydrogenases and a reduced inhibition by CO. Using FTIR spectroelectrochemistry and EPR spectroscopy, three redox states of the active site were identified. The spectroscopic signatures of the most oxidized state closely resemble those of the H ox state from the prototypical [FeFe] hydrogenases, while the FTIR spectra of both singly and doubly reduced states show large differences. The FTIR bands of both the reduced states are strongly red-shifted relative to the H ox state, indicating reduction at the diiron site, but with retention of the bridging CO ligand. The unique functional and spectroscopic features of HydS are discussed with regard to the possible role of altered amino acid residues influencing the electronic properties of the H-cluster.

The [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii has been studied using 1 H NMR spectroscopy identifying the paramagnetically shifted 1 H resonances associated with both the [4Fe-4S] H and the [2Fe] H subclusters of the active site "H-cluster". The signal pattern of the unmaturated HydA1 containing only [4Fe-4S] H is reminiscent of bacterial-type ferredoxins. The spectra of maturated HydA1, with a complete H-cluster in the active H ox and the CO-inhibited H ox -CO state, reveal additional upfield and downfield shifted 1 H resonances originating from the four methylene protons of the azadithiolate ligand in the [2Fe] H subsite. The two axial protons are affected by positive spin density, while the two equatorial protons experience negative spin density. These protons can be used as important probes sensing the effects of ligand-binding to the catalytic site of the H-cluster.

The catalytic cofactor of [FeFe]-hydrogenses (H-cluster) is composed of a generic cubane [4Fe-4S]-cluster (4Fe H ) linked to a binuclear iron-sulfur cluster (2Fe H ) that has an open coordination site at which the reversible conversion of protons to molecular hydrogen occurs. The (2Fe H ) subsite features a diatomic coordination sphere composed of three CO and two CN - ligands affecting its redox properties and providing excellent probes for FTIR spectroscopy. The CO stretch vibrations are very sensitive to the redox changes within the H-cluster occurring during the catalytic cycle, whereas the CN - signals seem to be relatively inert to these effects. This could be due to the more structural role of the CN - ligands tightly anchoring the (2Fe H ) unit to the protein environment through hydrogen bonding. In this work we explore the effects of structural changes within the secondary ligand sphere affecting the CN - ligands on FTIR spectroscopy and catalysis. By comparing the FTIR spectra of wild-type enzyme and two mutagenesis variants, we are able to assign the IR signals of the individual CN - ligands of the (2Fe H ) site for different redox states of the H-cluster. Moreover, protein film electrochemistry reveals that targeted manipulation of the secondary coordination sphere of the proximal CN - ligand (i.e., closest to the (4Fe H ) site) can affect the catalytic bias. These findings highlight the importance of the protein environment for re-adjusting the catalytic features of the H-cluster in individual enzymes and provide valuable information for the design of artificial hydrogenase mimics.

[FeFe]-hydrogenases are metalloenzymes that reversibly reduce protons to molecular hydrogen at exceptionally high rates. We have characterized the catalytically competent hydride state (H hyd ) in the [FeFe]-hydrogenases from both Chlamydomonas reinhardtii and Desulfovibrio desulfuricans using 57 Fe nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT). H/D exchange identified two Fe-H bending modes originating from the binuclear iron cofactor. DFT calculations show that these spectral features result from an iron-bound terminal hydride, and the Fe-H vibrational frequencies being highly dependent on interactions between the amine base of the catalytic cofactor with both hydride and the conserved cysteine terminating the proton transfer chain to the active site. The results indicate that H hyd is the catalytic state one step prior to H 2 formation. The observed vibrational spectrum, therefore, provides mechanistic insight into the reaction coordinate for H 2 bond formation by [FeFe]-hydrogenases.

[FeFe] hydrogenases catalyze proton reduction and hydrogen oxidation displaying high rates at low overpotential. Their active site is a complex cofactor consisting of a unique [2Fe] subcluster ([2Fe] H ) covalently bound to a canonical [4Fe-4S] cluster ([4Fe-4S] H ). The [FeFe] hydrogenase from Desulfovibrio desulfuricans is exceptionally active and bidirectional. This enzyme features two accessory [4Fe-4S] F clusters for exchanging electrons with the protein surface. A thorough understanding of the mechanism of this efficient enzyme will facilitate the development of synthetic molecular catalysts for hydrogen conversion. Here, it is demonstrated that the accessory clusters influence the catalytic properties of the enzyme through a strong redox interaction between the proximal [4Fe-4S] F cluster and the [4Fe-4S] H subcluster of the H-cluster. This interaction enhances proton-coupled electronic rearrangement within the H-cluster increasing the apparent pK a of its one electron reduced state. This may help to sustain H 2 production at high pH values. These results may apply to all [FeFe] hydrogenases containing accessory clusters.

Nature's water-splitting catalyst, an oxygen-bridged tetramanganese calcium (Mn 4 O 5 Ca) complex, sequentially activates two substrate water molecules generating molecular O 2 . Its reaction cycle is composed of five intermediate (S i ) states, where the index i indicates the number of oxidizing equivalents stored by the cofactor. After formation of the S 4 state, the product dioxygen is released and the cofactor returns to its lowest oxidation state, S 0 . Membrane-inlet mass spectrometry measurements suggest that at least one substrate is bound throughout the catalytic cycle, as the rate of 18 O-labeled water incorporation into the product O 2 is slow, on a millisecond to second time scale depending on the S state. Here, we demonstrate that the Mn 4 O 5 Ca complex poised in the S 0 state contains an exchangeable hydroxo bridge. On the basis of a combination of magnetic multiresonance (EPR) spectroscopies, comparison to biochemical models and theoretical calculations we assign this bridge to O5, the same bridge identified in the S 2 state as an exchangeable fully deprotonated oxo bridge [Pérez Navarro, M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 15561]. This oxygen species is the most probable candidate for the slowly exchanging substrate water in the S 0 state. Additional measurements provide new information on the Mn ions that constitute the catalyst. A structural model for the S 0 state is proposed that is consistent with available experimental data and explains the observed evolution of water exchange kinetics in the first three states of the catalytic cycle.

[FeFe] hydrogenases are extremely active and efficient H 2 -converting biocatalysts. Their active site comprises a unique [2Fe] subcluster bonded to a canonical [4Fe-4S] cluster. The [2Fe] subsite can be introduced into hydrogenases lacking an assembled H-cluster through incubation with a synthesized [2Fe] H precursor, which initially produces the CO-inhibited state of the enzyme. We present FTIR spectroelectrochemical studies on the CO-inhibited state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans, DdHydAB. At very negative potentials, disassembly of the H-cluster and dissociation of the [2Fe] subcluster is observed. Subsequently raising the potential allows cofactor rebinding and H-cluster reassembly. This demonstrates how the stability of the [2Fe]-[4Fe-4S] intercluster bond depends on the applied potential and the presence of an inhibiting CO ligand on the [2Fe] subcluster. These results provide insight into the mechanisms of CO inhibition and H-cluster assembly in [FeFe] hydrogenases. A fundamental understanding of these properties will provide clues for designing better H 2 -converting catalysts.

[FeFe]-hydrogenases catalyze the reversible reduction of protons to molecular hydrogen with extremely high efficiency. The active site ("H-cluster") consists of a [4Fe-4S] H cluster linked through a bridging cysteine to a [2Fe] H subsite coordinated by CN - and CO ligands featuring a dithiol-amine moiety that serves as proton shuttle between the protein proton channel and the catalytic distal iron site (Fe d ). Although there is broad consensus that an iron-bound terminal hydride species must occur in the catalytic mechanism, such a species has never been directly observed experimentally. Here, we present FTIR and nuclear resonance vibrational spectroscopy (NRVS) experiments in conjunction with density functional theory (DFT) calculations on an [FeFe]-hydrogenase variant lacking the amine proton shuttle which is stabilizing a putative hydride state. The NRVS spectra unequivocally show the bending modes of the terminal Fe-H species fully consistent with widely accepted models of the catalytic cycle.

The active site of [FeFe] hydrogenases, the H-cluster, consists of a [4Fe-4S] cluster connected via a bridging cysteine to a [2Fe] complex carrying CO and CN - ligands as well as a bridging aza-dithiolate ligand (ADT) of which the amine moiety serves as a proton shuttle between the protein and the H-cluster. During the catalytic cycle, the two subclusters change oxidation states: [4Fe-4S] H 2+ ⇔ [4Fe-4S] H + and [Fe(I)Fe(II)] H ⇔ [Fe(I)Fe(I)] H thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H + + 2e - ⇄ H 2 . Using FTIR spectro-electrochemistry on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) at different pH values, we resolve the redox and protonation events in the catalytic cycle and determine their intrinsic thermodynamic parameters. We show that the singly reduced state H red of the H-cluster actually consists of two species: H red = [4Fe-4S] H + - [Fe(I)Fe(II)] H and H red H + = [4Fe-4S] H 2+ - [Fe(I)Fe(I)] H (H + ) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe-4S] H subcluster at similar midpoint-potentials (-375 vs -418 mV); the protonation event (H red /H red H + ) has a pK a ≈ 7.2.

Time-resolved nanosecond mid-infrared spectroscopy is for the first time employed to study the [FeFe] hydrogenase from Chlamydomonas reinhardtii and to investigate relevant intermediates of the enzyme active site. An actinic 355 nm, 10 ns laser flash triggered photodissociation of a carbonyl group from the CO-inhibited state Hox-CO to form the state Hox, which is an intermediate of the catalytic proton reduction cycle. Time-resolved infrared spectroscopy allowed us to directly follow the subsequent rebinding of the carbonyl, re-forming Hox-CO, and determine the reaction half-life to be t1/2 ≈ 13 ± 5 ms at room temperature. This gives direct information on the dynamics of CO inhibition of the enzyme.

Iron-sulfur clusters form one of the largest and most diverse classes of enzyme cofactors in nature. They may serve as structural factors, form electron transfer chains between active sites and external redox partners, or form components of enzyme active sites. Their specific role is a consequence of the cluster type and the surrounding protein environment. The relative effects of these factors are not completely understood, and it is not yet possible to predict the properties of iron-sulfur clusters based on amino acid sequences or rationally tune their properties to generate proteins with new desirable functions. Here, we generate mutations in a [2Fe-2S] cluster protein, the TmHydC subunit of the trimeric [FeFe]-hydrogenase from Thermotoga maritima, to study the factors that affect its redox potential. Saturation mutagenesis of Val131 was used to tune the redox potential over a 135 mV range and revealed that cluster redox potential and electronic properties correlate with amino acid hydrophobicity and the ability to form hydrogen bonds to the cluster. Proline scanning mutagenesis between pairs of ligating cysteines was used to remove backbone amide hydrogen bonds to the cluster and decrease the redox potential by up to 132 mV, without large structural changes in most cases. However, substitution of Gly83 with proline caused a change of HydC to a [4Fe-4S] cluster protein with a redox potential of -526 mV. Together, these results confirm the importance of hydrogen bonding in tuning cluster redox potentials and demonstrate the versatility of iron-sulfur cluster protein folds at binding different types of clusters.

The development of semiquinone-based resorcin[4]arene cavitands expands the toolbox of switchable molecular grippers by introducing the first paramagnetic representatives. The semiquinone (SQ) states were generated electrochemically, chemically, and photochemically. We analyzed their electronic, conformational, and binding properties by cyclic voltammetry, ultraviolet/visible (UV/vis) spectroelectrochemistry, electron paramagnetic resonance (EPR) and transient absorption spectroscopy, in conjunction with density functional theory (DFT) calculations. The utility of UV/vis spectroelectrochemistry and EPR spectroscopy in evaluating the conformational features of resorcin[4]arene cavitands is demonstrated. Guest binding properties were found to be enhanced in the SQ state as compared to the quinone (Q) or the hydroquinone (HQ) states of the cavitands. Thus, these paramagnetic SQ intermediates open the way to six-state redox switches provided by two conformations (open and closed) in three redox states (Q, SQ, and HQ) possessing distinct binding ability. The switchable magnetic properties of these molecular grippers and their responsiveness to electrical stimuli has the potential for development of efficient molecular devices.

Bis(p-methoxyphenyl)carbene is the first carbene that at cryogenic temperatures can be isolated in both its lowest energy singlet and triplet states. At 3 K, both states coexist indefinitely under these conditions. The carbene is investigated in argon matrices by IR, UV-vis, and X-band EPR spectroscopy and in MTHF glasses by W-band EPR and Q-band ENDOR spectroscopy. UV (365 nm) irradiation of the system results in formation of predominantly the triplet carbene, whereas visible (450 nm) light shifts the photostationary equilibrium toward the singlet state. Upon annealing at higher temperatures (>10 K), the triplet is converted to the singlet; however, cooling back to 3 K does not restore the triplet. Therefore, depending on matrix temperature and irradiation conditions, matrices containing predominantly the triplet or singlet carbene can be generated. Controlling the magnetic and chemical properties of carbenes by using light of different wavelengths might be of general interest for applications such as information storage and radical-initiated polymerization processes.

In transition-metal complexes, the geometric structure is intimately connected with the spin state arising from magnetic coupling between the paramagnetic ions. The tetramanganese-calcium cofactor that catalyzes biological water oxidation in photosystem II cycles through five catalytic intermediates, each of which adopts a specific geometric and electronic structure and is thus characterized by a specific spin state. Here, we review spin-structure correlations in Nature's water-splitting catalyst. The catalytic cycle of the Mn4O5Ca cofactor can be described in terms of spin-dependent reactivity. The lower "inactive" S states of the catalyst, S0 and S1, are characterized by low-spin ground states, SGS = 1/2 and SGS = 0. This is connected to the "open cubane" topology of the inorganic core in these states. The S2 state exhibits structural and spin heterogeneity in the form of two interconvertible isomers and is identified as the spin-switching point of the catalytic cycle. The first S2 state form is an open cubane structure with a low-spin SGS = 1/2 ground state, whereas the other represents the first appearance of a closed cubane topology in the catalytic cycle that is associated with a higher-spin ground state of SGS = 5/2. It is only this higher-spin form of the S2 state that progresses to the "activated" S3 state of the catalyst. The structure of this final metastable catalytic state was resolved in a recent report, showing that all manganese ions are six-coordinate. The magnetic coupling is dominantly ferromagnetic, leading to a high-spin ground state of SGS = 3. The ability of the Mn4O5Ca cofactor to adopt two distinct structural and spin-state forms in the S2 state is critical for water binding in the S3 state, allowing spin-state crossing from the inactive, low-spin configuration of the catalyst to the activated, high-spin configuration. Here we describe how an understanding of the magnetic properties of the catalyst in all S states has allowed conclusions on the catalyst function to be reached. A summary of recent literature results is provided that constrains the sequence of molecular level events: catalyst/substrate deprotonation, manganese oxidation, and water molecule insertion.

A new class of synthetic models for the active site of [NiFe]-hydrogenases are described. The Ni(I/II)(SCys)2 and Fe(II)(CN)2CO sites are represented with (RC5H4)Ni(I/II) and Fe(II)(diphos)(CO) modules, where diphos = 1,2-C2H4(PPh2)2(dppe) or cis-1,2-C2H2(PPh2)2(dppv). The two bridging thiolate ligands are represented by CH2(CH2S)2(2-) (pdt(2-)), Me2C(CH2S)2(2-) (Me2pdt(2-)), and (C6H5S)2(2-). The reaction of Fe(pdt)(CO)2(dppe) and [(C5H5)3Ni2]BF4 affords [(C5H5)Ni(pdt)Fe(dppe)(CO)]BF4 ([1a]BF4). Monocarbonyl [1a]BF4 features an S = 0 Ni(II)Fe(II) center with five-coordinated iron, as proposed for the Ni-SIa state of the enzyme. One-electron reduction of [1a](+) affords the S = 1/2 derivative [1a](0), which, according to density functional theory (DFT) calculations and electron paramagnetic resonance and Mössbauer spectroscopies, is best described as a Ni(I)Fe(II) compound. The Ni(I)Fe(II) assignment matches that for the Ni-L state in [NiFe]-hydrogenase, unlike recently reported Ni(II)Fe(I)-based models. Compound [1a](0) reacts with strong acids to liberate 0.5 equiv of H2 and regenerate [1a](+), indicating that H2 evolution is catalyzed by [1a](0). DFT calculations were used to investigate the pathway for H2 evolution and revealed that the mechanism can proceed through two isomers of [1a](0) that differ in the stereochemistry of the Fe(dppe)CO center. Calculations suggest that protonation of [1a](0) (both isomers) affords Ni(III)-H-Fe(II) intermediates, which represent mimics of the Ni-C state of the enzyme.

The active site of [FeFe] hydrogenase contains a catalytic binuclear iron subsite coordinated by CN(-) and CO ligands as well as a unique azadithiolate (adt(2-)) bridging ligand. It has been established that this binuclear cofactor is synthesized and assembled by three maturation proteins HydE, -F, and -G. By means of in vitro maturation in the presence of (15)N- and (13)C-labeled tyrosine it has been shown that the CN(-) and CO ligands originate from tyrosine. The source of the bridging adt(2-) ligand, however, remains unknown. In order to identify the nitrogen of the bridging amine using HYSCORE spectroscopy and distinguish its spectroscopic signature from that of the CN(-) nitrogens, we studied three isotope-labeled variants of the H-cluster ((15)N-adt(2-)/C(14)N(-), (15)N-adt(2-)/C(15)N(-), and (14)N-adt(2-)/C(15)N(-)) and extracted accurate values of the hyperfine and quadrupole couplings of both CN(-) and adt(2-) nitrogens. This will allow an evaluation of isotopologues of the H-cluster generated by in vitro bioassembly in the presence of various (15)N-labeled potential precursors as possible sources of the bridging ligand.

The preparation and spectroscopic characterization of a CO-inhibited [FeFe] hydrogenase with a selectively (57)Fe-labeled binuclear subsite is described. The precursor [(57)Fe2(adt)(CN)2(CO)4](2-) was synthesized from the (57)Fe metal, S8, CO, (NEt4)CN, NH4Cl, and CH2O. (Et4N)2[(57)Fe2(adt)(CN)2(CO)4] was then used for the maturation of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii, to yield the enzyme selectively labeled at the [2Fe]H subcluster. Complementary (57)Fe enrichment of the [4Fe-4S]H cluster was realized by reconstitution with (57)FeCl3 and Na2S. The Hox-CO state of [2(57)Fe]H and [4(57)Fe-4S]H HydA1 was characterized by Mössbauer, HYSCORE, ENDOR, and nuclear resonance vibrational spectroscopy.

Diamagnetic iron chloro compounds [(P(Ph)2N(Ph)2)FeCp*Cl] [1Cl] and [(P(Cy)2N(Ph)2)FeCp*Cl] [2Cl] and the corresponding hydrido complexes [(P(Ph)2N(Ph)2)FeCp*H] [1H] and [(P(Cy)2N(Ph)2)FeCp*H] [2H] have been synthesized and characterized by NMR spectroscopy, electrochemical studies, electronic absorption, and (57)Fe Mössbauer spectroscopy (P(Ph)2N(Ph)2 = 1,3,5,7-tetraphenyl-1,5-diphospha-3,7-diazacyclooctane, P(Cy)2N(Ph)2 = 1,5-dicyclohexyl-3,7-diphenyl-1,5-diphospha-3,7-diazacyclooctane, Cp* = pentamethylcyclopentadienyl). Molecular structures of [2Cl], [1H], and [2H], derived from single-crystal X-ray diffraction, revealed that these compounds have a typical piano-stool geometry. The results show that the electronic properties of the hydrido complexes are strongly influenced by the substituents at the phosphorus donor atoms of the P(R)2N(Ph)2 ligand, whereas those of the chloro complexes are less affected. These results illustrate that the hydride is a strong-field ligand, as compared to chloride, and thus leads to a significant degree of covalent character of the iron hydride bonds. This is important in the context of possible catalytic intermediates of iron hydrido species, as proposed for the catalytic cycle of [FeFe] hydrogenases and other synthetic catalysts. Both hydrido compounds [1H] and [2H] show enhanced catalytic currents in cyclic voltammetry upon addition of the strong acid trifluoromethanesulfonimide [NHTf2] (pKa(MeCN) = 1.0). In contrast to the related complex [(P(tBu)N(Bn))2FeCp(C6F5)H], which was reported by Liu et al. (Nat. Chem. 2013, 5, 228-233) to be an electrocatalyst for hydrogen splitting, the here presented hydride complexes [1H] and [2H] show the tendency for electrocatalytic hydrogen production. Hence, the catalytic direction of this class of monoiron compounds can be reversed by specific ligand modifications.