Structural differences of oxidized iron-sulfur and nickel-iron cofactors in O2-tolerant and O2-sensitive hydrogenases studied by X-ray absorption spectroscopy.

The class of [NiFe]-hydrogenases comprises oxygen-sensitive periplasmic (PH) and oxygen-tolerant membrane-bound (MBH) enzymes. For three PHs and four MBHs from six bacterial species, structural features of the nickel-iron active site of hydrogen turnover and of the iron-sulfur clusters functioning in electron transfer were determined using X-ray absorption spectroscopy (XAS). Fe-XAS indicated surplus oxidized iron and a lower number of ~2.7 Å Fe-Fe distances plus additional shorter and longer distances in the oxidized MBHs compared to the oxidized PHs. This supported a double-oxidized and modified proximal FeS cluster in all MBHs with an apparent trimer-plus-monomer arrangement of its four iron atoms, in agreement with crystal data showing a [4Fe3S] cluster instead of a [4Fe4S] cubane as in the PHs. Ni-XAS indicated coordination of the nickel by the thiol group sulfurs of four conserved cysteines and at least one iron-oxygen bond in both MBH and PH proteins. Structural differences of the oxidized inactive [NiFe] cofactor of MBHs in the Ni-B state compared to PHs in the Ni-A state included a ~0.05 Å longer Ni-O bond, a two times larger spread of the Ni-S bond lengths, and a ~0.1 Å shorter Ni-Fe distance. The modified proximal [4Fe3S] cluster, weaker binding of the Ni-Fe bridging oxygen species, and an altered localization of reduced oxygen species at the active site may each contribute to O2 tolerance.

Rapid X-ray photoreduction of dimetal-oxygen cofactors in ribonucleotide reductase.

Prototypic dinuclear metal cofactors with varying metallation constitute a class of O2-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O2 activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques.

Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in Escherichia coli.

The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme.

Hydride binding to the active site of [FeFe]-hydrogenase.

[FeFe]-hydrogenase from green algae (HydA1) is the most efficient hydrogen (H2) producing enzyme in nature and of prime interest for (bio)technology. Its active site is a unique six-iron center (H-cluster) composed of a cubane cluster, [4Fe4S]H, cysteine-linked to a diiron unit, [2Fe]H, which carries unusual carbon monoxide (CO) and cyanide ligands and a bridging azadithiolate group. We have probed the molecular and electronic configurations of the H-cluster in functional oxidized, reduced, and super-reduced or CO-inhibited HydA1 protein, in particular searching for intermediates with iron-hydride bonds. Site-selective X-ray absorption and emission spectroscopy were used to distinguish between low- and high-spin iron sites in the two subcomplexes of the H-cluster. The experimental methods and spectral simulations were calibrated using synthetic model complexes with ligand variations and bound hydride species. Distinct X-ray spectroscopic signatures of electronic excitation or decay transitions in [4Fe4S]H and [2Fe]H were obtained, which were quantitatively reproduced by density functional theory calculations, thereby leading to specific H-cluster model structures. We show that iron-hydride bonds are absent in the reduced state, whereas only in the super-reduced state, ligand rotation facilitates hydride binding presumably to the Fe-Fe bridging position at [2Fe]H. These results are in agreement with a catalytic cycle involving three main intermediates and at least two protonation and electron transfer steps prior to the H2 formation chemistry in [FeFe]-hydrogenases.

Effect of exchange of the cysteine molybdenum ligand with selenocysteine on the structure and function of the active site in human sulfite oxidase.

Sulfite oxidase (SO) is an essential molybdoenzyme for humans, catalyzing the final step in the degradation of sulfur-containing amino acids and lipids, which is the oxidation of sulfite to sulfate. The catalytic site of SO consists of a molybdenum ion bound to the dithiolene sulfurs of one molybdopterin (MPT) molecule, carrying two oxygen ligands, and is further coordinated by the thiol sulfur of a conserved cysteine residue. We have exchanged four non-active site cysteines in the molybdenum cofactor (Moco) binding domain of human SO (SOMD) with serine using site-directed mutagenesis. This facilitated the specific replacement of the active site Cys207 with selenocysteine during protein expression in Escherichia coli. The sulfite oxidizing activity (kcat/KM) of SeSOMD4Ser was increased at least 1.5-fold, and the pH optimum was shifted to a more acidic value compared to those of SOMD4Ser and SOMD4Cys(wt). X-ray absorption spectroscopy revealed a Mo(VI)-Se bond length of 2.51 Å, likely caused by the specific binding of Sec207 to the molybdenum, and otherwise rather similar square-pyramidal S/Se(Cys)O2Mo(VI)S2(MPT) site structures in the three constructs. The low-pH form of the Mo(V) electron paramagnetic resonance (EPR) signal of SeSOMD4Ser was altered compared to those of SOMD4Ser and SOMD4Cys(wt), with g1 in particular shifted to a lower magnetic field, due to the Se ligation at the molybdenum. In contrast, the Mo(V) EPR signal of the high-pH form was unchanged. The substantially stronger effect of substituting selenocysteine for cysteine at low pH as compared to high pH is most likely due to the decreased covalency of the Mo-Se bond.

Molecular interference of Cd(2+) with Photosystem II.

Many heavy metals inhibit electron transfer reactions in Photosystem II (PSII). Cd(2+) is known to exchange, with high affinity in a slow reaction, for the Ca(2+) cofactor in the Ca/Mn cluster that constitutes the oxygen-evolving center. This results in inhibition of photosynthetic oxygen evolution. There are also indications that Cd(2+) binds to other sites in PSII, potentially to proton channels in analogy to heavy metal binding in photosynthetic reaction centers from purple bacteria. In search for the effects of Cd(2+)-binding to those sites, we have studied how Cd(2+) affects electron transfer reactions in PSII after short incubation times and in sites, which interact with Cd(2+) with low affinity. Overall electron transfer and partial electron transfer were studied by a combination of EPR spectroscopy of individual redox components, flash-induced variable fluorescence and steady state oxygen evolution measurements. Several effects of Cd(2+) were observed: (i) the amplitude of the flash-induced variable fluorescence was lost indicating that electron transfer from Y(Z) to P(680)(+) was inhibited; (ii) Q(A)(-) to Q(B) electron transfer was slowed down; (iii) the S(2) state multiline EPR signal was not observable; (iv) steady state oxygen evolution was inhibited in both a high-affinity and a low-affinity site; (v) the spectral shape of the EPR signal from Q(A)(-)Fe(2+) was modified but its amplitude was not sensitive to the presence of Cd(2+). In addition, the presence of both Ca(2+) and DCMU abolished Cd(2+)-induced effects partially and in different sites. The number of sites for Cd(2+) binding and the possible nature of these sites are discussed.

Improved arsenic(III) adsorption by Al2O3 nanoparticles and H2O2: evidence of oxidation to arsenic(V) from X-ray absorption spectroscopy.

We have investigated the oxidation of inorganic As(III) with H2O2 catalysed by Al2O3, using X-ray absorption near-edge structure and extended X-ray absorption fine structure spectroscopy. The effects of different reaction conditions (pH, time and initial H2O2 concentration) were also studied as were the kinetics of the oxidation reaction. We demonstrated that As(III) was oxidized to As(V) in the presence of H2O2 and Al2O3. Furthermore, all arsenic species found on the Al2O3 surface were in the As(V) state. The presence of both Al2O3 and H2O2 was necessary for oxidation of As(III) to take place within the period of time studied. The oxidation kinetics indicate a mechanism where reversible As(III) binding to the alumina surface is followed by irreversible oxidation by H2O2 leading to strongly bound As(V). Results from this study indicate that there is a surface-catalysed oxidation of As(III) on Al2O3 by H2O2, a reaction that can take place in nature and can be of help in the development of novel treatment systems for As(III) removal.

Bridging-hydride influence on the electronic structure of an [FeFe] hydrogenase active-site model complex revealed by XAES-DFT.

Two crystallized [FeFe] hydrogenase model complexes, 1 = (µ-pdt)[Fe(CO)(2)(PMe(3))](2) (pdt = SC1H2C2H2C3H2S), and their bridging-hydride (Hy) derivative, [1Hy](+) = [(µ-H)(µ-pdt)[Fe(CO)(2) (PMe(3))](2)](+) (BF(4)(−)), were studied by Fe K-edge X-ray absorption and emission spectroscopy, supported by density functional theory. Structural changes in [1Hy](+) compared to 1 involved small bond elongations (<0.03 Å) and more octahedral Fe geometries; the Fe­H bond at Fe1 (closer to pdt-C2) was ~0.03 Å longer than that at Fe2. Analyses of (1) pre-edge absorption spectra (core-to-valence transitions), (2) Kß(1,3), Kß', and Kß(2,5) emission spectra (valence-to-core transitions), and (3) resonant inelastic X-ray scattering data (valence-to-valence transitions) for resonant and non-resonant excitation and respective spectral simulations indicated the following: (1) the mean Fe oxidation state was similar in both complexes, due to electron density transfer from the ligands to Hy in [1Hy](+). Fe 1s→3d transitions remained at similar energies whereas delocalization of carbonyl AOs onto Fe and significant Hy-contributions to MOs caused an ~0.7 eV up-shift of Fe1s→(CO)s,p transitions in [1Hy](+). Fed-levels were delocalized over Fe1 and Fe2 and degeneracies biased to O(h)­Fe1 and C(4v)­Fe2 states for 1, but to O(h)­Fe1,2 states for [1Hy](+). (2) Electron-pairing of formal Fe(d(7)) ions in low-spin states in both complexes and a higher effective spin count for [1Hy](+) were suggested by comparison with iron reference compounds. Electronic decays from Fe d and ligand s,p MOs and spectral contributions from Hys,p→1s transitions even revealed limited site-selectivity for detection of Fe1 or Fe2 in [1Hy](+). The HOMO/LUMO energy gap for 1 was estimated as 3.0 ± 0.5 eV. (3) For [1Hy](+) compared to 1, increased Fed (x(2) − y(2)) − (z(2)) energy differences (~0.5 eV to ~0.9 eV) and Fed→d transition energies (~2.9 eV to ~3.7 eV) were assigned. These results reveal the specific impact of Hy-binding on the electronic structure of diiron compounds and provide guidelines for a directed search of hydride species in hydrogenases.