GATA3 expression in primary lung carcinomas: correlation with histopathologic features and TTF-1, napsin A, and p40 expression.

This study assessed the expression of GATA3 in primary lung carcinomas and correlated it with tumor histology and immunostains routinely utilized in the work up of primary lung cancers. Tissue microarrays (TMAs) were constructed from a cohort of 184 non-small cell carcinomas, stained with GATA3, p40, TTF-1, and napsin A, and analyzed semi-quantitatively. All TMA cases with GATA3 expression were further analyzed using corresponding whole slide sections. Positive GATA3 staining was present in 16 cases (9%), including 7 squamous cell carcinomas (SqCCs) (4%), 4 adenocarcinomas (AdCs) (2%), 2 adenosquamous carcinomas (AdSqCs) (1%), 2 large cell carcinomas (LCCs) (1%), and 1 sarcomatoid carcinoma (SC) (<1%). Among tumor histotypes, SqCC was more likely to stain with GATA3 (7/49, 14%), while AdC was less likely (4/111, 4%) (p = 0.04). In GATA3-positive cases, high-level expression was observed in 9 cases (56%), including 5 p40-positive SqCCs (3 were nonkeratinizing), 1 p40-positive AdSqC (negative for TTF-1 and napsin A), and 1 AdC (solid), 1 LCC, and 1 SC, each negative for p40, TTF-1, and napsin A). Low-level GATA3 expression was found in 3 AdCs (1 was lepidic and 2 were acinar predominant), 2 SqCCs (keratinizing), 1 AdSqC, and 1 LCC. These findings indicate that GATA3 expression occurs in a minor but significant proportion of primary non-small cell lung carcinomas, most often involves SqCC, and tends to show increasing levels of expression in more poorly differentiated subtypes. Caution should be exercised when interpreting GATA3 expression, and a panel of immunostains should be utilized when assigning tumor origin.

High-grade Neuroendocrine Carcinomas of the Vulva: A Clinicopathologic Study of 16 Cases.

Vulvar high-grade neuroendocrine carcinomas (HGNECs) are rare and primarily thought to represent Merkel cell carcinoma (MCC). We present the clinicopathologic features of 16 such cases, the largest series to date. Patients were most often White, postmenopausal, and symptomatic from a palpable vulvar mass/nodule. Tumors ranged from 0.7 to 6 cm and most commonly involved the labium majus. Majority of the cases were pure HGNECs (15/16) with small cell (SC) morphology (14/16); 2 were large cell neuroendocrine carcinomas, of which 1 was combined with moderately differentiated adenocarcinoma. All tumors expressed synaptophysin. Of the 14 HGNECs with SC morphology, 6 were CK20-positive MCCs with characteristic CAM5.2 and neurofilament (NF) expression. Five of these MCCs were positive for Merkel cell polyoma virus large T-antigen (MCPyVLTAg). In contrast, 6 HGNECs with SC morphology were negative for CK20, NF, and MCPyVLTAg and classified as SCNECs. High-risk human papilloma virus was positive in all SCNECs and negative in all MCCs. One case of HGNEC with SC morphology could not be entirely characterized due to lack of tissue for ancillary testing. Five of 12 (42%) cases had a discrepant diagnosis initially rendered. Most patients (10/15) presented with International Federation of Gynecology and Obstetrics stage III or IV disease. Usual sites of metastasis included inguinal lymph node, liver, bone, and lung. Twelve patients underwent surgery with adjuvant chemotherapy and/or radiation therapy, 1 received adjuvant immunotherapy, and 1 patient received neoadjuvant chemotherapy followed by surgery and adjuvant radiation therapy. Median overall survival was 24 months (range: 7 to 103 mo), and overall 5-year survival was 12% (95% confidence interval: 1% to 39%). In summary, vulvar HGNECs are rare, aggressive neoplasms that can be further subclassified into MCC, SCNEC, and large cell neuroendocrine carcinoma. CK20, CAM5.2, NF, TTF-1, MCPyVLTAg, and high-risk human papilloma virus facilitate the distinction of MCC from SCNEC. Proper identification of vulvar HGNECs is critical for patient management.

Histopathologic changes in cadaver eyes after MicroPulse and continuous wave transscleral cyclophotocoagulation.

OBJECTIVE: The purpose of this study was to compare the acute histological effects of MicroPulse transscleral cyclophotocoagulation (MPCPC) using the MicroPulse P3 Device and continuous wave transscleral cyclophotocoagulation (CWCPC) on the ciliary body and adjacent structures in human cadaver eyes. METHODS: Quadrants of 6 human cadaver eyes from 3 different donors were subjected to traditional CWCPC, slow burn CWCPC, MPCPC, or no treatment (internal control). Sutures were used to differentiate different treatment areas on each eye. Differential inking was applied after treatments to aid in microscopic correlation. All specimens were subject to standard histologic processing. Tissue sections were cut at 4 microns and stained with hematoxylin and eosin according to established protocols. Pathologic evaluation by light microscopy was confirmed by a senior pathologist blinded to treatment groups. RESULTS: In all 6 eyes, tissues treated with traditional and low burn CWCPC showed variable coagulative tissue damage to the ciliary body compared with untreated tissues. Minimal histologic changes were identified within the ciliary processes, although variable pigment clumping and streaming were noted within the pigmented ciliary epithelium. In contrast to CWCPC, MPCPC-treated tissues showed only minimal coagulative tissue damage to the ciliary body. Variable pigment clumping and streaming, however, were also noted in the pigmented ciliary epithelium in MPCPC-treated tissues. CONCLUSIONS: In human cadaver eyes, MPCPC treatment caused less tissue disruption to the ciliary body compared with traditional and low burn CWCPC treatments. MPCPC may be a less destructive and more selective method of cyclophotocoagulation when compared with traditional and low burn CWCPC.

Assessment of the intramolecular magnetic interactions in the highly saddled iron(iii) porphyrin π-radical cations: the change from planar to saddle conformations.

The intramolecular magnetic interactions in one-electron oxidized iron(iii) porphyrin π-radical cations, [Fe(OETPP˙)Cl][SbCl6] (1), [Fe(OMTPP˙)Cl][SbCl6] (2) and [Fe(TPP˙)Cl][SbCl6] (3), have been compared by means of X-ray crystallography, SQUID magnetometry, cyclic voltammetry, UV-Vis spectroelectrochemical analysis, NMR spectroscopy analysis and unrestricted DFT calculations. Unlike a generally recognized antiferromagnetic coupling dxy↑dxz↑dyz↑dz2↑dx2-y2↑P˙+(a2u)↓ (S = 2) state via a weak bonding interaction as in (3), we have disclosed that a strong bonding interaction among iron dx2-y2 and porphyrin a2u orbitals forms in (1) into a highly delocalized Ψπ = [P˙+(a2u) + FeIII(dx2-y2, dz2)] orbital that is able to accommodate two spin-paired electrons to form the Ψπ2dxy1dxz1dyz1, dz21 (S = 2) ground state. Concurrently, the spin polarization effect is exerted on the paired spins in the Ψπ orbital by magnetic induction from the remaining unpaired electrons in the iron d orbitals. The interpretation mentioned above is further verified by the diamagnetic nature of the saddled copper(ii) porphyrin π-cation radical, CuII(OETPP˙)(ClO4) (S = 0), where the strong bonding interaction leads to the Ψπ2dxy2dxz2dyz2dz22 (S = 0) ground state but no spin polarization exists. Thus, the magnetic nature of the iron(iii) porphyrin π-radical cation is tuneable by saddling the ring planarity.

A Mn(iv)-peroxo complex in the reactions with proton donors.

Protons play an important role in promoting O-O or M-O bond cleavage of metal-peroxo complexes. Treatment of side-on O2-bound [PPN][MnIV(TMSPS3)(O2)] (1, PPN = bis(triphenylphosphine)iminium and TMSPS3H3 = 2,2',2''-trimercapto-3,3',3''-tris(trimethylsilyl)triphenylphosphine) with perchloric acid (HClO4) in the presence of PR3 (R = phenyl or p-tolyl) results in the formation of neutral five-coordinate MnIII(OPR3)(TMSPS3) complexes (R = phenyl, 2a; p-tolyl, 2b), which are confirmed by X-ray crystallography. Isotope labelling experiments demonstrate that the oxygen atom in the phosphine oxide product derives from the peroxo ligand of 1. Reactions of 1 with weak proton donors, such as phenylthiol, phenol, substituted phenol and methanol, are also investigated to explore the reactivity of the MnIV-peroxo complex, leading to the isolation of a series of five-coordinate [MnIII(L)(TMSPS3)]- complexes (L = phenylthiolate, phenolate or methoxide). Mechanistic aspects of the reactions of the MnIV-peroxo complex with proton donors are discussed as well.

False-Positive Light Chain Clonal Restriction by Flow Cytometry in Patients Treated With Alemtuzumab: Potential Pitfalls for the Misdiagnosis of B-Cell Neoplasms.

Objectives: To increase awareness of potential diagnostic test interference associated with alemtuzumab, which is a therapeutic immunoglobulin G1 κ monoclonal antibody used in hematologic malignancies, autoimmune diseases, and transplant-related disorders. Methods: Bone marrow and blood from patients with T-cell prolymphocytic leukemia treated with alemtuzumab were evaluated by flow cytometry. Healthy donor blood was analyzed with or without in vitro treatment with alemtuzumab for comparison. Results: Immunophenotypic analysis of bone marrow collected 4 weeks after alemtuzumab treatment demonstrated artifactual surface κ light chain restriction in CD19+ B cells and CD3+ T cells. Similar findings were observed in blood from another patient in a specimen collected 3 days after alemtuzumab treatment. These findings were recapitulated in healthy donor blood incubated with alemtuzumab. Conclusions: Alemtuzumab can produce direct interference during flow cytometry analysis, resulting in false-positive evidence of light chain clonality. Clinicians and laboratorians should be cognizant of this risk to avoid misdiagnosis of B-cell neoplasms.

Vulvar Lesions in an 8-Year-Old Girl: Cutaneous Manifestations of Multisystem Langerhans Cell Histiocytosis.

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare localized or systemic disease characterized by proliferation of myeloid-derived dendritic cells. Vulvar lesions might be the herald symptom of LCH and might mimic other cutaneous lesions. Prognosis varies widely on the basis of the extent and spread of disease. CASE: An 8-year-old girl with a 4-month history of vulvar lesions resistant to topical steroids was referred by her pediatrician. Vulvar biopsy was diagnostic for LCH. Imaging studies revealed a left hip lesion consistent with LCH. The patient was subsequently treated for multisystem LCH with vinblastine and prednisone. SUMMARY AND CONCLUSION: Although rare, LCH might be diagnosed by gynecologic providers and should be included in the differential diagnosis of genital lesions. We recommend having a low threshold for performing biopsy of vulvar lesions.

Alkane Oxidation: Methane Monooxygenases, Related Enzymes, and Their Biomimetics.

Methane monooxygenases (MMOs) mediate the facile conversion of methane into methanol in methanotrophic bacteria with high efficiency under ambient conditions. Because the selective oxidation of methane is extremely challenging, there is considerable interest in understanding how these enzymes carry out this difficult chemistry. The impetus of these efforts is to learn from the microbes to develop a biomimetic catalyst to accomplish the same chemical transformation. Here, we review the progress made over the past two to three decades toward delineating the structures and functions of the catalytic sites in two MMOs: soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO). sMMO is a water-soluble three-component protein complex consisting of a hydroxylase with a nonheme diiron catalytic site; pMMO is a membrane-bound metalloenzyme with a unique tricopper cluster as the site of hydroxylation. The metal cluster in each of these MMOs harnesses O2 to functionalize the C-H bond using different chemistry. We highlight some of the common basic principles that they share. Finally, the development of functional models of the catalytic sites of MMOs is described. These efforts have culminated in the first successful biomimetic catalyst capable of efficient methane oxidation without overoxidation at room temperature.

The Mo-Mo Quintuple Bond as a Ligand to Stabilize Transition-Metal Complexes.

Herein, we report the employment of the Mo-Mo quintuple bonded amidinate complex to stabilize Group 10 metal fragments {(Et3P)2M} (M=Pd, Pt) and give rise to the isolation of the unprecedented δ complexes. X-ray analysis unambiguously revealed short contacts between Pd or Pt and two Mo atoms and a slight elongation of the Mo-Mo quintuple bond in these two compounds. Computational studies show donation of the Mo-Mo quintuple-bond δ electrons to an empty σ orbital on Pd or Pt, and back-donation from a filled Pd or Pt dπ orbital into the Mo-Mo δ* level (LUMO), consistent with the Dewar-Chatt-Duncanson model.

Copper-obatoclax derivative complexes mediate DNA cleavage and exhibit anti-cancer effects in hepatocellular carcinoma.

Obatoclax is an indole-pyrrole compound that induces cancer cell apoptosis through targeting the anti-apoptotic Bcl-2 protein family. Previously, we developed a series of obatoclax derivatives and studied their STAT3 inhibition-dependent activity against cancer cell lines. The obatoclax analog, prodigiosin, has been reported to mediate DNA cleavage in cancer cells by coordinating with copper complexes. To gain an understanding of copper-obatoclax complex activity, we applied obatoclax derivatives to examine their copper-mediated nuclease activity as a means to establish a basis for structure activity relationship. Replacement of the indole ring of obatoclax with furanyl, thiophenyl or Boc-indolyl rings reduced the DNA cleavage ability. The same effect was achieved through the replacement of the obatoclax pyrrolyl ring with thiazolidinedione and thioacetal. Among the compounds tested, we demonstrated that the complex of obatoclax or compound 7 with copper exhibited potent DNA strand scission which correlated with HCC cell growth inhibition.

The characterization of the saddle shaped nickel(III) porphyrin radical cation: an explicative NMR model for a ferromagnetically coupled metallo-porphyrin radical.

Ni(III)(OETPP˙)(Br)2 is the first Ni(III) porphyrin radical cation with structural and (1)H and (13)C paramagnetic NMR data for porphyrinate systems. Associating EPR and NMR analyses with DFT calculations as a new model is capable of clearly determining the dominant state from two controversial spin distributions in the ring to be the Ni(III) LS coupled with an a1u spin-up radical.

Myosin binding protein-C phosphorylation is the principal mediator of protein kinase A effects on thick filament structure in myocardium.

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a regulator of pump function in healthy hearts. However, the mechanisms of regulation by cAMP-dependent protein kinase (PKA)-mediated cMyBP-C phosphorylation have not been completely dissociated from other myofilament substrates for PKA, especially cardiac troponin I (cTnI). We have used synchrotron X-ray diffraction in skinned trabeculae to elucidate the roles of cMyBP-C and cTnI phosphorylation in myocardial inotropy and lusitropy. Myocardium in this study was isolated from four transgenic mouse lines in which the phosphorylation state of either cMyBP-C or cTnI was constitutively altered by site-specific mutagenesis. Analysis of peak intensities in X-ray diffraction patterns from trabeculae showed that cross-bridges are displaced similarly from the thick filament and toward actin (1) when both cMyBP-C and cTnI are phosphorylated, (2) when only cMyBP-C is phosphorylated, and (3) when cMyBP-C phosphorylation is mimicked by replacement with negative charge in its PKA sites. These findings suggest that phosphorylation of cMyBP-C relieves a constraint on cross-bridges, thereby increasing the proximity of myosin to binding sites on actin. Measurements of Ca(2+)-activated force in myocardium defined distinct molecular effects due to phosphorylation of cMyBP-C or co-phosphorylation with cTnI. Echocardiography revealed that mimicking the charge of cMyBP-C phosphorylation protects hearts from hypertrophy and systolic dysfunction that develops with constitutive dephosphorylation or genetic ablation, underscoring the importance of cMyBP-C phosphorylation for proper pump function.

Dissociation of structural and functional phenotypes in cardiac myosin-binding protein C conditional knockout mice.

BACKGROUND: Cardiac myosin-binding protein C (cMyBP-C) is a sarcomeric protein that dynamically regulates thick-filament structure and function. In constitutive cMyBP-C knockout (cMyBP-C(-/-)) mice, loss of cMyBP-C has been linked to left ventricular dilation, cardiac hypertrophy, and systolic and diastolic dysfunction, although the pathogenesis of these phenotypes remains unclear. METHODS AND RESULTS: We generated cMyBP-C conditional knockout (cMyBP-C-cKO) mice expressing floxed cMyBP-C alleles and a tamoxifen-inducible Cre-recombinase fused to 2 mutated estrogen receptors to study the onset and progression of structural and functional phenotypes caused by the loss of cMyBP-C. In adult cMyBP-C-cKO mice, knockdown of cMyBP-C over a 2-month period resulted in a corresponding impairment of diastolic function and a concomitant abbreviation of systolic ejection, although contractile function was largely preserved. No significant changes in cardiac structure or morphology were immediately evident; however, mild hypertrophy developed after near-complete knockdown of cMyBP-C. In response to pressure overload induced by transaortic constriction, cMyBP-C-cKO mice treated with tamoxifen also developed greater cardiac hypertrophy, left ventricular dilation, and reduced contractile function. CONCLUSIONS: These results indicate that myocardial dysfunction is largely caused by the removal of cMyBP-C and occurs before the onset of cytoarchitectural remodeling in tamoxifen-treated cMyBP-C-cKO myocardium. Moreover, near ablation of cMyBP-C in adult myocardium primarily leads to the development of hypertrophic cardiomyopathy in contrast to the dilated phenotype evident in cMyBP-C(-/-) mice, which highlights the importance of additional factors such as loading stress in determining the expression and progression of cMyBP-C-associated cardiomyopathy.

Dioxygen activation of a trinuclear Cu(I)Cu(I)Cu(I) cluster capable of mediating facile oxidation of organic substrates: competition between O-atom transfer and abortive intercomplex reduction.

The dioxygen activation of a series of Cu(I)Cu(I)Cu(I) complexes based on the ligands (L) 3,3'-(1,4-diazepane- 1,4-diyl)bis(1-{[2-(dimethylamino)ethyl](methyl)amino}propan-2-ol)(7-Me) or 3,3'-(1,4-diazepane-1,4-diyl)bis(1-{[2-(diethylamino)ethyl](ethyl)amino}propan-2-ol)(7-Et) forms an intermediate capable of mediating facile O-atom transfer to simple organic substrates at room temperature. To elucidate the dioxygen chemistry, we have examined the reactions of 7-Me, 7-Et, and 3,3'-(1,4-diazepane-1,4-diyl)bis[1-(4-methylpiperazin-1-yl)propan-2-ol] (7-N-Meppz) with dioxygen at -80, -55, and -35 °C in propionitrile (EtCN) by UV-visible, 77 K EPR, and X-ray absorption spectroscopy, and 7-N-Meppz and 7-Me with dioxygen at room temperature in acetonitrile (MeCN) by diode array spectrophotometry. At both -80 and -55 °C, the mixing of the starting [Cu(I)Cu(I)Cu(I)(L)](1+) complex (1) with O(2)-saturated propionitrile (EtCN) led to a bright green solution consisting of two paramagnetic species: the green dioxygen adduct [Cu(II)Cu(II)(µ-η(2):η(2)-peroxo)Cu(II)(L)](2+) (2) and the blue [Cu(II)Cu(II)(µ-O)Cu(II)(L)](2+) species (3). These observations are consistent with the initial formation of [Cu(II)Cu(II)(µ-O)(2)Cu(III)(L)](1+)(4), followed by rapid abortion of this highly reactive species by intercluster electron transfer from a second molecule of complex 1 to give the blue species 3 and subsequent oxygenation of the partially oxidized [Cu(II)Cu(I)Cu(I)(L)](2+)(5) to form the green dioxygen adduct 2. Assignment of 2 to [Cu(II)Cu(II)(µ-η(2):η(2)-peroxo)Cu(II)(L)](2+) is consistent with its reactivity with water to give H(2)O(2) and the blue species 3, as well as its propensity to be photoreduced in the X-ray beam during X-ray absorption experiments at room temperature. In light of these observations, the development of an oxidation catalyst based on the tricopper system requires consideration of the following design criteria: 1) rapid dioxygen chemistry; 2) facile O-atom transfer from the activated cluster to substrate; and 3) a suitable reductant to rapidly regenerate complex 1 to accomplish efficient catalytic turnover.

Protein kinase A-induced myofilament desensitization to Ca(2+) as a result of phosphorylation of cardiac myosin-binding protein C.

In skinned myocardium, cyclic AMP-dependent protein kinase A (PKA)-catalyzed phosphorylation of cardiac myosin-binding protein C (cMyBP-C) and cardiac troponin I (cTnI) is associated with a reduction in the Ca(2+) responsiveness of myofilaments and an acceleration in the kinetics of cross-bridge cycling, although the respective contribution of these two proteins remains controversial. To further examine the relative roles that cTnI and cMyBP-C phosphorylation play in altering myocardial function, we determined the Ca(2+) sensitivity of force (pCa(50)) and the activation dependence of the rate of force redevelopment (k(tr)) in control and PKA-treated mouse myocardium (isolated in the presence of 2,3-butanedione monoxime) expressing: (a) phosphorylatable cTnI and cMyBP-C (wild type [WT]), (b) phosphorylatable cTnI on a cMyBP-C-null background (cMyBP-C(-/-)), (c) nonphosphorylatable cTnI with serines(23/24/43/45) and threonine(144) mutated to alanines (cTnI(Ala5)), and (d) nonphosphorylatable cTnI on a cMyBP-C-null background (cTnI(Ala5)/cMyBP-C(-/-)). Here, PKA treatment decreased pCa(50) in WT, cTnI(Ala5), and cMyBP-C(-/-) myocardium by 0.13, 0.08, and 0.09 pCa units, respectively, but had no effect in cTnI(Ala5)/cMyBP-C(-/-) myocardium. In WT and cTnI(Ala5) myocardium, PKA treatment also increased k(tr) at submaximal levels of activation; however, PKA treatment did not have an effect on k(tr) in cMyBP-C(-/-) or cTnI(Ala5)/cMyBP-C(-/-) myocardium. In addition, reconstitution of cTnI(Ala5)/cMyBP-C(-/-) myocardium with recombinant cMyBP-C restored the effects of PKA treatment on pCa(50) and k(tr) reported in cTnI(Ala5) myocardium. Collectively, these results indicate that the attenuation in myofilament force response to PKA occurs as a result of both cTnI and cMyBP-C phosphorylation, and that the reduction in pCa(50) mediated by cMyBP-C phosphorylation most likely arises from an accelerated cross-bridge cycling kinetics partly as a result of an increased rate constant of cross-bridge detachment.

Transmural variation in myosin heavy chain isoform expression modulates the timing of myocardial force generation in porcine left ventricle.

Recent studies have shown that the sequence and timing of mechanical activation of myocardium vary across the ventricular wall. However, the contributions of variable expression of myofilament protein isoforms in mediating the timing of myocardial activation in ventricular systole are not well understood. To assess the functional consequences of transmural differences in myofilament protein expression, we studied the dynamic mechanical properties of multicellular skinned preparations isolated from the sub-endocardial and sub-epicardial regions of the porcine ventricular midwall. Compared to endocardial fibres, epicardial fibres exhibited significantly faster rates of stretch activation and force redevelopment (k(tr)), although the amount of force produced at a given [Ca2+] was not significantly different. Consistent with these results, SDS-PAGE analysis revealed significantly elevated expression of alpha myosin heavy chain (MHC) isoform in epicardial fibres (13 +/- 1%) versus endocardial fibres (3 +/- 1%). Linear regression analysis revealed that the apparent rates of delayed force development and force decay following stretch correlated with MHC isoform expression (r2 = 0.80 and r2 = 0.73, respectively, P < 0.05). No differences in the relative abundance or phosphorylation status of other myofilament proteins were detected. These data show that transmural differences in MHC isoform expression contribute to regional differences in dynamic mechanical function of porcine left ventricles, which in turn modulate the timing of force generation across the ventricular wall and work production during systole.

Facile O-atom insertion into C-C and C-H bonds by a trinuclear copper complex designed to harness a singlet oxene.

Two trinuclear copper [Cu(I)Cu(I)Cu(I)(L)](1+) complexes have been prepared with the multidentate ligands (L) 3,3'-(1,4-diazepane-1,4-diyl)bis(1-((2-(dimethylamino)ethyl)(methyl)amino)propan-2-ol) (7-Me) and (3,3'-(1,4-diazepane-1,4-diyl)bis(1-((2-(diethylamino) ethyl)(ethyl) amino)propan-2-ol) (7-Et) as models for the active site of the particulate methane monooxygenase (pMMO). The ligands were designed to form the proper spatial and electronic geometry to harness a "singlet oxene," according to the mechanism previously suggested by our laboratory. Consistent with the design strategy, both [Cu(I)Cu(I)Cu(I)(L)](1+) reacted with dioxygen to form a putative bis(mu(3)-oxo)Cu(II)Cu(II)Cu(III) species, capable of facile O-atom insertion across the central C-C bond of benzil and 2,3-butanedione at ambient temperature and pressure. These complexes also catalyze facile O-atom transfer to the C-H bond of CH(3)CN to form glycolonitrile. These results, together with our recent biochemical studies on pMMO, provide support for our hypothesis that the hydroxylation site of pMMO contains a trinuclear copper cluster that mediates C-H bond activation by a singlet oxene mechanism.

Theoretical modeling of the hydroxylation of methane as mediated by the particulate methane monooxygenase.

We present here the results of density functional theory (DFT) calculations directed toward elucidation of the CH bond activation mechanism that might be adopted by the particulate methane monooxygenase (pMMO) in the hydroxylation of methane and related small alkanes. In these calculations, we considered three of the most probable models for the transition metal active site mediating the "oxo-transfer": (i) the trinuclear copper cluster bis(mu(3)-oxo)trinuclear copper(II, II, III) complex 1, recently proposed by Chan et al. [S.I. Chan, K.H.-C. Chen, S.S.-F. Yu, C.-L. Chen, S.S.-J. Kuo, Biochemistry 43 (2004) 4421-4430.]; (ii) the most frequently used model complex, bis(mu-oxo)Cu(III)(2) complex 2; and (iii) the mixed-valence bis(mu-oxo)Cu(II)Cu(III) complex 3. The results obtained indicate that the methane hydroxylation chemistry mediated by the trinuclear copper cluster bis(mu(3)-oxo)trinuclear copper(II, II, III) complex 1 offers the most facile pathway for methane hydroxylation, and this model yields KIE values that are in good agreement with experiment. In this mechanism, the reaction proceeds along a "singlet" potential surface and a "singlet oxene" is directly inserted across a CH bond in a concerted manner. Kinetic isotope effects (k(H)/k(D) or KIE) associated with the concerted oxene insertion process mediated by complex 1 are calculated to be 5.2 at 300K when tunneling effects are included. Overall rate constants for the methane hydroxylation by the three models have been calculated as a function of temperature, and the rates are at least 5-6 orders of magnitude more facile when the chemistry is mediated by complex 1 compared to complex 2 or complex 3.