Characterization of the active site of catalytically inactive forms of [NiFe] hydrogenases by density functional theory

The inactive forms, unready (Ni-A, Ni-SU) and ready (Ni-B), of NiFe hydrogenases are modeled by examining the possibility of hydroxo, oxo, hydroperoxo, peroxo, and sulfenate groups in active-site models and comparing predicted IR frequencies and g tensors with those of the enzyme. The best models for Ni-A and Ni-SU have hydroxo (μ-OH) bridges between Fe and Ni and a terminal sulfenate [Ni–S(=O)Cys] group, although a hydroperoxo model for Ni-A is also quite viable, whereas the best model for Ni-B has only a μ-OH bridge. In addition, a mechanism for the activation of unready hydrogenase is proposed on the basis of the relative stabilities of sulfenate models versus peroxide models.     

Structural differences between the ready and unready oxidized states of [NiFe] hydrogenases

[NiFe] hydrogenases catalyze the reversible heterolytic cleavage of molecular hydrogen. Several oxidized, inactive states of these enzymes are known that are distinguishable by their very different activation properties. So far, the structural basis for this difference has not been understood because of lack of relevant crystallographic data. Here, we present the crystal structure of the ready Ni-B state of Desulfovibrio fructosovorans [NiFe] hydrogenase and show it to have a putative -hydroxo Ni–Fe bridging ligand at the active site. On the other hand, a new, improved refinement procedure of the X-ray diffraction data obtained for putative unready Ni-A/Ni-SU states resulted in a more elongated electron density for the bridging ligand, suggesting that it is a diatomic species. The slow activation of the Ni-A state, compared with the rapid activation of the Ni-B state, is therefore proposed to result from the different chemical nature of the ligands in the two oxidized species. Our results along with very recent electrochemical studies suggest that the diatomic ligand could be hydro–peroxide.An erratum to this article can be found at     

Activation process of [NiFe] hydrogenase elucidated by high-resolution X-ray analyses: conversion of the ready to the unready state

Hydrogenases catalyze oxidoreduction of molecular hydrogen and have potential applications for utilizing dihydrogen as an energy source. [NiFe] hydrogenase has two different oxidized states, Ni-A (unready, exhibits a lag phase in reductive activation) and Ni-B (ready). We have succeeded in converting Ni-B to Ni-A with the use of Na2S and O2 and determining the high-resolution crystal structures of both states. Ni-B possesses a monatomic nonprotein bridging ligand at the Ni-Fe active site, whereas Ni-A has a diatomic species. The terminal atom of the bridging species of Ni-A occupies a similar position as C of the exogenous CO in the CO complex (inhibited state). The common features of the enzyme structures at the unready (Ni-A) and inhibited (CO complex) states are proposed. These findings provide useful information on the design of new systems of biomimetic dihydrogen production and fuel cell devices.     

Single crystal EPR studies of the oxidized active site of [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F

The Ni-A and the Ni-B forms of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F have been studied in single crystals by continuous wave and pulsed EPR spectroscopy at different temperatures (280?K, 80?K, and 10?K). For the first time, the orientation of the g-tensor axes with respect to the recently published atomic structure of the active site at 1.8?Å resolution was elucidated for Ni-A and Ni-B. The determined g-tensors have a similar orientation. The configuration of the electronic ground state is proposed to be Ni(III) 3d ¹ _z2 for Ni-A and Ni-B. The g _z principal axis is close to the Ni-S(Cys549) direction; the g _x and the g _y axes are approximately along the Ni-S(Cys546) and Ni-S(Cys81) bonds, respectively. It is proposed that the difference between the Ni-A and Ni-B states lies in a protonation of the bridging ligand between the Ni and the Fe.     

Insight into the copper coordination environment in the prion protein through density functional theory calculations of EPR parameters

Density functional theory (DFT) calculations of Cu(II) electron paramagnetic resonance (EPR) parameters for the octarepeat unit of the prion protein were conducted. Model complexes were constructed and optimized using the crystal structure of the octarepeat unit of the prion protein. Copper g and A tensors and nitrogen hyperfine and quadrupole coupling constants were calculated using DFT. Solvent effects were incorporated using the conductor-like screening model as well as through the inclusion of explicit water molecules. Calculations using the model with an additional axial water molecule added to the coordination sphere of the Cu(II) metal center give the best qualitative agreement for the copper g and A tensors. The S-band experimental EPR spectra were interpreted in light of the DFT calculations of the directly coordinated nitrogen hyperfine coupling constants which indicate that the three directly coordinated nitrogen atoms in the octarepeat unit are not equivalent. These results demonstrate that DFT calculations of EPR parameters can provide important insight with respect to the structural interpretation of experimental EPR data. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Relativistic DFT calculation of the reaction cycle intermediates of [NiFe] hydrogenase: a contribution to understanding the enzymatic mechanism

Structures and spectroscopic observables of the paramagnetic intermediates of the enzymatic reaction cycle of the metalloenzyme [NiFe] hydrogenase were calculated using relativistic density functional theory (DFT) within the zero-order regular approximation (ZORA). By comparing experimental and calculated magnetic resonance parameters (g- and hyperfine tensors) for the states Ni-A, Ni-B, Ni-C, Ni-L, and Ni-CO the details of the atomic composition of these paramagnetic intermediates could be elucidated that are mostly not available from X-ray structure analysis. In general, good agreement between calculated and experimental observables could be obtained. A detailed picture of the changes of the active center during the catalytic cycle was deduced from the obtained structures. Based on these results, a consistent model for the sequence of redox states including protonation steps is proposed which is important for understanding the mechanism of the [NiFe] hydrogenase.     

A resonance Raman band assignable to the O–O stretching mode in the resting oxidized state of bovine heart cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

In the resting oxidized state (the fully oxidized “as-isolated” state) of cytochrome c oxidase (CcO) preparation, a resonance Raman band is observed at 755 cm^-1 upon 647.1 nm excitation in resonance with an absorption band at 655 nm. Addition of cyanide eliminates the Raman band concomitant with loss of the absorption band at 655 nm. These results strongly suggest that the Raman band at 755 cm^-1 originates from the O−O stretching mode of the bridging peroxide (Fe−O^-−O^-−Cu) in the O₂ reduction site of the fully oxidized “as-isolated” CcO. Although the peroxide bridged structure has been proposed on the basis of X-ray crystallography and reductive titration experiments, the present vibrational spectroscopic analyses reveal conclusively the chemical nature of the bridging ligand at the O₂ reduction site of the fully oxidized “as-isolated” bovine heart CcO.     

The elements of an electrocardiographic lead tensor theory

This report describes a method by which the concepts of the lead vector and the lead field may be extended to include equivalent cardiac singularities above the first order. The theoretical treatment results in generalization of the well-known lead vector principle into a lead tensor apparatus. The specific electrocardiographic contribution of annth order multipole is found to be the product of annth rank contravariant (“heart”) tensor by a covariant (“lead”) tensor of the same rank, and with identical indices. Methods are also described whereby lead tensor components may be cast into a form which relates them directly to the more usual, spherical harmonic notation of multipolar current generators. Some attention is devoted to the possibility of applying lead tensors to biomedical problems, particularly to the prospect of quantitatively evaluating the significant multipolar components of the human heart.     

Spin distribution of the H-cluster in the Hox–CO state of the [FeFe] hydrogenase from Desulfovibrio desulfuricans: HYSCORE and ENDOR study of 14N and 13C 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 ¹³CO and illuminated under white light at 273 K. In this case scrambling of the CO ligands occurs. Three ¹³C hyperfine couplings of 17.1, 7.4, and 3.8 MHz (isotropic part) were observed and assigned to ¹³CO at the extrinsic, the bridging, and the terminal CO-ligand positions of the distal iron, respectively. No ¹³CO 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, ¹⁴N nuclear spin interactions were measured. On the basis of the observed ¹⁴N 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.

Probing intermediates in the activation cycle of [NiFe] hydrogenase by infrared spectroscopy: the Ni-SIr state and its light sensitivity

The [NiFe] hydrogenase from the sulphate-reducing bacterium Desulfovibrio vulgaris Miyazaki F is reversibly inhibited in the presence of molecular oxygen. A key intermediate in the reactivation process, Ni-SI_r, provides the link between fully oxidized (Ni-A, Ni-B) and active (Ni-SI_a, Ni-C and Ni-R) forms of hydrogenase. In this work Ni-SI_r was found to be light-sensitive (T ≤ 110 K), similar to the active Ni-C and the CO-inhibited states. Transition to the final photoproduct state (Ni-SL) was shown to involve an additional transient light-induced state (Ni-SI₁₉₆₁). Rapid scan kinetic infrared measurements provided activation energies for the transition from Ni-SL to Ni-SI_r in protonated as well as in deuterated samples. The inhibitor CO was found not to react with the active site of the Ni-SL state. The wavelength dependence of the Ni-SI_r photoconversion was examined in the range between 410 and 680 nm. Light-induced effects were associated with a nickel-centred electronic transition, possibly involving a change in the spin state of nickel (Ni²⁺). In addition, at T ≤ 40 K the CN⁻ stretching vibrations of Ni-SL were found to be dependent on the colour of the monochromatic light used to irradiate the species, suggesting a change in the interaction of the hydrogen-bonding network of the surrounding amino acids. A possible mechanism for the photochemical process, involving displacement of the oxygen-based ligand, is discussed.     

Preference for bridging versus terminal ligands in magnesium dimers

Magnesium dimers play important roles in inorganic and organometallic chemistry. This study evaluates the inherent bridging ability of a range of different ligands in magnesium dimers. In the first part, the Cambridge Structural Database is interrogated to establish the frequency of different types of ligands found in bridging versus terminal positions in two key structural motifs: one in which there are two bridging ligands (the D _2h “Mg₂(μ-X₂)” structure); the other in which there are three bridging ligands (the C _3v “Mg₂(μ-X₃)” structure). The most striking finding from the database search is the overwhelming preference for magnesium dimers possessing two bridging ligands. The most common bridging ligands are C-, N-, and O-based. In the second part, DFT calculations (at the B3LYP/6-311+G(d) level of theory) are carried out to examine a wider range of structural types for dimers consisting of the stoichiometries Mg₂Cl₃R and Mg₂Cl₂R₂, where R = CH₃, SiH₃, NH₂, PH₂, OH, SH, CH₂CH₃, CH=CH₂, C≡CH, Ph, OAc, F and Br. Consistent with the database search, the most stable magnesium dimers are those that contain two bridging ligands. Furthermore, it was demonstrated that the electronic effect of the bridging ligands is important in influencing the stability of the magnesium dimers. The preference for a bridging ligand, which reflects its ability to stabilize a magnesium dimer, follows the order: OH > NH₂ > C≡CH > SH > Ph > Br > PH₂ = CH=CH₂ > CH₂CH₃ > CH₃ > SiH₃. Finally, the role that the ether solvent Me₂O has on the stability of isomeric Mg₂Cl₂Me₂ dimers was studied. It was found that the first solvent molecule stabilizes the dimers, while the second solvent molecule can either have a stabilizing or destabilizing effect, depending on the isomer structure.     

Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Ι) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at E _m = +197 mV (heme c) and −4.5 mV (heme b). Variable-temperature (4–120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe–4S]⁺ cluster and overlapping signals associated with at least three types of [4Fe–4S]⁺ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called “low-pH” and “high-pH,” changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

pH-Induced Molten Globule State of <Emphasis Type="Italic">Rhizopus niveus</Emphasis> Lipase is More Resistant Against Thermal and Chemical Denaturation Than Its Native State

Here, we have characterized four pH-dependent states: alkaline state, “B” (pH 9.0), native state, “N” (pH 7.4), acid-induced state, “A” (pH 2.2) and molten globule state, “MG” (pH 1.8) of Rhizopus niveus lipase (RNL) by CD, tryptophanyl fluorescence, ANS binding, DLS, and enzyme activity assay. This “MG” state lacks catalytic activity and tertiary structure but it has native-like significant secondary structure. The “R _h” of all the four states of RNL obtained from DLS study suggests that the molecular compactness of the protein increases as the pH of solution decreases. Kinetic analysis of RNL shows that it has maximum catalytic efficiency at state “B” which is 15-fold higher than state “N.” The CD and tryptophanyl fluorescence studies of RNL on GuHCl and temperature-induced unfolding reveal that the “MG” state is more stable than the other states. The DSC endotherms of RNL obtained at pH 9.0, 7.4, and 2.2 were with two transitions, while at pH 1.8 it showed only a single transition.     

Genome-wide investigation on the genetic variations of rice disease resistance genes

Exploitation of plant disease resistance (R) gene in breeding programs has been proven to be the most efficient strategy for coping with the threat of pathogens. An understanding of R-gene variation is the basis for this strategy. Here we report a genome-wide investigation on the variation of NBS-LRR-encoding genes, the common type of R genes, between two sequenced rice genomes, Oryza sativa L. var. Nipponbare and 93–11. We show that the allelic nucleotide diversity in 65.0% of 397 least-divergent pairs is not high (0.344% on average), while the remaining 35% display a greater diversity (5.4% on average). The majority of conserved R genes is single-copy and/or located as a singleton. The clustered, particularly the complex-clustered, R-genes contribute greatly to the rich genetic variation. Surprisingly only 11.2% of R-genes have remarkably high ratios of non-synonymous to synonymous rates, which is much less than the 17.4% observed between Arabidopsis genomes. Noticeable “artificially selective sweeping” could be detected in a large proportion of the conserved R-genes, a scenario described in the “arms race” co-evolutionary model. Based on our study, a variation pattern of R-genes is proposed and confirmed by the analysis of R-genes from other rice lines, indicating that the observed variation pattern may be common in all rice lines.Electronic Supplementary Material Supplementary material is available for this article at     

Stereotyped sequence of mating behavior in the Far Eastern catfish, Silurus asotus, from Lake Biwa

 Mating behavior of the Far Eastern catfish, Silurus asotus (Siluriformes: Siluridae), was observed in a ricefield system facing the shore of Lake Biwa in mid-May to early June in 1990–1997. A set behavioral sequence similar to those of two other silurid fishes, S. biwaensis and S. lithophilus, both endemic to the Lake Biwa system, was observed: “chasing,”“clinging,”“enfolding” while “squeezing” by the male, and “circling” by the spawning pair. This form of mating behavior is quite different from that of S. asotus reported from the Ooi River system in Kyoto Prefecture, which mainly spawns in running water in ditches. Received: April 10, 2001 / Revised: November 5, 2001 / Accetped: November 20, 2001     

The Crystal Structure of the [NiFe] Hydrogenase from the Photosynthetic Bacterium Allochromatium vinosum: Characterization of the Oxidized Enzyme (Ni-A State)

The crystal structure of the membrane-associated [NiFe] hydrogenase from Allochromatium vinosum has been determined to 2.1 Å resolution. Electron paramagnetic resonance (EPR) and Fourier transform infrared spectroscopy on dissolved crystals showed that it is present in the Ni-A state (> 90%). The structure of the A. vinosum [NiFe] hydrogenase shows significant similarities with [NiFe] hydrogenase structures derived from Desulfovibrio species. The amino acid sequence identity is ∼ 50%. The bimetallic [NiFe] active site is located in the large subunit of the heterodimer and possesses three diatomic non-protein ligands coordinated to the Fe (two CN⁻ , one CO). Ni is bound to the protein backbone via four cysteine thiolates; two of them also bridge the two metals. One of the bridging cysteines (Cys64) exhibits a modified thiolate in part of the sample. A mono-oxo bridging ligand was assigned between the metal ions of the catalytic center. This is in contrast to a proposal for Desulfovibrio sp. hydrogenases that show a di-oxo species in this position for the Ni-A state. The additional metal site located in the large subunit appears to be a Mg²⁺ ion. Three iron-sulfur clusters were found in the small subunit that forms the electron transfer chain connecting the catalytic site with the molecular surface. The calculated anomalous Fourier map indicates a distorted proximal iron-sulfur cluster in part of the crystals. This altered proximal cluster is supposed to be paramagnetic and is exchange coupled to the Ni³⁺ ion and the medial [Fe₃S₄]⁺ cluster that are both EPR active (S = 1/2 species). This finding of a modified proximal cluster in the [NiFe] hydrogenase might explain the observation of split EPR signals that are occasionally detected in the oxidized state of membrane-bound [NiFe] hydrogenases as from A. vinosum.     

Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck)

Citrus is an important fruit crop as regards accumulation of carotenoids. In plant carotenoid biosynthesis, phytoene synthase gene (Psy) plays a key role in catalyzing the head-to-head condensation of geranylgeranyl diphosphate molecules to produce colorless phytoene. In the present paper, we reported the phytoene contents determination and characterization of Psy during fruit ripening of “Washington” navel orange and its red-fleshed mutant “Cara Cara”. Results showed that phytoene was exclusively accumulated in peel and pulp of “Cara Cara”. Although phytoene was observed accumulating with fruit ripening of “Cara Cara”, the contents in pulp were 10 times higher than those in peel. The isolated two Psy cDNAs were both 1520 bp in full length, containing 436 deduced amino acid residues, with a different amino acid at 412th. Genomic hybridization results showed that one or two copies might be present in “Cara Cara” and “Washington” genomes. During “Cara Cara” and “Washington” fruit coloration, expression of Psy was observed to be up-regulated, as revealed by tissue specific profiles in the flavedo, albedo, segment membrane and juice sacs. However, Psy expression in albedo of “Cara Cara” was higher than that in “Washington”, as evidenced by phytoene accumulation in the peel.     

Estimation of relative contribution of “mobile phycobilisome” and “energy spillover” in the light–dark induced state transition in Spirulina platensis

Previously, it was clarified that phycobilisome (PBS) mobility and energy spillover were both involved in light-to-dark induced state transitions of intact Spirulina platensis cells. In this work, by taking advantage of the characteristic fluorescence spectra of photosystem I (PSI) trimers and monomers as indicators, the relative contributions for the “mobile PBS” and “energy spillover” are quantitatively estimated by separating the fluorescence contribution of PBS mobility from that of PSI oligomeric change. Above the phase transition temperature (T _PT) of the membrane lipids, the relative proportion of the contributions is invariable with 65% of “mobile PBS” and 35% of “energy spillover”. Below T _PT, the proportion for the “mobile PBS” becomes larger under lowering temperature even reaching 95% with 5% “energy spillover” at 0°C. It is known that lower temperature leads to a further light state due to a more reduced or oxidized PQ pool. Based on the current result, it can be deduced that disequilibrium of the redox state of the PQ pool will trigger PBS movement instead of change in the PSI oligomeric state.     

Community effects following the deletion of a habitat-forming alga from rocky marine shores

Habitat-forming species increase spatial complexity and alter local environmental conditions, often facilitating a diversified assemblage of plants and animals. Removal of dominant species, therefore, can potentially lead to pronounced changes in diversity and community structure through a series of negative and positive interactions involving several components of the community. Here we test community responses to the deletion of the dominant, canopy-forming alga Hormosira banksii from the mid-intertidal zone of wave-protected rocky shores in southern New Zealand. This species was removed in winter (July) from three 3×3-m areas at each of two platforms (Kaikoura and Moeraki) on the east coast of the South Island. Initially, 59 taxa occurred in stands, but there were only four algal species with greater than 5% cover and three mobile invertebrate species with more than five individuals per 0.25 m². By 6 months after Hormosira removal, most fucoid and coralline algae had burned off, and there were blooms of ephemeral algae in the removal plots, but almost no change within controls. After 2 years, diversity declined by 44% relative to controls at Kaikoura and 36% at Moeraki, and the amount of bare space had increased by tenfold at Kaikoura and twofold at Moeraki. Few sessile or mobile invertebrates were present. Recruitment of Hormosira occurred after 14 months in the removal plots. At this time, a “press” disturbance was initiated into one half of each removal plot to test the effects of continued removal of Hormosira on diversity. Similar “end-points” of the control and “press” removal plots were not reached after 2 years, and even after Hormosira recruitment into the original “pulse” experiment there was little recovery of the community. In this mid-intertidal system with considerable thermal stress, and perhaps in others with few perennial species, diversity and community structure can critically depend on positive associations with a single dominant species.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Determination of the residue-specific 15N CSA tensor principal components using multiple alignment media

The individual components of the backbone ¹⁵N CSA tensor, σ₁₁, σ₂₂, σ₃₃, and the orientation of σ₁₁ relative to the NH bond described by the angle β have been determined for uniformly labeled ¹⁵N, ¹³C ubiquitin from partial alignment in phospholipid bicelles, Pf1 phage, and poly(ethylene glycol) by measuring the residue-specific residual dipolar couplings and chemical shift deviations. No strong correlation between any of the CSA tensor components is observed with any single structural feature. However, the experimentally determined tensor components agree with the previously determined average CSA principal components [Cornilescu and Bax (2000) J. Am. Chem. Soc. 122, 10143–10154]. Significant deviations from the averages coincide with residues in β-strand or extended regions, while α-helical residue tensor components cluster close to the average values.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .