Thermodynamic equilibrium between blue and green copper sites and the role of the protein in controlling function
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: 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: 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: 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, 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: Management of Crohn’s disease from efficacy, quality of life and health economic perspectives
abstract: The expectations of the physician and patient in the modern management of Crohn’s disease have moved on from alleviation of symptoms whenever relapses occur, to sustained maintenance of remission and mucosal healing. The majority of treatment costs are as a consequence of hospitalizations and surgery. These heightened therapeutic goals are leading to expectations that management costs will decrease. Health-related quality of life can be measured by validated disease-specific tools in Crohn’s disease, and such measurements are now forming common outcome measures in clinical trials of new therapies in Crohn’s disease. This review gives an overview of patient-focused management of Crohn’s disease and the rapid evolution in therapy, with pharmacoeconomic analysis where available.

title: Fatal Outcome Despite Full Lympho-Hematopoietic Reconstitution After Allogeneic Stem Cell Transplantation in Atypical Ataxia Telangiectasia
abstract: Allogeneic hematopoietic stem cell transplantation (HSCT) has not been a therapeutic option in ataxia telangiectasia (AT) due to overwhelming toxicity of conditioning in the context of the global DNA repair deficiency. Furthermore HSCT is unable to cure neurological involvement of AT. We report on a Turkish child with a Hyper IgM phenotype disorder, in which clinical aspects of AT were absent and thus, AT not diagnosed. He was transplanted with a reduced toxicity, but full intensity conditioning regimen comprising treosulfan, fludarabine and ATG. The peritransplant period was uneventful and the patient was discharged at day +57. 8 months after HSCT, the patient developed hepatopathy with monoclonal gammopathy of unclear significance and died due to hepatic failure and encephalopathy at the age of 32 months. Post mortem high throughput sequencing revealed a mutation in the ATM gene.

title: Regeneration of lower and upper jaws in urodeles is differentially affected by retinoic acid.
abstract: The vitamin A derivative retinoic acid (RA) is a powerful teratogen which can induce severe craniofacial and limb malformations if administered at certain stages of gestation. In addition this compound has been shown to affect patterning in regenerating systems. A classical example is the induction of supernumerary structures along the proximodistal axis of the regenerating amphibian limb. We have investigated the effect of RA on other regenerating systems, the amphibian lower and upper jaws, both in developing and adult animals. We report here that RA does not induce formation of extra structures either in the lower or in the upper jaw of adult newts under experimental conditions where duplications of the regenerating limb occur. However, RA selectively induces severe malformations in the upper jaw regenerate that resemble those induced in avian and mammalian embryos. Analysis of the expression of the newt retinoic acid receptors RAR alpha and delta in upper and lower jaws showed that RAR alpha was expressed at a significant level in the wound epidermis, but not in blastemal cells, whereas no RAR delta could be detected in the regenerate either by in situ hybridization or by using an anti-RAR delta antibody. Therefore, unlike in the limb, in jaws RAR delta is not up-regulated following amputation, and this difference in expression may be causally related to the different effects induced by RA on jaws and limbs. In order to establish whether retinoids affected regeneration of developing jaws in a similar fashion, their effects were studied in animals whose jaws had been amputated at different developmental stages. Under the experimental conditions used overall growth retardation and head defects were observed in the majority of embryos which had been amputated and treated with retinol palmitate (RP) between stages 26-28 and 38-39. In contrast, patterning of upper jaw regenerates in larvae amputated at stage 26-28 and 38-39. In contrast, patterning of upper jaw regenerates in larvae amputated at stage 45 was not significantly affected by the treatment, although the early phase of regeneration was slower than in controls. The different responses to retinoids of regenerating facial structures in embryos, larvae and adults will be discussed.

title: Histone-like protein HU from Deinococcus radiodurans binds preferentially to four-way DNA junctions.
abstract: The histone-like protein HU from Escherichia coli is involved in DNA compaction and in processes such as DNA repair and recombination. Its participation in these events is reflected in its ability to bend DNA and in its preferred binding to DNA junctions and DNA with single-strand breaks. Deinococcus radiodurans is unique in its ability to reconstitute its genome from double strand breaks incurred after exposure to ionizing radiation. Using electrophoretic mobility shift assays (EMSA), we show that D.radiodurans HU (DrHU) binds preferentially only to DNA junctions, with half-maximal saturation of 18 nM. In distinct contrast to E.coli HU, DrHU does not exhibit a marked preference for DNA with nicks or gaps compared to perfect duplex DNA, nor is it able to mediate circularization of linear duplex DNA. These unexpected properties identify DrHU as the first member of the HU protein family not to serve an architectural role and point to its potential participation in DNA recombination events. Our data also point to a mechanism whereby differential target site selection by HU proteins is achieved and suggest that the substrate specificity of HU proteins should be expected to vary as a consequence of their individual capacity for inducing the required DNA bend.

title: Domain specific association of small fluorescent probe trans-3-(4-monomethylaminophenyl)-acrylonitrile (MMAPA) with bovine serum albumin (BSA) and its dissociation from protein binding sites by Ag nanoparticles: spectroscopic and molecular docking study.
abstract: Photoinduced intramolecular charge transfer produced a polar excited state in trans-3-(4-monomethylaminophenyl)acrylonitrile (MMAPA), rendering the resulting emission sensitive to the medium polarity. Strong binding interaction of silver nanoparticles with the probe was observed, causing fluorescence quenching through the static quenching process. The probe MMAPA was found to bind to the less polar hydrophobic, restricted proteinous environment of bovine serum albumin (BSA) resulting in the blue shift of the emission maximum with an increase in emission intensity and fluorescence anisotropy. Studies using site markers of flufenamic acid and phenylbutazone coupled with molecular docking results predicted that the binding site of the probe is in between subdomains IIIA and IB of BSA and is different from the conventional Sudlow sites. The denaturation of the probe-bound BSA by urea or heat released the probe from this proteinous environment to water marked by exactly reverse spectral changes. On the interaction of silver nanoparticles with the probe bound protein, the probe was observed to move from its binding site in the protein to the Ag(0) nanoparticle surface involving conformational changes of the protein near the probe binding site.