EPR/ENDOR, Mössbauer, and quantum-chemical investigations of diiron complexes mimicking the active oxidized state of [FeFe]hydrogenase.

Understanding the catalytic process of the heterolytic splitting and formation of molecular hydrogen is one of the key topics for the development of a future hydrogen economy. With an interest in elucidating the enzymatic mechanism of the [Fe(2)(S(2)C(2)H(4)NH)(CN)(2)(CO)(2)(µ-CO)] active center uniquely found in [FeFe]hydrogenases, we present a detailed spectroscopic and theoretical analysis of its inorganic model [Fe(2)(S(2)X)(CO)(3)(dppv)(PMe(3))](+) [dppv = cis-1,2-bis(diphenylphosphino)ethylene] in two forms with S(2)X = ethanedithiolate (1edt) and azadithiolate (1adt). These complexes represent models for the oxidized mixed-valent Fe(I)Fe(II) state analogous to the active oxidized "H(ox)" state of the native H-cluster. For both complexes, the (31)P hyperfine interactions were determined by pulse electron paramagnetic resonance and electron nuclear double resonance (ENDOR) methods. For 1edt, the (57)Fe parameters were measured by electron spin-echo envelope modulation and Mössbauer spectroscopy, while for 1adt, (14)N and selected (1)H couplings could be obtained by ENDOR and hyperfine sublevel correlation spectroscopy. The spin density was found to be predominantly localized on the Fe(dppv) site. This spin distribution is different from that of the H-cluster, where both the spin and charge densities are delocalized over the two Fe centers. This difference is attributed to the influence of the "native" cubane subcluster that is lacking in the inorganic models. The degree and character of the unpaired spin delocalization was found to vary from 1edt, with an abiological dithiolate, to 1adt, which features the authentic cofactor. For 1adt, we find two (14)N signals, which are indicative for two possible isomers of the azadithiolate, demonstrating its high flexibility. All interaction parameters were also evaluated through density functional theory calculations at various levels.

Empirical and computational design of iron-sulfur cluster proteins.

Here, we compare two approaches of protein design. A computational approach was used in the design of the coiled-coil iron-sulfur protein, CCIS, as a four helix bundle binding an iron-sulfur cluster within its hydrophobic core. An empirical approach was used for designing the redox-chain maquette, RCM as a four-helix bundle assembling iron-sulfur clusters within loops and one heme in the middle of its hydrophobic core. We demonstrate that both ways of design yielded the desired proteins in terms of secondary structure and cofactors assembly. Both approaches, however, still have much to improve in predicting conformational changes in the presence of bound cofactors, controlling oligomerization tendency and stabilizing the bound iron-sulfur clusters in the reduced state. Lessons from both ways of design and future directions of development are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Spectroscopic characterization of the molybdenum cofactor of the sulfane dehydrogenase SoxCD from Paracoccus pantotrophus.

The bacterial sulfane dehydrogenase SoxCD is a distantly related member of the sulfite oxidase (SO) enzyme family that is proposed to oxidize protein-bound sulfide (sulfane) of SoxY as part of a multienzyme mechanism of thiosulfate metabolism. This study characterized the molybdenum cofactor of SoxCD1, comprising the catalytic molybdopterin subunit SoxC and the truncated c-type cytochrome subunit SoxD1. Electron paramagnetic resonance spectroscopy of the Mo(V) intermediate generated by dithionite reduction revealed low- and high-pH species with g and A((95,97)Mo) matrices nearly identical to those of SO, indicating a similar pentacoordinate active site in SoxCD1. However, no sulfite-induced reduction to Mo(V) was detected, nor could a strongly coupled (1)H signal or a phosphate-inhibited species be generated. This indicates that the outer coordination sphere controls substrate binding in SoxCD, permitting access only to protein-bound sulfur via the C-terminal tail of SoxY.

Advanced electron paramagnetic resonance and density functional theory study of a {2Fe3S} cluster mimicking the active site of [FeFe] hydrogenase.

Despite extensive investigations of the active site of the [FeFe] hydrogenases, many details concerning the properties of the "hydrogen converting cluster" are not yet fully understood. The complexity of the so-called H-cluster is one of the main difficulties in studying the properties of its components. The present study is aimed at the mixed-valence EPR active [Fe2(µ-CO)(CO)3(CN)2{MeSCH2C(Me)(CH2S)2}](1-) that is structurally closely related to the redox active binuclear part of the H-cluster in its CO-inhibited oxidized state. In this work, we present a characterization of this compound by advanced pulse EPR methods. The accurate determination of the (57)Fe, (1)H, (2)H, (14)N, and (15)N electron nuclear hyperfine interactions provided a very detailed picture of the electronic structure of this complex. A theoretical study using density functional theory (DFT) calculations identified possible isomers of the compound and further refined the knowledge about its properties. It was found that upon one electron oxidation of the parent Fe(I)-Fe(I) complex, the dominant mixed-valence Fe(I)-Fe(II) species is the one in which the CN ligand of the iron center that is distal to the thioether moves from the basal to the apical position. The unpaired spin distribution of the model complex is found to be clearly different from that of the native H-cluster. These differences are discussed and provide new insight into the functional features of the [FeFe] hydrogenase active site.

De novo design of a non-natural fold for an iron-sulfur protein: alpha-helical coiled-coil with a four-iron four-sulfur cluster binding site in its central core.

Using a 'metal-first' approach, we computationally designed, prepared, and characterized a four-iron four-sulfur (Fe(4)S(4)) cluster protein with a non-natural alpha-helical coiled-coil fold. The novelty of this fold lies in the placement of a Fe(4)S(4) cluster within the hydrophobic core of a four-helix bundle, making it unique among previous iron-sulfur (FeS) protein designs, and different from known natural FeS proteins. The apoprotein, recombinantly expressed and purified from E. coli, readily self-assembles with Fe(4)S(4) clusters in vitro. UV-Vis absorption and CD spectroscopy, elemental analysis, gel filtration, and analytical ultracentrifugation confirm that the protein is folded and assembled as designed, namely, alpha-helical coiled-coil binding a single Fe(4)S(4) cluster. Dithionite-reduced holoprotein samples have characteristic rhombic EPR spectra, typical of low-potential, [Fe(4)S(4)](+) (S=1/2), with g values of g(zy)=(1.970, 1.975), and g(x)=2.053. The temperature, and power dependence of the signal intensity were also characteristic of [Fe(4)S(4)](+) clusters with very efficient spin relaxation, but almost without any interaction between adjacent clusters. The new design is very promising although optimization is required, particularly for preventing aggregation, and adding second shell interactions to stabilize the reduced state. Its main advantage is its extendibility into a multi-FeS cluster protein by simply duplicating and translating the binding site along the coiled-coil axis. This opens new possibilities for designing protein-embedded redox chains that may be used as "wires" for coupling any given set of redox enzymes.

Ligand-centred reactivity of bis(picolyl)amine iridium: sequential deprotonation, oxidation and oxygenation of a "non-innocent" ligand.

Treatment of [Ir(bpa)(cod)](+) complex [1](+) with a strong base (e.g., tBuO(-)) led to unexpected double deprotonation to form the anionic [Ir(bpa-2H)(cod)](-) species [3](-), via the mono-deprotonated neutral amido complex [Ir(bpa-H)(cod)] as an isolable intermediate. A certain degree of aromaticity of the obtained metal-chelate ring may explain the favourable double deprotonation. The rhodium analogue [4](-) was prepared in situ. The new species [M(bpa-2H)(cod)](-) (M = Rh, Ir) are best described as two-electron reduced analogues of the cationic imine complexes [M(I)(cod)(Py-CH(2)-N=CH-Py)](+). One-electron oxidation of [3](-) and [4](-) produced the ligand radical complexes [3](*) and [4](*). Oxygenation of [3](-) with O(2) gave the neutral carboxamido complex [Ir(cod)(py-CH(2)-N-CO-py)] via the ligand radical complex [3](*) as a detectable intermediate.

(14)N HYSCORE investigation of the H-cluster of [FeFe] hydrogenase: evidence for a nitrogen in the dithiol bridge.

Hydrogenases are enzymes catalyzing the reversible heterolytic splitting of molecular hydrogen. Despite extensive investigations of this class of enzymes its catalytic mechanism is not yet well understood. In this paper spectroscopic investigations of the active site of [FeFe] hydrogenase are presented. The so-called H-cluster consists of a bi-nuclear catalytically active subcluster connected to a [4Fe4S] ferredoxin-like unit via a Cys-thiol bridge. An important feature of the H-cluster is that both irons in the bi-nuclear subcluster are coordinated by CN and CO ligands. The bi-nuclear site also carries a dithiol bridge, whose central atom has not yet been identified. Nitrogen and oxygen are the most probable candidates from a mechanistic point of view. Here we present a study of the (14)N nuclear quadrupole and hyperfine interactions of the active oxidized state of the H-cluster using advanced EPR methods. In total three (14)N nuclei with quadrupole couplings of 0.95 MHz, 0.35 MHz and 1.23 MHz were detected using hyperfine sublevel correlation spectroscopy (HYSCORE). The assignment of the signals is based on their (14)N quadrupole couplings in combination with DFT calculations. One signal is assigned to the CN ligand of the distal iron, one to a Lys side chain nitrogen and one to the putative nitrogen of the dithiol bridge. Hence, these results provide the first experimental evidence for a di-(thiomethyl)amine ligand (-S-CH(2)-NH-CH(2)-S-) in the bi-nuclear subcluster. This finding is important for understanding the mechanism of [FeFe] hydrogenases, since the nitrogen is likely to act as an internal base facilitating the heterolytic splitting/formation of H(2).

Spectroelectrochemical characterization of the active site of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii.

Hydrogenases catalyze the reversible oxidation of molecular hydrogen. The active site of the [FeFe] hydrogenases (H-cluster) contains a catalytically active binuclear subcluster ([2Fe](H)) connected to a "cubane" [4Fe4S](H) subcluster. Here we present an IR spectroelectrochemical study of the [FeFe] hydrogenase HydA1 isolated from the green alga Chlamydomonas reinhardtii. The enzyme shows IR bands similar to those observed for bacterial [FeFe] hydrogenases. They are assigned to the stretching vibrations of the CN(-) and CO ligands on both irons of the [2Fe](H) subcluster. By following changes in frequencies of the IR bands during electrochemical titrations, two one-electron redox processes of the active enzyme could be distinguished. The reduction of the oxidized state (H(ox)) occurred at a midpoint potential of -400 mV vs NHE (H(ox)/H(red) transition) and relates to a change of the formal oxidation state of the binuclear subcluster. A subsequent reduction (H(red)/H(sred) transition) was determined to have a midpoint potential of -460 mV vs NHE. On the basis of the IR spectra, it is suggested that the oxidation state of the binuclear subcluster does not change in this transition. Tentatively, a reduction of the [4Fe4S](H) cluster has been proposed. In contrast to the bacterial [FeFe] hydrogenases, where the bridging CO ligand becomes terminal when going from H(ox) to H(red), in HydA1 the bridging CO is present in both the H(ox) and H(red) state. The removal of the bridging CO moiety has been observed in the H(red) to H(sred) transition. The significance of this result for the hydrogen conversion mechanism of this class of enzymes is discussed.

Spin distribution of the H-cluster in the H(ox)-CO state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans: HYSCORE and ENDOR study of (14)N and (13)C nuclear interactions.

Hydrogenases are enzymes which catalyze the reversible cleavage of molecular hydrogen into protons and electrons. In [FeFe] hydrogenases the active center is a 6Fe6S cluster, referred to as the "H-cluster." It consists of the redox-active binuclear subcluster ([2Fe](H)) coordinated by CN(-) and CO ligands and the cubane-like [4Fe-4S](H) subcluster which is connected to the protein via Cys ligands. One of these Cys ligands bridges to the [2Fe](H) subcluster. The CO-inhibited form of [FeFe] hydrogenase isolated from Desulfovibrio desulfuricans was studied using advanced EPR methods. In the H(ox)-CO state the open coordination site at the [2Fe](H) subcluster is blocked by extrinsic CO, giving rise to an EPR-active S = 1/2 species. The CO inhibited state was prepared with (13)CO and illuminated under white light at 273 K. In this case scrambling of the CO ligands occurs. Three (13)C hyperfine couplings of 17.1, 7.4, and 3.8 MHz (isotropic part) were observed and assigned to (13)CO at the extrinsic, the bridging, and the terminal CO-ligand positions of the distal iron, respectively. No (13)CO exchange of the CO ligand to the proximal iron was observed. The hyperfine interactions detected indicate a rather large distribution of the spin density over the terminal and bridging CO ligands attached to the distal iron. Furthermore, (14)N nuclear spin interactions were measured. On the basis of the observed (14)N hyperfine couplings, which result from the CN(-) ligands of the [2Fe](H) subcluster, it has been concluded that there is very little unpaired spin density on the cyanides of the binuclear subcluster.

The electronic structure of the H-cluster in the [FeFe]-hydrogenase from Desulfovibrio desulfuricans: a Q-band 57Fe-ENDOR and HYSCORE study.

The active site of the (57)Fe-enriched [FeFe]-hydrogenase (i.e., the "H-cluster") from Desulfovibrio desulfuricans has been examined using advanced pulse EPR methods at X- and Q-band frequencies. For both the active oxidized state (H(ox)) and the CO inhibited form (H(ox)-CO) all six (57)Fe hyperfine couplings were detected. The analysis shows that the apparent spin density extends over the whole H-cluster. The investigations revealed different hyperfine couplings of all six (57)Fe nuclei in the H-cluster of the H(ox)-CO state. Four large 57Fe hyperfine couplings in the range 20-40 MHz were found (using pulse ENDOR and TRIPLE methods) and were assigned to the [4Fe-4S](H) (cubane) subcluster. Two weak (57)Fe hyperfine couplings below 5 MHz were identified using Q-band HYSCORE spectroscopy and were assigned to the [2Fe](H) subcluster. For the H(ox) state only two different 57Fe hyperfine couplings in the range 10-13 MHz were detected using pulse ENDOR. An (57)Fe line broadening analysis of the X-band CW EPR spectrum indicated, however, that all six (57)Fe nuclei in the H-cluster are contributing to the hyperfine pattern. It is concluded that in both states the binuclear subcluster [2Fe](H) assumes a [Fe(I)Fe(II)] redox configuration where the paramagnetic Fe(I) atom is attached to the [4Fe-4S](H) subcluster. The (57)Fe hyperfine interactions of the formally diamagnetic [4Fe-4S](H) are due to an exchange interaction between the two subclusters as has been discussed earlier by Popescu and Münck [Popescu, C.V.; Münck, E., J. Am. Chem. Soc. 1999, 121, 7877-7884]. This exchange coupling is strongly enhanced by binding of the extrinsic CO ligand. Binding of the dihydrogen substrate may induce a similar effect, and it is therefore proposed that the observed modulation of the electronic structure by the changing ligand surrounding plays an important role in the catalytic mechanism of [FeFe]-hydrogenase.

Hydrogen-atom transfer in open-shell organometallic chemistry: the reactivity of Rh(II)(cod) and Ir(II)(cod) radicals.

A series of new metalloradical rhodium and iridium complexes [M(II)(cod)(N-ligand)](2+) in the uncommon oxidation state +II were synthesized by one-electron oxidation of their [M(I)(cod)(N-ligand)](+) precursors (M=Rh, Ir; cod=(Z,Z)-1,5-cyclooctadiene; and N-ligand is a podal bis(pyridyl)amine ligand: N,N-bis(2-pyridylmethyl)amine (dpa), N-(2-pyridylmethyl)-N-(6-methyl-2-pyridylmethyl)amine (pla), or N-benzyl-N,N-bis(6-methyl-2-pyridylmethyl)amine (Bn-dla). EPR spectroscopy, X-ray diffraction, and DFT calculations reveal that each of these [M(II)(cod)(N-ligand)](2+) species adopts a square-pyramidal geometry with the two cod double bonds and the two pyridine fragments in the basal plane and the N(amine) donor at the apical position. The unpaired electron of these species mainly resides at the metal center, but the apical N(amine) donor also carries a considerable fraction of the total spin density (15-18 %). Density functional calculations proved a valuable tool for the analysis and simulation of the experimental EPR spectra. Whereas the M(II)(olefin) complexes are quite stable as solids, in solution they spontaneously transform into a 1:1 mixture of M(III)(allyl) species and protonated M(I)(olefin) complexes (in the forms [M(I)(olefin)(protonated N-ligand)](2+) for M=Rh and [M(III)(H)(olefin)(N-ligand)](2+) for M=Ir). Similar reactions were observed for the related propene complex [M(II)(propene)(Me(2)tpa)](2+) (Me(2)tpa=N,N,N-tris(6-methyl-2-pyridylmethyl)amine). The decomposition rate of the [M(II)(cod)(N-ligand)](2+) species decreases with increasing N-ligand bulk in the following order: dpa>pla>Bn-dla. Decomposition of the most hindered [M(II)(cod)(Bn-dla)](2+) complexes proceeds by a second-order process. The kinetic rate expression v=k(obs)[M(II)](2) in acetone with k(obs)=k'[H(+)][S], where [S] is the concentration of additional coordinating reagents (MeCN), is in agreement with ligand-assisted dissociation of one of the pyridine donors. Solvent coordination results in formation of more open, reactive species. Protonation of the noncoordinating pyridyl group increases the concentration of this species, and thus [H(+)] appears in the kinetic rate expression. The kinetic data are in agreement with bimolecular hydrogen-atom transfer from M(II)(cod) to another M(II) species (DeltaH( not equal)=11.5+/-2 kcal mol(-1), DeltaS( not equal)=-27+/-10 cal K(-1) mol(-1), and DeltaG( not equal)(298 K)=19.5+/-5 kcal mol(-1)).

EPR analysis of multiple forms of [4Fe-4S](3+) clusters in HiPIPs.

The electron paramagnetic resonance (EPR) spectrum from the [4Fe-4S](3+) cluster in several high-potential iron-sulfur proteins (HiPIPs) is complex: it is not the pattern of a single, isolated S=1/2 system. Multifrequency EPR from 9 to 130 GHz reveals that the apparent peak positions (g values) are frequency-independent: the spectrum is dominated by the Zeeman interaction plus g-strain broadening. The spectra taken at frequencies above the X-band are increasingly sensitive to rapid-passage effects; therefore, the X-band data, which are slightly additionally broadened by dipolar interaction, were used for quantitative spectral analysis. For a single geometrical [4Fe-4S](3+) structure the (Fe-Fe)(5+) mixed-valence dimer can be assigned in six different ways to a pair of iron ions, and this defines six valence isomers. Systematic multicomponent g-strain simulation shows that the [4Fe-4S](3+) paramagnets in seven HiPIPs from different bacteria each consist of three to four discernible species, and these are assigned to valence isomers of the clusters. This interpretation builds on previous EPR analyzes of [4Fe-4S](3+) model compounds, and it constitutes a high-resolution extension of the current literature model, proposed from paramagnetic NMR studies.

IrII(ethene): metal or carbon radical?

One-electron oxidation of [(Me(n)tpa)Ir(I)(ethene)]+ complexes (Me(3)tpa = N,N,N-tri(6-methyl-2-pyridylmethyl)amine; Me(2)tpa = N-(2-pyridylmethyl)-N,N,-di[(6-methyl-2-pyridyl)methyl]-amine) results in relatively stable, five-coordinate Ir(II)-olefin species [(Me(n)tpa)Ir(II)(ethene)](2+) (1(2+): n = 3; 2(2+): n = 2). These contain a "vacant site" at iridium and a "non-innocent" ethene fragment, allowing radical type addition reactions at both the metal and the ethene ligand. The balance between metal- and ligand-centered radical behavior is influenced by the donor capacity of the solvent. In weakly coordinating solvents, 1(2+) and 2(2+) behave as moderately reactive metallo-radicals. Radical coupling of 1(2+) with NO in acetone occurs at the metal, resulting in dissociation of ethene and formation of the stable nitrosyl complex [(Me(3)tpa)Ir(NO)](2+) (6(2+)). In the coordinating solvent MeCN, 1(2+) generates more reactive radicals; [(Me(3)tpa)Ir(MeCN)(ethene)](2+) (9(2+)) by MeCN coordination, and [(Me(3)tpa)Ir(II)(MeCN)](2+) (10(2+)) by substitution of MeCN for ethene. Complex 10(2+) is a metallo-radical, like 1(2+) but more reactive. DFT calculations indicate that 9(2+) is intermediate between the slipped-olefin Ir(II)(CH(2)=CH(2)) and ethyl radical Ir(III)-CH(2)-CH(2). resonance structures, of which the latter prevails. The ethyl radical character of 9(2+) allows radical type addition reactions at the ethene ligand. Complex 2(2+) behaves similarly in MeCN. In the absence of further reagents, 1(2+) and 2(2+) convert to the ethylene bridged species [(Me(n)tpa)(MeCN)Ir(III)(mu(2)-C(2)H(4))Ir(III)(MeCN)(Me(3)tpa)](4+) (n = 3: 3(4+); n = 2: 4(4+)) in MeCN. In the presence of TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxo), formation of 3(4+) from 1(2+) in MeCN is completely suppressed and only [(Me(3)tpa)Ir(III)(TEMPO(-))(MeCN)](2+) (7(2+)) is formed. This is thought to proceed via radical coupling of TEMPO at the metal center of 10(2+). In the presence of water, hydrolysis of the coordinated acetonitrile fragment of 7(2+) results in the acetamido complex [(Me(3)tpa)Ir(III)(NHC(O)CH(3)))(TEMPOH)](2+) (8(2+)).

Hydrogen bond geometries from electron paramagnetic resonance and electron-nuclear double resonance parameters: density functional study of quinone radical anion-solvent interactions.

Density functional theory was used to study the impact of hydrogen bonding on the p-benzosemiquinone radical anion BQ(*-) in coordination with water or alcohol molecules. After complete geometry optimizations, (1)H, (13)C, and (17)O hyperfine as well as (2)H nuclear quadrupole coupling constants and the g-tensor were computed. The suitability of different model systems with one, two, four, and 20 water molecules was tested; best agreement between theory and experiment could be obtained for the largest model system. Q-band pulse (2)H electron-nuclear double resonance (ENDOR) experiments were performed on BQ(*-) in D(2)O. They compare very well with the spectra simulated by use of the theoretical values from density functional theory. For BQ(*-) in coordination with four water or alcohol molecules, rather similar hydrogen-bond lengths between 1.75 and 1.78 A were calculated. Thus, the computed electron paramagnetic resonance (EPR) parameters are hardly distinguishable for the different solvents, in agreement with experimental findings. Furthermore, the distance dependence of the EPR parameters on the hydrogen-bond length was studied. The nuclear quadrupole and the dipolar hyperfine coupling constants of the bridging hydrogens show the expected dependencies on the H-bond length R(O.H). A correlation was obtained for the g-tensor. It is shown that the point-dipole model is suitable for the estimation of hydrogen-bond lengths from anisotropic hyperfine coupling constants of the bridging (1)H nuclei for H-bond lengths larger than approximately 1.7 A. Furthermore, the estimation of H-bond lengths from (2)H nuclear quadrupole coupling constants of bridging deuterium nuclei by empirical relations is discussed.