Activation of N2O reduction by the fully reduced micro4-sulfide bridged tetranuclear Cu Z cluster in nitrous oxide reductase.
The tetranuclear CuZ cluster catalyzes the two-electron reduction of N2O to N2 and H2O in the enzyme nitrous oxide reductase. This study shows that the fully reduced 4CuI form of the cluster correlates with the catalytic activity of the enzyme. This is the first demonstration that the S = 1/2 form of CuZ can be further reduced. Complementary DFT calculations support the experimental findings and demonstrate that N2O binding in a bent mu-1,3-bridging mode to the 4CuI form is most efficient due to strong back-bonding from two reduced copper atoms. This back-donation activates N2O for electrophilic attack by a proton.

title: Spectroscopic and electronic structure studies of phenolate Cu(II) complexes: phenolate ring orientation and activation related to cofactor biogenesis.
abstract: A combination of spectroscopies and DFT calculations have been used to define the electronic structures of two crystallographically defined Cu(II)-phenolate complexes. These complexes differ in the orientation of the phenolate ring which results in different bonding interactions of the phenolate donor orbitals with the Cu(II), which are reflected in the very different spectroscopic properties of the two complexes. These differences in electronic structures lead to significant differences in DFT calculated reactivities with oxygen. These calculations suggest that oxygen activation via a Cu(I) phenoxyl ligand-to-metal charge transfer complex is highly endergonic (>50 kcal/mol), hence an unlikely pathway. Rather, the two-electron oxidation of the phenolate forming a bridging Cu(II) peroxoquinone complex is more favorable (11.3 kcal/mol). The role of the oxidized metal in mediating this two-electron oxidation of the coordinated phenolate and its relevance to the biogenesis of the covalently bound topa quinone in amine oxidase are discussed.

title: Spectroscopic and computational studies of nitrite reductase: proton induced electron transfer and backbonding contributions to reactivity.
abstract: A combination of spectroscopy and DFT calculations has been used to define the geometric and electronic structure of the nitrite bound type 2 (T2) copper site at high and low pH in nitrite reductase from Rhodobacter sphaeroides. At high pH there is no electron transfer from reduced type 1 (T1) to the nitrite bound T2 copper, while protonation triggers T1 --> T2 electron transfer and generation of NO. The DFT calculated reaction coordinate for the N-O bond cleavage in nitrite reduction by the reduced T2 copper suggests that the process is best described as proton transfer triggering electron transfer. Bidentate nitrite binding to copper is calculated to play a major role in activating the reductive cleavage of the nitrite bond through backbonding combined with stabilization of the (-)OH product by coordination to the Cu(2+).

title: Resolution of the spectroscopy versus crystallography issue for NO intermediates of nitrite reductase from Rhodobacter sphaeroides.
abstract: Copper nitrite reductase (NiR) is a homotrimeric enzyme, containing a T1 copper site, which transfers electrons to the T2 catalytic site, where nitrite is reduced by one electron to nitric oxide (NO2− + 2H+ + e− → NO + H2O).1–3 Recently a side-on bound copper nitrosyl complex of NiR has been crystallized by reacting the reduced enzyme with excess NO.4 Based on the EPR spectrum of the species generated by the reaction of reduced NiR with saturated NO in solution, it has been assigned as an η 2-NO−Cu2+species. However, spectroscopic data on several Cu-nitrosyl model complexes5 and an NO adduct formed by reacting reduced NiR with nitrite6 and DFT calculations on an η2-NOCu species7 describe these complexes as Cu+NO•. In this study, we define the species generated in solution by reacting reduced NiR with NO, using EPR, MCD and ENDOR spectroscopy and correlate these with that observed by crystallography. 
 
Reduced NiR reacted with saturated NO solution generates the EPR spectrum in Figure 1A red, which is equivalent to the spectrum reported by Tocheva et al.4 This species has an EPR signal characteristic of an oxidized T2 Cu (g|| = 2.30 and A|| = 117 G). The MCD spectrum  has low energy d-d bands, also indicative of a T2 cupric complex. Both EPR and MCD spectra show a negligible T1 contribution indicating that this site is reduced . 
 
 
 
Figure 1 
 
Overlay of reduced NiR with excess NO (red) and nitrite bound NiR (blue) A) EPR B) MCD, C) N14 ENDOR, D) H1 ENDOR, with exchangeable features with 8.5 MHz coupling. Parts A and B have nitrite bound to T2 site with the T1 Cu reduced to remove intense overlaying ... 
 
 
 
Nitrite binds to the T2 site of the resting WT enzyme,8,9 but the EPR and, in particular, the MCD features of the nitrite bound T2 site are obscured by the dominant T1 Cu signals. However under these conditions (nitrite bound), the T1 Cu could be selectively reduced with acsorbate. The EPR spectrum of the nitrite bound T2 species  is identical to that produced above by reacting reduced NiR with excess NO. The MCD spectrum  of the T1 reduced T2-nitrite bound form is also identical to that generated by the reduced enzyme with NO. The T2 EPR spectrum and the MCD d-d bands of the resting WT enzyme are very sensitive to nitrite binding . Thus the EPR and MCD data strongly indicate that the species generated by the reaction of excess NO with reduced NiR has T1 reduced and T2 oxidized with nitrite bound. 
 
Further N14 and H1 ENDOR data for this species generated by the reaction of reduced NiR with an NO solution  and those for a form with T1+T2 oxidized and nitrite bound (collected at g=2.266 where there is no contribution from T1, Fig. 1C, D, blue) have the same features. The nitrogen features  from Cu2+NO2− bound T2 are from its histidines, and these features all show lower ENDOR frequencies and smaller hyperfine couplings due to the change of the T2 axial ligand from aquo to nitrite.10 
 
From the above results the reaction of reduced NiR with excess NO gives T1 reduced T2 Cu2+-NO2−. However, the reaction of the reduced enzyme with limited NO has an EPR signal , characteristic of a Cu+NO•  This species lacks d-d bands in the MCD spectrum , further confirming that the Cu is reduced. Therefore, reduced NiR forms a Cu+NO• species at low concentrations of NO. 
 
 
 
Figure 2 
 
EPR spectra A) following the reaction of reduced NiR with sub-stoichiometric NO solution (10:1 enzyme: NO) trapped at 1 second (green) 4 second (red) and 18 seconds (purple). B) following the reaction of the resting oxidized enzyme (black) with NO gas ... 
 
 
 
The reaction of reduced NiR with excess NO involves two one-electron oxidations; reduced T2 → oxidized T2, and NO → NO2−. A similar reaction has been observed for the coupled binuclear Cu active sites in hemocyanins11 (Scheme S1). Deoxyhemocyanin (2Cu+) reacts with NO generating N2O, and a met (2Cu2+) form, which further reacts with NO to generate the half-met (Cu2+Cu+) nitrite bound form. N2O evolution is in fact detected during the reaction of reduced NiR with excess NO, using nitrous oxide reductase (35 μmols N2O reduced min−1mg−1of enzyme), which requires N2O as its substrate.12 Reaction of the resting WT NiR (analogous to the met form of hemocyanin) with NO generates the T1 reduced, T2 oxidized form of the enzyme (g||=2.30, A||=150G), which converts to the nitrite bound T2 form , indicating NO is oxidized to nitrite and nitrite binding is slow. 
 
 
 
Scheme 1 
 
Proposed mechanism for the formation of nitrite bound T2 NiR from reduced enzyme with excess NO. Red boxes = species observed. 
 
 
 
To evaluate the entire reaction mechanism, reduced NiR was reacted with limited NO solutions and intermediates were trapped at different times. We were able to trap the Cu+NO• form at shorter times, leading to the formation of the T1 reduced, T2 oxidized form, and ultimately generating the T1 reduced, T2 oxidized nitrite bound form  at longer times. A reaction mechanism consistent with these results is presented in Scheme 1. Note that in the scheme, intermediate E is not observed because of rapid ET from T2 → T1.13 The experimental details and kinetic rates are provided in the supporting information. 
 
There is still one issue to resolve on correlating the spectroscopy with crystallography. The Cu-NO species trapped in the crystal structure has a side-on  DFT calculations indicate that this species is best described as a Cu+-NO• species.7 However an end-on Cu-NO species is also trapped after limited exposure to NO gas (B, in scheme 1) which is equivalent to the species generated by reacting reduced NiR with nitrite (η1-CuNO).6 Geometry optimized DFT calculations (B3LYP/6–311g* on Cu, N, O and 6–31g* on C, H)14 of both side-on  and end-on  Cu-NO adducts indicate that both forms have a Cu+NO• electronic configuration and that the end-on structure is more stable by 7 kcal/mol. However the orientation of the 2nd sphere aspartic acid residue (Asp129)9,15 appears to play a key role in determining the geometry of the Cu-NO species. In the side-on adduct this residue is perpendicular to the Cu-N-O plane and H-bonds to the O of N- O  while in the end-on structure the Asp129 residue H-bonds to the O of NO and is parallel to the Cu-N-O plane . Both orientations of the Asp129 residue have been observed in crystal structures.15 The Asp129 residue exists in the perpendicular orientation in the crystal structures of the oxidized and reduced NiR and H-bonds to the axial ligand. However, to form the more stable end-on Cu+NO• adduct this aspartate residue has to rotate 90° which can occur in solution but may not be feasible in a crystal. 
 
 
 
Figure 3 
 
Overlay of the DFT optimized side-on bound NO (red) and end-on bound NO (blue) structures indicating the different orientations of the aspartate residue. 
 
 
 
In summary, the species generated in solution during the reaction of reduced NiR with excess NO is the T1 reduced, T2 oxidized nitrite bound form and not the side-on Cu-NO species observed in the crystal structure. The latter is best described as Cu+NO• rather than Cu2+NO−. The mechanism for the reaction of reduced NiR with excess NO involves its two electron oxidation, generating N2O. This oxidized form of the enzyme oxidizes NO generating nitrite bound to the oxidized T2 site generated by ET from T2 → T1. DFT calculations provide insight into factors which favor the η2-NO•Cu+ complex in the crystal structure.

title: Thermodynamic equilibrium between blue and green copper sites and the role of the protein in controlling function
abstract: A combination of spectroscopies and density functional theory calculations indicate that there are large temperature-dependent absorption spectral changes present in green nitrite reductases (NiRs) due to a thermodynamic equilibrium between a green and a blue type 1 (T1) copper site. The axial methionine (Met) ligand is unconstrained in the oxidized NiRs, which results in an enthalpically favored (ΔH ≈4.6 kcal/mol) Met-bound green copper site at low temperatures, and an entropically favored (TΔS ≈4.5 kcal/mol, at room temperature) Met-elongated blue copper site at elevated temperatures. In contrast to the NiRs, the classic blue copper sites in plastocyanin and azurin show no temperature-dependent behavior, indicating that a single species is present at all temperatures. For these blue copper proteins, the polypeptide matrix opposes the gain in entropy that would be associated with the loss of the weak axial Met ligand at physiological temperatures by constraining its coordination to copper. The potential energy surfaces of Met binding indicate that it stabilizes the oxidized state more than the reduced state. This provides a mechanism to tune down the reduction potential of blue copper sites by >200 mV.

title: Spectroscopic, computational, and kinetic studies of the mu4-sulfide-bridged tetranuclear CuZ cluster in N2O reductase: pH effect on the edge ligand and its contribution to reactivity.
abstract: A combination of spectroscopy and density functional theory (DFT) calculations has been used to evaluate the pH effect at the CuZ site in Pseudomonas nautica (Pn) nitrous oxide reductase (N2OR) and Achromobacter cycloclastes (Ac) N2OR and its relevance to catalysis. Absorption, magnetic circular dichroism, and electron paramagnetic resonance with sulfur K-edge X-ray absorption spectra of the enzymes at high and low pH show minor changes. However, resonance Raman (rR) spectroscopy of PnN2OR at high pH shows that the 415 cm-1 Cu-S vibration (observed at low pH) shifts to higher frequency, loses intensity, and obtains a 9 cm-1 18O shift, implying significant Cu-O character, demonstrating the presence of a OH- ligand at the CuICuIV edge. From DFT calculations, protonation of either the OH- to H2O or the mu4-S2- to mu4-SH- would produce large spectral changes which are not observed. Alternatively, DFT calculations including a lysine residue at an H-bonding distance from the CuICuIV edge ligand show that the position of the OH- ligand depends on the protonation state of the lysine. This would change the coupling of the Cu-(OH) stretch with the Cu-S stretch, as observed in the rR spectrum. Thus, the observed pH effect (pKa approximately 9.2) likely reflects protonation equilibrium of the lysine residue, which would both raise E degrees and provide a proton for lowering the barrier for the N-O cleavage and for reduction of the [Cu4S(im)7OH]2+ to the fully reduced 4CuI active form for turnover.

title: Endotoxin-induced organ injury.
abstract: To review the effects of endotoxemia on the major organ systems of the body and discuss potential mechanisms of tissue injury.



Appraisal of 60 articles representing a cross section of studies relating to in vivo and in vitro responses to endotoxin.



Cell cultures, isolated tissue preparations, animal and human studies.



Endotoxemia results in the activation of numerous cellular and hematogenous mediators. These mediators range from prostaglandins, thromboxanes, and leukotrienes, to complement components. Tumor necrosis factor may be responsible for initiating many of the observed responses to endotoxin. Species and tissue specificity are a prominent feature of the response to endotoxin.



No single agent can yet be implicated as the common mediator of endotoxin-induced organ injury. Endotoxin initiates the elaboration of a cascade of secondary mediators that amplify the response to the initial insult. The relative importance of individual agents as mediators of the response to endotoxin varies with the experimental model studied.

title: Linkage mapping of quantitative trait loci in humans: an overview.
abstract: In this article, we provide an overview of the different statistical procedures that have been developed for linkage mapping of quantitative trait loci. We outline the model assumptions, the data requirements and the underlying tests for linkage for the different methods.

title: Comparison of electrical thresholds for evoking movements from sensori-motor areas of the cat cerebral cortex and its relation to motor training
abstract: Motor maps and electrical thresholds for evoking movements from motor areas of the cerebral cortex were evaluated in normal cats by using intracortical microstimulation techniques. Stainless steel chambers were implanted over craniotomies in adult cats trained to perform reaching and retrieval movements with their forelimbs. Prehensile motor training was continued and movement performance monitored for about 6–10 weeks during which the cortex was progressively explored with sharp tungsten electrodes inserted into cortical gyri (anterior and posterior sigmoid, and coronal) and the banks of sulci (cruciate, presylvian and coronal). Twice weekly, under light general anaesthesia, 3–4 tracks were made in either hemisphere till about 50 tracks were made in each hemisphere. Mean thresholds for evoking forelimb movements from different cytoarchitectonic areas (4γ, 4δ, 6aγ and 3a) were compared and no consistent or significant differences were observed between the different areas. In the animals (4/6) which used either forelimb to perform the tasks, there were no consistent differences in the mean thresholds for evoking forelimb movements from the two hemispheres. However, in 2 animals, which used their right forelimbs predominantly or exclusively to perform all the tasks, mean thresholds for evoking forelimb movements was significantly higher in areas 4γ and 6aγ of the left hemisphere (compared to the right); no consistent differences in the mean thresholds for evoking hindlimb or facial movements were observed between the two hemispheres. These findings suggest that ICMS thresholds for evoking forelimb movements may be similar in different sensorimotor areas of the cat cerebral cortex, and these thresholds could be influenced by motor training.

title: IKKbeta mediates cell shape-induced aromatase expression and estrogen biosynthesis in adipose stromal cells.
abstract: Aromatase (Cyp19) is a key enzyme in estrogen biosynthesis and an important target in breast cancer therapy. Within tumor microenvironment, tumor cells stimulate aromatase expression in adipose stromal cells (ASCs), which in turn promotes estrogen-dependent growth of estrogen receptor (ER)-positive tumor cells. However, it is not clear how aromatase transcription and estrogen biosynthesis are regulated in ASCs under a precancerous condition. Here we demonstrate that cell shape change alone is sufficient to induce aromatase expression in primary ASCs from cancer-free individuals. The activation of aromatase transcription is mediated by IkappaB kinase-beta (IKKbeta), a kinase previously known for its cancer-promoting activity in tumor cells. Activation of IKKbeta leads to elevated expression of transcription factor CCAAT/enhancer-binding protein-beta (C/EBPbeta), which binds to and stimulates two breast cancer-associated promoters of the aromatase gene. We also show that shape-induced estrogen production in ASCs can stimulate estrogen-dependent transcription in ER-positive breast tumor cells. We suggest that IKKbeta-dependent aromatase induction due to changes in cellular architecture in adipose tissue may contribute to the breast cancer risks associated with high mammagraphic density and obesity.

title: Study of properties of cholinephosphotransferase from fetal guinea pig lung mitochondria and microsomes
abstract: SummaryWe have reported earlier that cholinephosphotransferase (EC 2.7.8.2) is present in both mitochondria and microsomes of fetal guinea pig lung. This study was designed to compare the properties of mitochondrial and microsomal cholinephosphotransferase in fetal guinea pig lung. Various parameters, such as substrate specificity, Km values, sensitivity to N-ethylmaleimide, dithiothreitol and trypsin were measured. Both showed significant preference for unsaturated diacylglycerols over saturated diacylglycerols. Data on Km and Vmax indicate that the affinity of this enzyme for different diacylglycerols varies between the two forms. The ID50 values for N-ethylmaleimide were 20 mM and 12.5 mM for the mitochondrial and microsomal form of the enzyme, respectively. Dithiothreitol showed an inhibitory effect on both; however, the mitochondrial form was inhibited less than the microsomal form. The effects of N-ethylmaleimide and dithiothreitol on both forms of enzyme indicated that the microsomal cholinephosphotransferase requires a higher concentration of -SH for its activity than the mitochondrial enzyme does. The enzyme was inhibited by trypsin in both mitochondria and microsome under isotonic condition suggesting that this enzyme is on the outside of the membrane in both endoplasmic reticulum and mitochondria.