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Appropriate biomarkers are required to predict the clinical outcome of triple‐negative breast cancer (TNBC). In this study, we focused on the clinical importance of two representative tumor‐associated proteins, Bcl‐2 and p53. Bcl‐2 expression is usually related to estrogen receptor expression and a favorable outcome in breast cancer. TNBC has been reported to show a high frequency of p53 positivity suggesting TP53 mutations. The expressions of Bcl‐2 and p53 were immunohistochemically examined in TNBC involving two age groups of postmenopausal women (≥75 y/o, n = 75; 55–64 y/o, n = 47), who underwent surgery without neoadjuvant therapy. We examined their associations with each other, or with clinicopathological factors including the outcome. Bcl‐2 expression was inversely correlated with androgen receptor, apocrine morphology, and p53 expressions, and was an independent predictor of a poor outcome in total or in younger women. p53 positivity was associated with a more favorable outcome than p53 negativity in the younger group. In combined analyzes, none of the twenty Bcl‐2‐negative/p53‐positive cases in the younger group exhibited recurrence, resulting in the independent favorable predictive value of Bcl‐2‐negative/p53‐positive. The anti‐apoptotic nature of Bcl‐2 may be apparent in TNBC. The excellent outcome of Bcl‐2‐negative/p53‐positive cases in the younger group warrants further combined investigation of Bcl‐2/p53 in TNBC. [ABSTRACT FROM AUTHOR]

Purpose: Paget disease of the breast is a rare cancer that originates from the nipple–areolar complex. It is often overlooked and misdiagnosed as benign chronic eczema of the nipple. We aimed to retrospectively verify whether blood flow analysis using Doppler sonography was useful for detecting the presence of Paget disease. Methods: In this retrospective study, 12 patients with pathologically proven unilateral nipple eczematous lesions (seven with Paget disease and five with simple dermatitis) were included. Nipple blood flow signal was observed using Doppler sonography, and the detected blood flow signals were quantified using digitally recorded images. Quantified blood flow ratio and pathologically examined capillary density were evaluated between affected and unaffected nipples. Findings of mammography, grayscale sonography, and contrast-enhanced magnetic resonance imaging (CE-MRI) were reviewed. Results: In patients with Paget disease, Doppler effects in the affected nipple were more clearly visible than those in the unaffected nipple. These effects were sufficiently visible to identify Paget disease. No obvious effects were observed in the affected and unaffected nipples of simple dermatitis. The quantified blood flow ratio and pathologically examined capillary density were significantly higher for the Paget lesion than those for the non-Paget lesion. The sensitivity of CE-MRI and Doppler sonography was markedly correlated, revealing blood flow changes in the nipple lesions of Paget disease. Conclusion: Doppler sonography visualized the proliferation of blood vessels in Paget lesions. The visualization of increased nipple blood flow using Doppler sonography is a simple and low-cost method that provides useful data for identifying Paget disease during routine medical care. [ABSTRACT FROM AUTHOR]

Hydrogenases catalyze the reversible conversion of molecular hydrogen to protons and electrons via a heterolytic splitting mechanism. The active sites of [NiFe] hydrogenases comprise a dinuclear Ni-Fe center carrying CO and CN– ligands. The catalytic activity of the standard (O2-sensitive) [NiFe] hydrogenases vanishes under aerobic conditions. The O2-tolerant [NiFe] hydrogenases can sustain H2 oxidation activity under atmospheric conditions. These hydrogenases have very similar active site structures that change the ligand sphere during the activation/catalytic process. An important structural difference between these hydrogenases has been found for the proximal iron-sulphur cluster located in the vicinity of the active site. This unprecedented [4Fe-3S]-6Cys cluster can supply two electrons, which lead to rapid recovery of the O2 inactivation, to the [NiFe] active site. [ABSTRACT FROM AUTHOR]

Introduction: Invasive lobular carcinomas have an increased propensity for distant metastases, particularly to the peritoneum, ovaries, and uterus. In contrast, distant metastases of nonpalpable lobular carcinomas are extremely rare, and the causes of underlying symptoms of primary carcinomas remain unclear. We report a case of an asymptomatic invasive lobular carcinoma with a primary mammary lesion in a patient with rectal stenosis. Case presentation: A 69-year-old Japanese woman presented to our hospital for treatment of constipation. Although rectal stenosis was confirmed, thorough testing of her lower digestive tract did not identify its cause. Thus, an exploratory laparotomy and tissue biopsy was performed, and the presence of an invasive lobular carcinoma was confirmed. Subsequent breast examinations showed that the invasive lobular carcinoma that led to the rectal stenosis was a metastatic lesion from a primary lesion of the breast duct. As the present breast lobular carcinoma was asymptomatic and nonpalpable, we did not initially consider metastatic breast cancer as a cause of her symptoms, and the final diagnosis was delayed. Conclusions: Peritoneal metastasis from nonpalpable invasive lobular carcinomas is very rare. However, breast cancer metastasis should be considered when carcinomatous peritonitis is present in a patient with an unknown primary cancer. [ABSTRACT FROM AUTHOR]

The enzyme hydrogenase reversibly converts dihydrogen to protons and electrons at a metal catalyst. The location of the abundant hydrogens is of key importance for understanding structure and function of the protein. However, in protein X-ray crystallography the detection of hydrogen atoms is one of the major problems, since they display only weak contributions to diffraction and the quality of the single crystals is often insufficient to obtain sub-ångström resolution. Here we report the crystal structure of a standard [NiFe] hydrogenase (∼91.3 kDa molecular mass) at 0.89 Å resolution. The strictly anoxically isolated hydrogenase has been obtained in a specific spectroscopic state, the active reduced Ni-R (subform Ni-R1) state. The high resolution, proper refinement strategy and careful modelling allow the positioning of a large part of the hydrogen atoms in the structure. This has led to the direct detection of the products of the heterolytic splitting of dihydrogen into a hydride (H−) bridging the Ni and Fe and a proton (H+) attached to the sulphur of a cysteine ligand. The Ni-H− and Fe-H− bond lengths are 1.58 Å and 1.78Å, respectively. Furthermore, we can assign the Fe-CO and Fe-CN− ligands at the active site, and can obtain the hydrogen-bond networks and the preferred proton transfer pathway in the hydrogenase. Our results demonstrate the precise comprehensive information available from ultra-high-resolution structures of proteins as an alternative to neutron diffraction and other methods such as NMR structural analysis. [ABSTRACT FROM AUTHOR]

The article presents a study on hydrogenases. It mentions that the group of metalloenzymes catalyze dihydrogen conversion into electrons and protons as well as its reverse reaction. It also notes their provision of energy for organisms through molecular hydrogen oxidation along with balancing cell redox potential.

Membrane type 1 matrix metalloproteinase (MT1-MMP) belongs to the large family of zinc-dependent endopeptidases termed MMPs that are located in the extracellular matrix. MT1-MMP was crystallized at 277 K using the vapour-diffusion method with PEG as a precipitating agent. Data sets for MT1-MMP were collected to 2.24 Å resolution at 100 K. The crystals belonged to space group P43212, with unit-cell parameters a = 62.99, c = 122.60 Å. The crystal contained one molecule per asymmetric unit, with a Matthews coefficient ( VM) of 2.90 Å3 Da−1; the solvent content is estimated to be 57.6%. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

The X-ray crystallographic structure of the native R2F subunit of the ribonucleotide reductase (RNR) of Corynebacterium ammoniagenes ATCC 6872 is reported, with a resolution of 1.36 Å. The metal site contains an oxo/hydroxo-bridged manganese dimer, located near a tyrosine residue (Y115). The coordination of the manganese dimer and its distance to a nearby tyrosine residue resemble the di-iron metalloradical cofactor of class I RNR from Escherichia coli. Multifrequency EPR measurements of the highly active C. ammoniagenes R2F subunit show that the metal site contains a ferromagnetically exchange-coupled MnIIIMnIII dimer weakly coupled to a tyrosyl radical. A mechanism for the metalloradical cofactor (MnIIIMnIIIY•) generation is proposed. H2O2 (HO2-) instead of O2 is hypothesized as physiological oxidant for the Mn dimer which in turn oxidizes the tyrosine Y115. Changes in the ligand sphere of both manganese ions during metalloradical generation direct the complex formation of this cofactor, disfavoring alternate reaction pathways such as H2O2 dismutation, as observed for manganese catalase, a structural analogue of the R2F metal site. The presented results demonstrate the importance of manganese for radical formation in this RNR and confirm the assignment of this enzyme to class Ib. [ABSTRACT FROM AUTHOR]

The interaction of ferriheme proteins with nitrite has recently attracted interest as a source for NO or other nitrogen oxides in mammalian physiology. However, met-hemoglobin (metHb), which was suggested as a key player in this process, does not convert nitrite unless small amounts of NO are added in parallel. We have recently reported that, in contrast, nitrophorins (NPs) convert nitrite as the sole substrate to form NO even at pH 7.5. which is an unprecedented case among ferrihemes [He. C.. and Knipp. M. (2009) J. Am. Clien,. Soc. /31, 12042-I 2043j. NP5, which comprise a class of unique heme b proteins from the saliva of the bloodsucking insect Rhodnius prolixus, appear in a number of concomitant isoproteins. Herein, the first spectroscopic characterization of the initial complexes of the two isoproteins NP4 and NP7 with nitrite is presented and compared to the data reported for metHb and met-myoglobin (metMb). Because upon nitrite binding, NPs, in contrast to metHb and metMb, continue to react with nitrite, resonance Raman spectroscopy and continuous wave electron paramagnetic resonance spectroscopy were applied to frozen samples. As a result. the existence of two six-coordinate ferriheme low-spin complexes was established. Furthermore. X-ray crystallography of NP4 crystals soaked with nitrite revealed the formation ofan η1-N nitro complex, which is in contrast to the 9'-O-bound nitrite in metMb and metHb. Stopped-flow kinetic experiments show that although the ligand dissociation constants of NP4 and NP7 (15-190 M-1)are comparable to those of metHb and metMb. the rates of ligand binding and release are significantly slower. Moreover, not only the reaction kinetics but also electron paramagnetic resonance spectroscopy reveals notable differences between the two isoproteins. [ABSTRACT FROM AUTHOR]

Abstract: X-ray crystallographic studies [Ogata et al., J. Am. Chem. Soc. 124 (2002) 11628–11635] have shown that carbon monoxide binds to the nickel ion at the active site of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F and inhibits its catalytic function. In the present work spectroscopic aspects of the CO inhibition for this bacterial organism are reported for the first time and enable a direct comparison with the existing crystallographic data. The binding affinity of each specific redox state for CO is probed by FTIR spectro-electrochemistry. It is shown that only the physiological state Ni–SIa reacts with CO. The CO-inhibited product state is EPR-silent (Ni2+) and exists in two forms, Ni–SCO and Ni–SCOred. At very negative potentials, the exogenous CO is electrochemically detached from the active site and the active Ni–R states are obtained. At temperatures below 100 K, photodissociation of the extrinsic CO from the Ni–SCO state results in Ni–SIa that is identified to be the only light-induced state. In the dark, rebinding of CO takes place; the recombination rate constants are of biexponential character and the activation barrier is determined to be approximately 9 kJ mol−1. In addition, formation of a paramagnetic CO-inhibited state (Ni–CO) was observed that results from the interaction of carbon monoxide with the Ni–L state. It is proposed that the nickel in Ni–CO is in a formal monovalent state (Ni1+). [Copyright &y& Elsevier]

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-SIr, provides the link between fully oxidized (Ni-A, Ni-B) and active (Ni-SIa, Ni-C and Ni-R) forms of hydrogenase. In this work Ni-SIr 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-SIr 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-SIr 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. [ABSTRACT FROM AUTHOR]

Summary: 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. [Copyright &y& Elsevier]

Characterizes the carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F. Use of X-ray crystallography and absorption and resonance Raman spectroscopy; Determination of the nine crystals of hydrogenase; Observation of distinct changes in electron density distribution.

Annexin V is a Ca2+-dependent phospholipid binding protein. Although it has been shown to inhibit protein kinase C (PKC) in cell-free systems, its role in the intact cell is unclear. A stable MCF-7 human breast cancer cell overexpression system was established to investigate the function of annexin V. In these cells, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation and kinase activity of ERK1/2 were suppressed. Morphological changes induced by TPA were reduced by annexin V overexpression as well as by the pan-PKC inhibitor, bisindolylmaleimide I, and by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) inhibitor, PD98059. TPA-induced MEK1/2 and Raf-1 phosphorylation were reduced in these cells. The TPA-enhanced active Ras, and its association with Raf-1, were reduced. TPA treatment of MCF-7 cells caused an increased association of Shc with Grb2. However, this increased association was prevented in the annexin V-overexpressors. p21WAF/CIP1 is responsible for inhibition of cell cycle progression in MCF-7 cells. TPA induced the expression of p21WAF/CIP1 to a greater extent in MCF-7 parent and control plasmid cells than in annexin V overexpressors. PD98059 inhibited this increase, suggesting that TPA upregulation of p21WAF/CIP1 occurs via the MEK pathway, and that annexin V overexpression blunts it. This work shows that annexin V overexpression suppresses the TPA-induced Ras/ERK signaling by inhibiting at/or upstream of Shc, possibly through the inhibition of PKCs. Oncogene (2000) 19, 2904–2912 [ABSTRACT FROM AUTHOR]

Dissimilatory sulfite reductase (Dsr) plays an important role in sulfate respiration in many sulfate-reducing bacteria. Dsr from Desulfovibrio vulgaris Miyazaki F has been purified and crystallized at 277 K using the sitting-drop vapour-diffusion method with PEG 3350 and potassium thiocyanate as precipitants. A data set was collected to 3.7 Å resolution from a single crystal at 100 K using synchrotron radiation. The Dsr crystal belonged to space group P41212, with unit-cell parameters a = b = 163.26, c = 435.32 Å. The crystal structure of Dsr was determined by the molecular-replacement method based on the three-dimensional structure of Dsr from D. vulgaris Hildenborough. The crystal contained three α2β2γ2 units per asymmetric unit, with a Matthews coefficient ( VM) of 2.35 Å3 Da−1; the solvent content was estimated to be 47.7%. [ABSTRACT FROM AUTHOR]

Ribonucleotide reduction, the unique step in DNA-precursor biosynthesis, involves radical-dependent redox chemistry and diverse metallo-cofactors. The metallo-cofactor (R2F) encoded by the nrdF ( nucleotide re duction) gene in Corynebacterium ammoniagenes ATCC 6872 was isolated after homologous expression and a new crystal form of ribonucleotide reductase R2F was obtained. R2F was crystallized at 277 K using the vapour-diffusion method with PEG as the precipitating agent. A data set was collected to 1.36 Å resolution from a single crystal at 100 K using synchrotron radiation. The crystal belonged to space group C2, with unit-cell parameters a = 96.21, b = 87.68, c = 83.25 Å, β = 99.29°. The crystal contained two molecules per asymmetric unit, with a Matthews coefficient ( VM) of 2.69 Å3 Da−1; the solvent content was estimated to be 54.3%. X-ray fluorescence spectroscopy and MAD diffraction data indicated the presence of manganese in the molecule and the absence of iron. [ABSTRACT FROM AUTHOR]

Light-oxygen-voltage (LOV) proteins play an important role in blue-light-dependent physiological processes in many organisms. The LOV domain of the blue-light receptor YtvA from Bacillus amyloliquefaciens FZB42 has been purified and crystallized at 277 K using the sitting-drop vapour-diffusion method with 2-ethoxyethanol as a precipitant. A data set was collected to 1.60 Å resolution from a single crystal at 100 K using synchrotron radiation. The LOV domain of YtvA crystallized in space group C2221, with unit-cell parameters a = 64.95, b = 83.76, c = 55.81 Å. The crystal structure of the LOV domain of YtvA was determined by the molecular-replacement method. The crystal contained one molecule per asymmetric unit, with a Matthews coefficient ( VM) of 3.04 Å3 Da−1; the solvent content was estimated to be 59.5%. [ABSTRACT FROM AUTHOR]

Sulfur in its various oxidation states is used for energy conservation in many microorganisms. Adenylylsulfate reductase is a key enzyme in the sulfur-reduction pathway of sulfate-reducing bacteria. The adenylylsulfate reductase from Desulfovibrio vulgaris Miyazaki F has been purified and crystallized at 277 K using the vapour-diffusion method with ammonium sulfate as the precipitating agent. A data set was collected to 1.7 Å resolution from a single crystal at 100 K using synchrotron radiation. The crystal belonged to space group P31, with unit-cell parameters a = b = 125.93, c = 164.24 Å. The crystal contained two molecules per asymmetric unit, with a Matthews coefficient ( VM) of 4.02 Å3 Da−1; the solvent content was estimated to be 69.4%. [ABSTRACT FROM AUTHOR]

The catalytic domain of death-associated protein kinase (DAPK) has been overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method with PEG 8000 and magnesium acetate as precipitants. Complexes with the inhibitor staurosporine and its analogue BDB402 were also crystallized in the presence of PEG 400 and PEG 8000, respectively. Diffraction data were collected to 2.4 Å for the native catalytic domain, to 2.9 Å for the staurosporine complex and to 2.7 Å for the BDB402 complex. All three crystals belong to space group P212121, with unit-cell parameters a = 77.992, b = 109.909, c = 50.063 Å for the catalytic domain, a = 78.911, b = 113.162, c = 50.658 Å for the staurosporine complex and a = 77.337, b = 108.869, c = 50.186 Å for the BDB402 complex. In both complexes the inhibitor molecule was clearly assigned in the difference Fourier map calculated on the basis of the phases obtained from the structure of the catalytic domain. [ABSTRACT FROM AUTHOR]

A new perspective on enzymes: Free‐electron lasers are increasingly used to obtain crystal structures of interesting enzymes like photosystem II from nanocrystals at room temperature, to avoid radiation damage, and to detect structural differences between specific states in the catalytic cycle. [ABSTRACT FROM AUTHOR]

Neuer Blick auf Enzyme: Freie ‐ Elektronen ‐ Laser werden zunehmend genutzt, um Strukturen von Nanokristallen interessanter Enzyme wie dem Photosystem II bei Raumtemperatur aufzuklären, Strahlungsschäden zu vermeiden und Strukturunterschiede spezifischer Zustände während der Katalyse zu detektieren. [ABSTRACT FROM AUTHOR]

Background: We examined the vascularity of mammary Paget disease histologically to confirm the increased blood flow observed previously by clinical imaging. The relationships among blood vessel density (BVD), histopathological parameters of blood flow in the nipple, and the expression of angiogenic factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor A (VEGFA) were examined. Methods: We calculated the average CD34-positive BVD and podoplanin (D2–40)-positive lymphatic vessel density (LVD) and the proportion of proliferating of endothelial cells in 14 Paget disease, 3 dermatitis biopsy, and 14 age-matched control cases. As a parameter related to blood flow in the nipple, the total CD34-positive blood vessel lumen area relative to the entire nipple area was measured in each Paget disease and control case using an automated image analysis system. Immunohistochemical expression of bFGF and VEGFA in Paget cells was also examined. Results: The average BVD and LVD were significantly higher in the Paget disease cases than in the dermatitis (p = 0.003) and control (p < 0.001) cases. The proportion of proliferating endothelial cells was also increased in the Paget disease cases. The ratio of the CD34-positive blood vessel lumen area to nipple area was also significantly higher in the Paget disease than control cases (p = 0.003). The average BVD was correlated with the average LVD (r = 0.734, p < 0.001) and ratio of the blood vessel lumen area to nipple area (r = 0.692, p < 0.001). Immunohistochemical expression of bFGF was strong in all Paget disease cases, with a significantly higher expression score in the Paget disease than dermatitis (p = 0.003) and control (p < 0.001) cases. The bFGF, but not VEGFA, expression score, was strongly correlated with the average BVD (r = 0.818, p < 0.001) and ratio of the blood vessel lumen area to nipple area (r = 0.503, p = 0.006). Conclusion: These results provide direct histopathological evidence of a marked increase in nipple blood flow in Paget disease detected by clinical imaging. bFGF is considered to play a pivotal role in angiogenesis in mammary Paget disease. [ABSTRACT FROM AUTHOR]

Background: Nitrophorins (NPs) are NO transporting proteins from the saliva of the blood-sucking insect Rhodnius prolixus. The delivery of NO is accomplished by storage on the protein’s ferriheme cofactor and the gas molecule is released when the protein is injected into a victim’s tissue. However, we demonstrated that nitrophorins can also produce NO from nitrite as the only substrate, i.e., in this reaction nitrite serves as both electron donor and electron acceptor. The enzymatic activity was termed nitrite dismutase activity and is now classified with the EC number 1.7.6.1. This study provides insight into the structural features required for the catalytic activity. Methods: Proteins were recombinantly expressed in Escherichia coli cells. They were characterized by X-ray crystallography, EPR spectroscopy, UV/vis spectroscopy, resonance Raman spectroscopy, and NMR spectroscopy. The enzymatic reaction was studied by absorbance detected stopped-flow kinetics. Results: The binding mode of nitrite to the NP Fe (III) is clearly different compared to that of the globins, i.e., N-bound. Contrary to the globins, the insertion of an H-donating residue (NP4(L130R)) into the protein pocket does not affect the nitrite binding mode; however, it cancels the enzymatic activity. On the other hand, mutation of the proximal Asp70 residue, which is H-bonded via a water to the iron coordinating His59, also affects the enzymatic activity, indicating that the charge distribution on the heme cofactor has a major influence on the iron reactivity. Conclusion: The nitrophorins are good model systems for the characterization of the nitrite dismutase catalysis. The absence of H-donating residues and the charge density on the proximal His were identified as important structural features that are required for the nitrite dismutase reaction. Among the many heme proteins expressed in the human organism some might bear a similar catalytic activity. We suggest the group of heme peroxidases as potential candidates for enzymatic “nitrite-only” NO production. Disclosure: Supported by the Deutsche Forschungsgemeinschaft (DFG), Grants KN 951-1/1 and 2. [ABSTRACT FROM AUTHOR]

Direct spectroscopic evidence for a hydride bridge in the Ni-R form of [NiFe] hydrogenase has been obtained using iron-specific nuclear resonance vibrational spectroscopy (NRVS). The Ni-H-Fe wag mode at 675 cm−1 is the first spectroscopic evidence for a bridging hydride in Ni-R as well as the first iron-hydride-related NRVS feature observed for a biological system. Although density function theory (DFT) calculation assisted the determination of the Ni-R structure, it did not predict the Ni-H-Fe wag mode at ∼675 cm−1 before NRVS. Instead, the observed Ni-H-Fe mode provided a critical reference for the DFT calculations. While the overall science about Ni-R is presented and discussed elsewhere, this article focuses on the long and strenuous experimental journey to search for and experimentally identify the Ni-H-Fe wag mode in a Ni-R sample. As a methodology, the results presented here will go beyond Ni-R and hydrogenase research and will also be of interest to other scientists who use synchrotron radiation for measuring dilute samples or weak spectroscopic features. [ABSTRACT FROM AUTHOR]

Gases like H2, N2, CO2, and CO are increasingly recognized as critical feedstock in "green" energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N2, CO2, and CO and the production of H2 require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur. Such systems are studied to understand the biocatalytic principles of gas conversion including N2 fixation by nitrogenase and H2 production by hydrogenase as well as CO2 and CO conversion by formate dehydrogenase, carbon monoxide dehydrogenase, and nitrogenase. In this review, we emphasize the importance of the cofactor/protein interface, discussing how second and outer coordination sphere effects determine, modulate, and optimize the catalytic activity of GPMs. These may comprise ionic interactions in the second coordination sphere that shape the electron density distribution across the cofactor, hydrogen bonding changes, and allosteric effects. In the outer coordination sphere, proton transfer and electron transfer are discussed, alongside the role of hydrophobic substrate channels and protein structural changes. Combining the information gained from structural biology, enzyme kinetics, and various spectroscopic techniques, we aim toward a comprehensive understanding of catalysis beyond the first coordination sphere. [ABSTRACT FROM AUTHOR]

Hydrogenases catalyze the reversible oxidation of H2. Carbon monoxide (CO) is known to be a competitive inhibitor of O2‐sensitive [NiFe]‐hydrogenases. Although the activities of some O2‐tolerant [NiFe]‐hydrogenases are unaffected by CO, the partially O2‐tolerant [NiFe]‐hydrogenase from Citrobacter sp. S‐77 (S77‐HYB) is inhibited by CO. In this work, the CO‐bound state of S77‐HYB was characterized by activity assays, spectroscopic techniques and X‐ray crystallography. Electron paramagnetic resonance spectroscopy showed a diamagnetic Ni2+ state, and Fourier‐transform infrared spectroscopy revealed the stretching vibration of the exogenous CO ligand. The crystal structure determined at 1.77 Å resolution revealed that CO binds weakly to the nickel ion in the Ni–Fe active site of S77‐HYB. These results suggest a positive correlation between O2 and CO tolerance in [NiFe]‐hydrogenases. [ABSTRACT FROM AUTHOR]

Abstract: Hydrogenase proteins catalyze the reversible conversion of molecular hydrogen to protons and electrons. The most abundant hydrogenases contain a [NiFe] active site; these proteins are generally biased towards hydrogen oxidation activity and are reversibly inhibited by oxygen. However, there are [NiFe] hydrogenase that exhibit unique properties, including aerobic hydrogen oxidation and preferential hydrogen production activity; these proteins are highly relevant in the context of biotechnological devices. This review describes four classes of these “nonstandard” [NiFe] hydrogenases and discusses the electrochemical, spectroscopic, and structural studies that have been used to understand the mechanisms behind this exceptional behavior. A revised classification protocol is suggested in the conclusions, particularly with respect to the term “oxygen-tolerance”. This article is part of a special issue entitled: metals in bioenergetics and biomimetics systems. [Copyright &y& Elsevier]

NO is an important signaling molecule in human tissue. However, the mechanisms by which this molecule is controlled and directed are currently little understood. Nitrophorins (NPs) comprise a group of ferriheme proteins originating from blood-sucking insects that are tailored to protect and deliver NO via coordination to and release from the heme iron. Therefore, the kinetics of the association and dissociation reactions were studied in this work using the ferroheme—CO complexes of NP4, NP4(D30N), and NP7 as isoelectronic models for the ferriheme-NO complexes. The kinetic measurements performed by nanosecond laser-flash-photolysis and stopped-flow are accompanied by resonance Raman and PT- JR spectroscopy to characterize the carbonyl species. Careful analysis of the CO rebinding kinetics reveals that in NP4 and, to a larger extent, NP7 internal gas binding cavities are located, which temporarily trap photodissociated ligands. Moreover, changes in the free energy barriers throughout the rebinding and release pathway upon increase of the pH are surprisingly small in case of NP4. Also in case of NP4, a heterogeneous kinetic trace is obtained at pH 7.5, which corresponds to the presence of two carbonyl species in the heme cavity that are seen in vibrational spectroscopy and that are due to the change of the distal heme pocket polarity. Quantification of the two species from FT-JR spectra allowed the fitting of the kinetic traces as two processes, corresponding to the previously reported open and closed conformation of the A-B and G-H loops. With the use of the A-B loop mutant NP4(D30N), it was confirmed that the kinetic heterogeneity is controlled by pH through the disruption of the H-bond between the Asp30 side chain and the Leul30 backbone carbonyl. Overall, this first study on the slow phase of the dynamics of diatomic gas molecule interaction with NPs comprises an important experimental contribution for the understanding of the dynamics involved in the binding/release processes of NO/CO in NPs. [ABSTRACT FROM AUTHOR]

Purpose. We hypothesized that innate immune response pathways might be involved in thyroid carcinogenesis. To investigate this hypothesis, we aimed at analyzing the expression of several receptors and molecules in the innate immune system in papillary thyroid carcinoma (PTC) and anaplastic thyroid carcinoma (ATC) tissues. Methods. Of the surgically resected specimens, 11 ATC tissues, 25 PTC tissues, and 8 nodular hyperplasia (NH) tissues were selected and examined for the expression of toll-like receptor (TLR) 2, TLR3, TLR4, TLR5, TLR7, TLR9, the myeloid differentiation primary response gene 88 (MyD88), and toll-interleukin-1 receptor domain-containing adaptor inducing INF-β (TRIF) by immunohistochemistry (IHC). Results. Several TLRs were expressed in each tissue. TLR3 was strongly expressed in all tissues. In contrast, TLR4 was not detected in any tissues. While TLR5 was moderately expressed in NH but significantly reduced in PTC and ATC, TLR9 was absent in NH tissue but moderately expressed in both PTC and ATC. On MyD88 expression, no significant difference was found between PTC and ATC. TRIF was significantly upregulated in PTC and ATC compared to NH. Surprisingly, PTC and ATC tissues exhibited similar expression patterns of TLRs, MyD88, and TRIF. Conclusion. These data suggest the involvement of the innate immune system in both PTC and ATC. Specifically, TLR3-mediated TRIF activation was confirmed in PTC and ATC. This provides new insight into thyroid carcinogenesis. [ABSTRACT FROM AUTHOR]

The heterotrimeric electron-bifurcating [FeFe] hydrogenase (HydABC) from Thermotoga maritima (Tm) couples the endergonic reduction of protons (H+) by dihydronicotinamide adenine dinucleotide (NADH) (∆G0 ≈ 18 kJ mol−1) to the exergonic reduction of H+ by reduced ferredoxin (Fdred) (∆G0 ≈ − 16 kJ mol−1). The specific mechanism by which HydABC functions is not understood. In the current study, we describe the biochemical and spectroscopic characterization of TmHydABC recombinantly produced in Escherichia coli and artificially maturated with a synthetic diiron cofactor. We found that TmHydABC catalyzed the hydrogen (H2)-dependent reduction of nicotinamide adenine dinucleotide (NAD+) in the presence of oxidized ferredoxin (Fdox) at a rate of ≈17 μmol NADH min−1 mg−1. Our data suggest that only one flavin is present in the enzyme and is not likely to be the site of electron bifurcation. FTIR and EPR spectroscopy, as well as FTIR spectroelectrochemistry, demonstrated that the active site for H2 conversion, the H-cluster, in TmHydABC behaves essentially the same as in prototypical [FeFe] hydrogenases, and is most likely also not the site of electron bifurcation. The implications of these results are discussed with respect to the current hypotheses on the electron bifurcation mechanism of [FeFe] hydrogenases. Overall, the results provide insight into the electron-bifurcating mechanism and present a well-defined system for further investigations of this fascinating class of [FeFe] hydrogenases. [ABSTRACT FROM AUTHOR]

Purpose: To clarify the profile of adverse events from endocrine therapies in older patients.Methods: We surveyed 15 subjective symptoms including hot flashes, sweating, knuckle stiffness, knee/shoulder joint pain, limb numbness, lethargy, forgetfulness, depressive state, irritated state, genital bleeding, leukorrhea increase, vaginal dryness, bone fracture, and weight gain by a questionnaire among 2044 patients over 55 years old (total number of answered sheets, 8875) and compared the results according to age (56-69 years old vs. ≥ 70 years old) and type of therapy (aromatase inhibitors (AIs) vs. selective estrogen receptor modulators (SERMs)). Among patients 56-69 years old, 6093 and 314 responses were from patients treated with AIs (1477 patients) and SERMs (123 patients), respectively, and 2292 and 176 responses were from those ≥ 70 years old treated with AIs (581 patients) and SERMs (51 patients), respectively.Results: In patients ≥ 70 years old, sweating, knuckle stiffness, knee/shoulder joint pain, limb numbness, and lethargy were significantly more frequent/severe with AIs than with SERMs. In those aged 56-69, knuckle stiffness and vaginal dryness were significantly more frequent with AIs than with SERMs, but the opposite occurred for hot flashes, leukorrhea increase, genital bleeding, and weight gain.Conclusions: Among patients ≥ 70 years old, many symptoms were significantly more frequent/severe with AIs than with SERMs, compared with those aged 56-69, which suggests a difference in the profile of adverse events according to the type of endocrine therapy and the patient's age. It is important to consider the benefits and risks of each treatment to optimize endocrine therapy for older patients. [ABSTRACT FROM AUTHOR]

Sensory type [FeFe] hydrogenases are predicted to play a role in transcriptional regulation by detecting the H2 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 Hox 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 Hox 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. [ABSTRACT FROM AUTHOR]

Background: Breast cancer-related lymphedema often causes cellulitis and is one of the most common complications after breast cancer surgery. Streptococci are the major pathogens underlying such cellulitis. Among the streptococci, the importance of the Lancefield groups C and G is underappreciated; most cases involve Streptococcus dysgalactiae subspecies equisimilis. Despite having a relatively weak toxicity compared with group A streptococci, Streptococcus dysgalactiae subspecies equisimilis is associated with a mortality rate that is as high as that of group A streptococci in cases of invasive infection because Streptococcus dysgalactiae subspecies equisimilis mainly affects elderly individuals who already have various comorbidities.Case Presentation: An 83-year-old Japanese woman with breast cancer-related lymphedema in her left upper limb was referred to our hospital with high fever and acute pain with erythema in her left arm. She showed septic shock with disseminated intravascular coagulation. Blood culture showed positive results for Streptococcus dysgalactiae subspecies equisimilis, confirming a diagnosis of streptococcal toxic-shock syndrome. She survived after successful intensive care.Conclusions: To the best of our knowledge, this case represents the first report of Streptococcus dysgalactiae subspecies equisimilis-induced streptococcal toxic-shock syndrome in a patient with breast cancer-related lymphedema. Breast cancer-related lymphedema is a common problem, and we must pay attention to invasive streptococcal soft tissue infections, particularly in elderly patients with chronic disease. [ABSTRACT FROM AUTHOR]

The sensitivity and specificity of a new automated electrochemiluminescence immunoassay system, Elecsys® Anti-p53 (Elecsys), were compared with that of the conventional serum anti-p53 antibody (s-p53-Ab) enzyme-linked immunosorbent assay kit [MESACUP anti-p53 test (MESACUP)]. Elecsys and MESACUP were used to analyze the levels of s-p53-Abs in patients with esophageal, colorectal and breast cancer. A total of 532 controls and 288, 235 and 329 patients with esophageal, colorectal and breast cancer, respectively, were enrolled. Additionally, the sera of patients with benign diseases of the esophagus, colorectal system and breast, patients with autoimmune diseases and healthy volunteers were analyzed as controls. Sensitivity and specificity were compared between the two assay systems. Positive agreement rates were 58.7% in all samples, 71.2% in esophageal samples, 73.6% in colorectal samples and 35.1% in breast samples. Negative agreement rates for the different cancer types were ≥97.1% and the overall agreement rates were ≥92.3%. When the specificities of the two assays were aligned for all samples, Elecsys demonstrated higher sensitivities for all types of analyzed cancer together, as well as for esophageal, colorectal and breast cancer, respectively. Although positive concordance between the two assay systems was low in terms of specificity, Elecsys had a higher sensitivity than the MESACUP. [ABSTRACT FROM AUTHOR]

Abstract: Nitrophorins are proteins occurring in the saliva of the blood-sucking insect Rhodnius prolixus to carry NO as a vasodilator and blood-coagulation inhibitor into the victim''s tissue. It was suggested that the rate of NO release can be enhanced by the blood-plasma component l-cysteine [J.M.C.Ribeiro, Insect Biochem. Mol. Biol. 26 (1996) 899–905]. However, the mechanism of the reaction is not clear. In the attempt to exploit the reaction in detail, complexes of nitrophorin 4 (NP4) with the thiols 2-mercaptoethanol, l-cysteine, and l-homocysteine and with HS− were formed and characterized under anaerobic conditions using absorption spectroscopy, X-ray crystallography, and EPR spectroscopy. In contrast to met-myoglobin, which is reduced by l-cysteine, all four compounds form low-spin FeIII complexes with NP4. The weak equilibration constants (167–5200M−1) neither support significant complexation nor the simple displacement of NO in vivo. Both amino acid based thiols form additional H-bonds with side chains of the heme pocket entry. Glutathione and l-methionine did not form a complex, indicating the specificity of the complexes with l-cysteine and l-homocysteine. Continuous wave EPR spectroscopy reveals the simultaneous existence of three low-spin systems in each case that are attributed to various protonation and/or conformational stages in the heme pocket. Electron nuclear double resonance (ENDOR) spectroscopy demonstrates that the thiol sulfurs are, at least in part, protonated. Overall, the results not only demonstrate the good accessibility of the NP4 heme center by biologically relevant thiols, but also represent the first structural characterization of a ferriheme protein in complex with l-cysteine l-homocysteine. [Copyright &y& Elsevier]

Nuclear inelastic scattering of 57Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign FeCO and FeCN bending and stretching vibrations of the active site outside the spectral range of the FeS cluster normal modes. [ABSTRACT FROM AUTHOR]

Blick ins Zentrum: Die Methode der inelastischen kernresonanten Streuung (NRVS) wurde genutzt, um 57Fe ‐ markierte [NiFe] ‐ Hydrogenase in unterschiedlichen Zuständen des Enzyms zu untersuchen. Damit gelang es, die Fe ‐ CO ‐ und Fe ‐ CN ‐ Beuge ‐ und ‐ Streckschwingung außerhalb des Spektralbereichs der Normalmoden der Fe ‐ S ‐ Cluster zu detektieren und zuzuordnen (siehe Spektrum). [ABSTRACT FROM AUTHOR]

High-Tc superconductor (HTS) rotating machine with melt-textured Gd-123 bulk pole-field magnets was assembled and tested. The axial gap type motor with neither brush/slip ring nor iron core was composed of a kind of multiple rotor, i.e., a twinned rotor. On each rotor disk, there are eight Gd-123 HTS bulk magnets. The bulk magnets were cooled down with circulating liquid nitrogen supplied inside the rotors. Separately, triple-layered armature copper coils cooled with liquid nitrogen were fixed for pulsed current magnetization for the bulks. Rotation of the twinned rotor provide a better single-phase waveform in 200 rpm compared to in single rotor operation. Magnetic flux density and its distribution on each bulk are stable with averaged decay of 4% for seven hours continuous synchronous operation. The results give a poof on the realization of strong flux collection motor. [ABSTRACT FROM AUTHOR]

An untreated 66-year-old woman with chronic myelogenous leukaemia (CML) in the chronic phase was initially given imatinib mesylate, rapidly achieving a good cytogenetic response with treatment. However, acute promyelocytic leukaemia complicated by a disseminated intravascular coagulation occurred 9 months after beginning imatinib treatment. Promyelocytic crisis of CML was diagnosed by demonstration of both BCR/ABL and PML/RARα chimeric genes in leukaemic cells by karyotypic and fluorescence in situ hybridization analysis. Clonal evolution with addition of the PML/RARα translocation may have arisen in the early chronic phase of CML, with expansion of this clone during imatinib treatment. Promyelocytic crisis of CML is rare; furthermore, we know of no previous report of promyelocytic crisis occurring during treatment with imatinib. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We report a 66-year-old man with hepatitis C virus (HCV)-related cirrhosis and simultaneous hepatic relapse of non-Hodgkin’s lymphoma (NHL) and of hepatocellular carcinoma (HCC). Although the liver is frequently involved by NHL, hepatic colocalization of NHL and HCC is rarely detected by imaging techniques. HCV has been suggested to be lymphotrophic as well as hepatotrophic, and therefore has attracted speculation about a causative role in some cases of lymphoma. The patient had a past history of cutaneous diffuse large B cell lymphoma (DLBCL) in concurrence with HCC 32 months previously. Complete remission (CR) had been maintained for both diseases until February 2004, when ultrasonography and computed tomography (CT) showed multiple liver tumors. Two of these, appearing hyperattenuating in the arterial phase of contrast-enhanced CT, were diagnosed histopathologically as HCC, and treated with radiofrequency ablation. The other tumors, hypoattenuating in the portal phase CT, were diagnosed histopathologically as DLBCL, and treated with cyclophosphamide, tetrahydropyranyl-Adriamycin, vincristine and prednisolone (THP-COP) in combination with rituximab. CR was achieved for both DLBCL and HCC. Given the previously demonstrated immune system tropism and perturbation by HCV, the virus might have contributed to the occurrence of the NHL as well as the HCC. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Cytochrome c3 isolated from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F, is a tetraheme protein. Its physiological partner, [NiFe] hydrogenase, catalyzes the reversible oxidoreduction of molecular hydrogen. To elucidate the mechanism of electron transfer between cytochrome c3 and [NiFe] hydrogenase, the transient complex formation by these proteins was investigated by means of NMR. All NH signals of uniformly 15N-labeled ferric cytochrome c3 except N-terminus, Pro, and Gly73 were assigned. ¹H-15N HSQC spectra were recorded for 15N-labeled ferric and ferrous cytochrome c3, in the absence and presence of hydrogenase. Chemical shift perturbations were observed in the region around heme 4 in both oxidation states. Additionally, the region between hemes 1 and 3 in ferrous cytochrome c3 was affected in the presence of hydrogenase, suggesting that the mode of interaction is different in each redox state. Heme 3 is probably the electron gate for ferrous cytochrome c3. To investigate the transient complex of cytochrome c3 and hydrogenase in detail, modeling of the complex was performed for the oxidized proteins using a docking program, ZDOCK 2.3, and NMR data. Furthermore, the roles of lysine residues of cytochrome c3 in the interaction with hydrogenase were investigated by site-directed mutagenesis. When the lysine residues around heme 4 were replaced by an uncharged residue, methionine, one by one, the Km of the electron-transfer kinetics increased. The results showed that the positive charges of Lys60, Lys72, Lys95, and Lys101 around heme 4 are important for formation of the transient complex with [NiFe] hydrogenase in the initial stage of the cytochrome c3 reduction. This finding is consistent with the most possible structure of the transient complex obtained by modeling. [ABSTRACT FROM AUTHOR]

The catalytic center of the [NiFe] hydrogenase of Desulfovibrio vulgaris Miyazaki F in the oxidized states was investigated by electron paramagnetic resonance and electron–nuclear double resonance spectroscopy applied to single crystals of the enzyme. The experimental results were compared with density functional theory (DFT) calculations. For the Ni-B state, three hyperfine tensors could be determined. Two tensors have large isotropic hyperfine coupling constants and are assigned to the β-CH2 protons of the Cys-549 that provides one of the bridging sulfur ligands between Ni and Fe in the active center. From a comparison of the orientation of the third hyperfine tensor with the tensor obtained from DFT calculations an OH− bridging ligand has been identified in the Ni-B state. For the Ni-A state broader signals were observed. The signals of the third proton, as observed for the “ready” state Ni-B, were not observed at the same spectral position for Ni-A, confirming a structural difference involving the bridging ligand in the “unready” state of the enzyme. [ABSTRACT FROM AUTHOR]

The gene encoding an enolase from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 2.1-kb DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with PstI and BamHI, contained an enolase gene (eno) and part of the methylenetetrahydrofolate dehydrogenase gene (folD). The nucleotide sequence of eno indicates that the protein monomer is composed of 434 amino acids. An expression system for eno under control of the T7 promoter was constructed in E. coli. The purified His-tagged enolase formed a homooctamer and was active in the formation of phosphoenolpyruvate (PEP) as well as in the reverse reaction, the formation of d-(+)-2-phosphoglyceric acid (2-PGA). The pH dependence and kinetic properties of the recombinant enolase from the sulfate-reducing bacterium were also studied. The amounts of eno mRNA when the bacterium was grown on glycerol or glucose were compared to that when D. vulgaris was grown on lactate. [Copyright &y& Elsevier]

In the catalytic cycle of [NiFe] hydrogenase the paramagnetic Ni-C intermediate is of key importance, since it is believed to carry the substrate hydrogen, albeit in a yet unknown geometry. Upon illumination at low temperatures, Ni-C is converted to the so-called Ni-L state with markedly different spectroscopic parameters. It is suspected that Ni-L has lost the "substrate hydrogen". In this work, both paramagnetic states have been generated in single crystals obtained from the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. Evaluation of the orientation dependent spectra yielded the magnitudes of the g tensors and their orientations in the crystal axes system for both Ni-C and Ni-L. The g tensors could further be related to the atomic structure by comparison with the X-ray crystallographic structure of the reduced enzyme. Although the gtensor magnitudes of Ni-C and Ni-L are quite different, the orientations of the resulting g tensors are very similar but differ from those obtained earlier for Ni-A and Ni-B (Trofanchuk et al. J. Biol. Inorg. Chem. 2000, 5, 36-44). The g tensors were also calculated by density functional theory (DFT) methods using various structural models of the active site. The calculated g tensor of Ni-C is, concerning magnitudes and orientation, in good agreement with the experimental one for a formal Ni(III) oxidation state with a hydride (H-) bridge between the Ni and the Fe atom. Satisfying agreement is obtained for the Ni-L state when a formal Ni(I) oxidation state is assumed for this species with a proton (H[sup +]) removed from the bridge between the nickel and the iron atom. [ABSTRACT FROM AUTHOR]