Long-term analysis of humoral responses and spike-specific T cell memory to Omicron variants after different COVID-19 vaccine regimens.

Background: The emergence of SARS-CoV-2 variants has raised concerns about the sustainability of vaccine-induced immunity. Little is known about the long-term humoral responses and spike-specific T cell memory to Omicron variants, with specific attention to BA.4/5, BQ.1.1, and XBB.1. Methods: We assessed immune responses in 50 uninfected individuals who received varying three-dose vaccination combinations (2X AstraZeneca + 1X Moderna, 1X AstraZeneca + 2X Moderna, and 3X Moderna) against wild-type (WT) and Omicron variants at eight months post-vaccination. The serum antibody titers were analyzed by enzyme-linked immunosorbent assays (ELISA), and neutralizing activities were examined by pseudovirus and infectious SARS-CoV-2 neutralization assays. T cell reactivities and their memory phenotypes were determined by flow cytometry. Results: We found that RBD-specific antibody titers, neutralizing activities, and CD4+ T cell reactivities were reduced against Omicron variants compared to WT. In contrast, CD8+ T cell responses, central memory, effector memory, and CD45RA+ effector memory T cells remained unaffected upon stimulation with the Omicron peptide pool. Notably, CD4+ effector memory T cells even exhibited a higher proportion of reactivity against Omicron variants. Furthermore, participants who received three doses of the Moderna showed a more robust response regarding neutralization and CD8+ T cell reactions than other three-dose vaccination groups. Conclusion: Reduction of humoral and CD4+ T cell responses against Omicron variants in vaccinees suggested that vaccine effectiveness after eight months may not have sufficient protection against the new emerging variants, which provides valuable information for future vaccination strategies such as receiving BA.4/5 or XBB.1-based bivalent vaccines.

[Pollution Characteristics and Health Risk Assessment of Heavy Metals in the Water of Lijiang River Basin].

In order to clarify the pollution levels of heavy metals in the drinking water sources of the Lijiang River Basin, surface water samples were collected from 62 sites throughout the Lijiang River during May 2019. Heavy metals, including As, Cd, Cr, Mn, Cu, Zn, Hg, Co, and Sb, in the water samples were analysed. Health risk assessments associated with these nine heavy metals were conducted using the health risk assessment model from the US EPA. The results indicated that the order of the average concentrations of heavy metals in the water samples were Mn > Zn > As > Cr > Cu > Sb > Co > Cd > Hg. No heavy metals exceeded the limit values of the drinking water health standards in China (GB 5749-2006), and the concentrations were lower than the limitations of Grade Ⅰ level in the environmental quality standards for surface water (GB 3838-2002). According to the spatial distribution, the high contents areas of As, Cr, Zn, and Sb were predominantly distributed downstream of the Lijiang River, while the high contents areas of Cd, Cu, Hg, Co, and Mn were mostly distributed in the upper reaches. Multivariate analysis indicated that Cd, Mn, Cu, and Co were primarily from agricultural production; Cr, Zn, and Sb were mainly from tourism transportation; As was predominantly from the weathering of rock parent material and soil erosion; Hg was mainly from the improper disposal of domestic garbage and atmospheric deposition. The results of the health risk assessment indicated that children were more susceptible to the threat of heavy metal pollution than adults, and the average annual risk of carcinogenic heavy metals to human health through drinking water ingestion were higher than those of non-carcinogenic metals. The maximum personal average annual health risk of Cr was higher than the maximum allowance levels recommended by the International Commission on Radiological Protection (5×10-5 a-1). The average annual risk of non-carcinogenic heavy metals (10-14-10-9 a-1) decreased in the order of Co > Cu > Hg > Zn > Sb > Mn, which were far below the maximum allowance levels recommended by the ICRP.

Long Non-Coding RNA NEAT1 Regulates Pyroptosis in Diabetic Nephropathy via Mediating the miR-34c/NLRP3 Axis.

INTRODUCTION: Diabetic nephropathy (DN) is a serious complication of diabetes mellitus and is considered to be a sterile inflammatory disease. Increasing evidence suggest that pyroptosis and subsequent inflammatory response play a key role in the pathogenesis of DN. However, the underlying cellular and molecular mechanisms responsible for pyroptosis in DN are largely unknown. METHODS: The rat models of DN were successfully established by single 65 mg/kg streptozotocin treatment. Glomerular mesangial cells were exposed to 30 mmol/L high glucose media for 48 h to mimic the DN environment in vitro. Gene and protein expressions were determined by quantitative real-time PCR and Western blot. Cell viability and pyroptosis were measured by MTT assay and flow cytometry analysis, respectively. The relationship between lncRNA NEAT1, miR-34c, and Nod-like receptor protein-3 (NLRP3) was confirmed by luciferase reporter assay. RESULTS: We found that upregulation of NEAT1 was associated with the increase of pyroptosis in DN models. miR-34c, as a target gene of NEAT1, mediated the effect of NEAT1 on pyroptosis in DN by regulating the expression of NLRP3 as well as the expressions of caspase-1 and interleukin-1ß. Either miR-34c inhibition or NLRP3 overexpression could reverse the accentuation of pyroptosis and inflammation by sh-NEAT1 transfection in the in vitro model of DN. CONCLUSIONS: Our findings suggested NEAT1 and its target gene miR-34c regulated cell pyroptosis via mediating NLRP3 in DN, providing new insights into understanding the molecular mechanisms of pyroptosis in the pathogenesis of DN.

CDK4/6 inhibition versus mTOR blockade as second-line strategy in postmenopausal patients with hormone receptor-positive advanced breast cancer: A network meta-analysis.

BACKGROUND: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors (palbociclib and abemaciclib) and mammalian target of rapamycin (mTOR) inhibitors (everolimus) are effective agents for restoring endocrine sensitivity in patients with advanced breast cancer progression on prior aromatase inhibitors. We conducted a network meta-analysis to compare these treatments in terms of progression-free survival (PFS), objective response rate (ORR), and clinical benefit rate (CBR). METHODS: The PubMed and Embase databases were searched for relevant publications between January 2000 and June 2018. Treatments were ranked based on a network meta-analysis. Ranking was determined by P-score. A random-effect model was used when heterogeneity was detected; otherwise, a fixed-effect model was used. RESULTS: Six trials comprising 4063 patients formed the comparison network. Compared with everolimus plus exemestane, the combinations of palbociclib or abemaciclib with fulvestrant showed similar efficacies in PFS and no differences in ORR. For the CBR, palbociclib demonstrated improvement, while abemaciclib did not. Incidences of severe adverse events did not significantly differ. A total of 29%, 15.9%, and 4% of patients discontinued everolimus, abemaciclib, and palbociclib, respectively, due to toxicity. CONCLUSION: These results suggest similar efficacies between CDK4/6 inhibition and mTOR blockade; however, CDK4/6 inhibitors were associated with favorable toxicity profiles.

Association of increased urine brain derived neurotrophic factor with lower urinary tract symptoms in men with benign prostatic hyperplasia.

Urinary brain-derived neurotrophic factor (BDNF), an ubiquitous neurotrophin, was found to rise in patients with benign prostatic hyperplasia (BPH). We hypothesized that the urinary level of BDNF could be a potential biomarker for lower urinary tract symptoms (LUTS) in patients with BPH. Totally, 76 patients with BPH-caused LUTS and 32 male control subjects without BPH were enrolled. International Prostate Symptom Score (IPSS) was applied to assess the symptom severity of LUTS. Urodynamic tests were performed for the diagnosis of underlying detrusor overactivity (DO) in the patients with BPH. Urine samples were collected from all subjects. Urinary BDNF levels were measured using enzyme-linked immunosorbent assays and normalized by urinary creatinine (Cr) levels. Seventy-six BPH patients were divided into moderate LUTS group (n=51, 720) according to the IPSS. Of the 76 BPH patients, DO was present in 34 (44.7%) according to the urodynamic test. The urinary BDNF/Cr levels were significantly higher in BPH patients with moderate LUTS (8.29±3.635, P<0.0001) and severe LUTS (11.8±6.44, P<0.0001) than normal controls (1.71±0.555). Patients with severe LUTS tended to have higher urinary BDNF/Cr levels than patients with moderate LUTS (11.8±6.44 vs. 8.29±3.635, P=0.000). The conditions of BPH with LUTS correlated with elevated urinary BDNF levels, and urinary BDNF levels were even higher in BPH-DO patients. The results of this study have provided evidence to suggest that urinary BDNF level test could evaluate the severity of LUTS in BPH patients, and BDNF level can be used as a biomarker for the diagnosis of DO in BPH patients.

Association of Increased Urine Brain Derived Neurotrophic Factor with Lower Urinary Tract Symptoms in Men with Benign Prostatic Hyperplasia / 华中科技大学学报（医学）（英德文版）

Urinary brain-derived neurotrophic factor (BDNF),an ubiquitous neurotrophin,was found to rise in patients with benign prostatic hyperplasia (BPH).We hypothesized that the urinary level of BDNF could be a potential biomarker for lower urinary tract symptoms (LUTS) in patients with BPH.Totally,76 patients with BPH-caused LUTS and 32 male control subjects without BPH were enrolled.International Prostate Symptom Score (IPSS) was applied to assess the symptom severity of LUTS.Urodynamic tests were performed for the diagnosis of underlying detrusor overactivity (DO) in the patients with BPH.Urine samples were collected from all subjects.Urinary BDNF levels were measured using enzyme-linked immunosorbent assays and normalized by urinary creatinine (Cr) levels.Seventy-six BPH patients were divided into moderate LUTS group (n=51,7＜IPSS ≤ 20) and severe LUTS group (n=25,IPSS＞20) according to the IPSS.Of the 76 BPH patients,DO was present in 34 (44.7％)according to the urodynamic test.The urinary BDNF/Cr levels were significantly higher in BPH patients with moderate LUTS (8.29±3.635,P＜0.0001) and severe LUTS (11.8±6.44,P＜0.0001) than normal controls (1.71±0.555).Patients with severe LUTS tended to have higher urinary BDNF/Cr levels than patients with moderate LUTS (11.8±6.44 vs.8.29±3.635,P=0.000).The conditions of BPH with LUTS correlated with elevated urinary BDNF levels,and urinary BDNF levels were even higher in BPH-DO patients.The results of this study have provided evidence to suggest that urinary BDNF level test could evaluate the severity of LUTS in BPH patients,and BDNF level can be used as a biornarker for the diagnosis of DO in BPH patients.

Overweight, Obesity, and Risk of Age-Related Macular Degeneration.

PURPOSE: The aim of this study was to quantify the relationship between categories of body mass index (BMI) and age-related macular degeneration (AMD) risk in different stages. METHODS: MEDLINE, EMBASE, and ISI Web of Science were searched for all eligible studies on the relationship between BMI and incident early or late AMD. The analyses were based on data extracted from study reports. The pooled relative risks (RRs) with 95% confidence intervals (CIs) were calculated to evaluate the strength of this association, and dose-response relationship was assessed by restricted cubic spline. RESULTS: Seven prospective cohort studies with 1613 cases identified among 31,151 subjects were included. For overweight, the relationship remained insignificant for its association with both early AMD (RR = 0.92, 95% CI: 0.68-1.15; P = 0.54) and late AMD (RR = 1.09, 95% CI: 0.93-1.25; P = 0.18). A marked 32% increase in the risk of developing late AMD was noted among obese individuals (RR = 1.32, 95% CI: 1.11-1.53, P < 0.01), while obesity showed no significant association with early AMD (RR = 0.91, 95% CI: 0.74-1.08; P = 0.67). Furthermore, elevated BMI showed a linear dose-response relation with AMD risk (Pnonlinearity = 0.17), and the AMD risk increased by 2% (RR = 1.02, 95% CI: 1.01-1.04) for each 1 kg/m2 increase in BMI within the overweight and obese BMI ranges. CONCLUSIONS: Excess body weight was weakly associated with increase in the risk of AMD in a dose-dependent fashion, especially for its late stage, indicating that keeping normal body weight and avoiding further weight gain may confer potential protection against this disease.

[Modified urethral pull-through procedure for posterior urethral stricture or atresia].

OBJECTIVE: To evaluate the feasibility and safety of the modified urethral pull-through procedure for the treatment of posterior urethral stricture or atresia. METHODS: We retrospectively analyzed 212 cases of posterior urethral stricture or atresia treated by the modified urethral pull-through procedure. The length of the stricture or atresia was 1.5 - 12 cm, and 66 cases had experienced 1 - 4 previous unsuccessful urethral repairs. Simple transperineal approach was adopted in 208 cases and transperineal-inferiorpubic approach in the other 4. And 15 of the patients underwent urethral construction with grafts. RESULTS: Satisfactory voiding was achieved in 198 (93.4%) of the patients, of whom 16 received 3 - 15 urethral dilations. Of the 14 cases that failed, 10 succeeded after a second and 2 after a third operation. Of the 15 cases that underwent substitution urethroplasty, 14 achieved satisfactory voiding, and only 1 needed repeat dilation. No serious complications were observed in any of the patients. CONCLUSION: Modified urethral pull-through procedure, with its advantages of safety, mini-invasiveness, simple operation and high success rate, is feasible for the treatment of posterior urethral stricture or atresia, while for that with the length >5 cm, substitution urethroplasty should be considered.

(4-Chloro-2-{[(pyridin-2-ylmeth-yl)imino]-meth-yl}phenolato)iodido(methanol)zinc(II).

The title Schiff base zinc(II) complex, [Zn(C(13)H(10)ClN(2)O)I(CH(3)OH)], was synthesized by the reaction of 5-chloro-salicyl-aldehyde, 2-amino-methyl-pyridine and zinc iodide in methanol. The Zn(II) atom is five-coordinated by one phenolate O atom, one imine and one pyridine N atom of the Schiff base ligand, one methanol O atom and one I atom, forming a distorted square-pyramidal geometry, with the I atom at the apical site. The dihedral angle between the benzene and pyridine rings is 22.9 (2)°. In the crystal, centrosymmetrically related mol-ecules are linked through inter-molecular O-H⋯O hydrogen bonds, forming dimers.

N'-(2,4-Dichloro-benzyl-idene)-4-hy-droxy-benzohydrazide.

The title hydrazone compound, C(14)H(10)Cl(2)N(2)O(2), was synthesized by the reaction of 2,4-dichloro-benzaldehyde and 4-hy-droxy-benzohydrazide. The mol-ecule adopts an E geometry with respect to the azomethine group and the dihedral angle between the aromatic rings is 7.0 (2)°. In the crystal, mol-ecules are linked through inter-molecular N-H⋯Cl and O-H⋯O hydrogen bonds, forming a three-dimensional network.

[Cytokine in synovial monocytic cells from rheumatoid arthritis patients].

OBJECTIVE: To evaluate the inhibitory effect of blockade of Rho kinase upon mediating the secretion of proinflammatory cytokine in monocytic cells from rheumatoid arthritis (RA) patients. METHODS: Synovial fluid (SF) monocytic cells and peripheral blood monocytes (PB) from active RA patients were treated with TNFalpha or LPS respectively in the presence or absence of a specific ROK inhibitor, Y27632. ROK activity was assessed by Western blot and cytokine secretion measured by ELISA. RESULTS: Elevated ROK activity was found in synovial fluid monocytic cells from active RA patients. ROK activity was correlated with DAS, an index of disease activity of RA patients. ROK inhibitor Y27632 reduced the secretion of TNFalpha, IL-1beta and IL-6 in RA SF monocytic cells, but had no effect upon the secretion of IL-10, an anti-inflammatory cytokine. CONCLUSION: The present study provides novel evidence that ROK mediates the secretion of proinflammatory cytokines in monocytic cells from RA synovial fluids, suggesting a critical role of ROK in macrophage-mediated synovial inflammation of RA. Thus inhibition of ROK may be a new therapeutic target for RA.

[Expression of lymphotactin in renal tuberculosis].

AIM: To study the expression of lymphotactin in normal kidney and renal tuberculosis and the arrangement of lymphotactin and infiltrating CD4(+) T cells and CD8(+) T cells in renal tuberculosis. METHODS: HLptn cDNA of tissues from six cases with normal kidneys and ten cases with tuberculous kidneys was amplified by RT-PCR. The RT-PCR products were separated with 2% of gel. The cDNA was cloned to vector pGM-T Easy and was completely sequenced. The immunohistochemical staining method was used to examine the expression of lymphotactin in normal kidneys and tuberculous kidneys and the expression of CD4 and CD8 in tuberculous kidneys. RESULTS: The sequence of cloned hLptn cDNA was confirmed and it was identical with the sequence of NO.U23772 published in GenBank. Both normal kidneys and tuberculous kidneys expressed hLptn mRNA. HLptn was detected not only in the cells of normal renal glomerulus and renal tubule but also in the cells of remaining renal glomerulus and renal tubule of tuberculous kidneys. The cells expressing surface antigens CD4 and CD8 scattered in granulomas. CONCLUSION: The constructive expression of hLptn is in the cells of renal glomerulus and renal tubule of normal kidney and tuberculous kidney. The accumulation of CD4(+) T cells and CD8(+) T cells in granulomas may not depend on hLptn.

Further insights into the spectroscopic properties, electronic structure, and kinetics of formation of the heme-peroxo-copper complex [(F8TPP)FeIII-(O2(2-)-CuII(TMPA)]+.

In the further development and understanding of heme-copper O2-reduction chemistry inspired by the active-site chemistry in cytochrome c oxidase, we describe a dioxygen adduct, [(F8TPP)FeIII-(O22-)-CuII(TMPA)](ClO4) (3), formed by addition of O2 to a 1:1 mixture of the porphyrinate-iron(II) complex (F8TPP)FeII (1a) {F8TPP = tetrakis(2,6-difluorophenyl)porphyrinate dianion} and the copper(I) complex [(TMPA)CuI(MeCN)](ClO4) (1b) {TMPA = tris(2-pyridylmethyl)amine}. Complex 3 forms in preference to heme-only or copper-only binuclear products, is remarkably stable {t1/2 (RT; MeCN) approximately 20 min; lambda max = 412 (Soret), 558 nm; EPR silent}, and is formulated as a peroxo complex on the basis of manometry {1a/1b/O2 = 1:1:1}, MALDI-TOF mass spectrometry {16O2, m/z 1239 [(3 + MeCN)+]; 18O2, m/z 1243}, and resonance Raman spectroscopy {nu(O-O) = 808 cm-1; Delta16O2/18O2 = 46 cm-1; Delta16O2/16/18O2 = 23 cm-1}. Consistent with a mu-eta2:eta1 bridging peroxide ligand, two metal-O stretching frequencies are observed {nu(Fe-O) = 533 cm-1, nu(Fe-O-Cu) = 511 cm-1}, and supporting normal coordinate analysis is presented. 2H and 19F NMR spectroscopies reveal that 3 is high-spin {also muB = 5.1 +/- 0.2, Evans method} with downfield-shifted pyrrole and upfield-shifted TMPA resonances, similar to the pattern observed for the structurally characterized mu-oxo complex [(F8TPP)FeIII-O-CuII(TMPA)]+ (4) (known S = 2 system, antiferromagnetically coupled high-spin FeIII and CuII). Mössbauer spectroscopy exhibits a sharp quadrupole doublet (zero field; delta = 0.57 mm/s, |DeltaEQ| = 1.14 mm/s) for 3, with isomer shift and magnetic field dependence data indicative of a peroxide ligand and S = 2 formulation. Both UV-visible-monitored stopped-flow kinetics and Mössbauer spectroscopic studies reveal the formation of heme-only superoxide complex (S)(F8TPP)FeIII-(O2-) (2a) (S = solvent molecule) prior to 3. Thermal decomposition of mu-peroxo complex 3 yields mu-oxo complex 4 with concomitant release of approximately 0.5 mol O2 per mol 3. Characterization of the reaction 1a/1b + O2 --> 2 --> 3 --> 4, presented here, advances our understanding and provides new insights to heme/Cu dioxygen-binding and reduction.

[Preliminary study on lymphotactin expression of monocyte-derived dendritic cells].

AIM: To induce dendritic cells (DCs) from human peripheral blood monocytes in vitro and to investigate the kinetics lymphotactin (Lptn) mRNA expression. METHODS: Monocytes were isolated from normal human peripheral blood cells by density gradient centrifugation and the plastic-adherent cells were cultured with the cytokines (rhGM-CSF, rhIL-4, rhTNF-alpha). The immature and mature DCs' surface antigens CD1a and CD83 were analyzed by flow cytometry (FCM). The morphology of mature DCs induced by rhTNF-alpha was observed under electron microscope. Lptn cDNA was amplified by RT-PCR, cloned to vector pGM-T Easy and completely sequenced. The RT-PCR products of cells were separated on gel and analyzed by semi-quantification. RESULTS: The cells cultured for 7 d exhibited special dendritic morphology of mature DCs and highly expressed CD83. The sequence of cDNA was confirmed and was identical to that of NO. U23772 published in GenBank. The Lptn mRNA expression of DCs was stronger in DCs cultured for 7 d than in those for 5 d, whereas it was not detected in those for 3 d. CONCLUSION: The monocyte-derived dendritic cells can express Lptn mRNA in a maturation-dependent manner.

Heme oxidation in a chimeric protein of the alpha-selective Neisseriae meningitidis heme oxygenase with the distal helix of the delta-selective Pseudomonas aeruginosa.

Heme oxygenases from the bacterial pathogens Neisseriae meningitidis (nm-HO) and Pseudomonas aeruginosa (pa-HO) share significant sequence identity (37%). In nm-HO, biliverdin IXalpha is the sole product of the reaction, whereas pa-HO yields predominantly biliverdin IXdelta. We have previously shown by NMR that the in-plane conformation of the heme in pa-HO is significantly different from that of nm-HO as a result of distinct interactions of the heme propionates with the protein scaffold [Caignan, G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman, M. A., and Rivera, M. (2002) J. Am. Chem. Soc. 124, 14879-14892]. In the report presented here, we have extended these studies to investigate the role of the distal helix by preparing a chimera of nm-HO (nm-HOch), in which distal helix residues 107-142 of nm-HO have been replaced with the corresponding residues of the delta-regioselective pa-HO (112-147). Electronic absorption spectra, resonance Raman and FTIR spectroscopic studies confirm that the orientation and hydrogen bonding properties of the proximal His ligand are not significantly altered in the chimera relative those of the wild-type proteins. The catalytic turnover of the nm-HOch-heme complex yields almost exclusively alpha-biliverdin and a small but reproducible amount of delta-biliverdin. NMR spectroscopic studies reveal that the altered regioselectivity in the chimeric protein likely stems from a dynamic equilibrium between two alternate in-plane conformations of the heme (in-plane heme disorder). Replacement of K16 with Ala and Met31 with Lys in the chimeric protein in an effort to tune key polypeptide-heme propionate contacts largely stabilizes the in-plane conformer conducive to delta-meso hydroxylation.

Point mutations at the type I Cu ligands, Cys457 and Met467, and at the putative proton donor, Asp105, in Myrothecium verrucaria bilirubin oxidase and reactions with dioxygen.

The type I Cu site in the Cys457Ser mutant of Myrothecium verrucaria bilirubin oxidase was vacant, but the trinuclear center composed of a type II Cu and a pair of type III Cu's was fully occupied by three Cu ions. Cys457Ser could react with dioxygen, affording reaction intermediate I with absorption maxima at 340, 470, and 675 nm. This intermediate corresponds to that obtained from laccase, whose type I Cu is cupric and type II and III Cu's are cuprous [Zoppellaro, G., Sakurai, T., and Huang, H. (2001) J. Biochem. 129, 949-953] or whose type I Cu is substituted with Hg [Palmer, A. E., Lee, S. K., and Solomon, E. I. (2001) J. Am. Chem. Soc. 123, 6591-6599]. Another type I Cu mutant, Met467Gln, with modified spectroscopic properties and redox potential, afforded reaction intermediate II with absorption maxima at 355 and 450 nm. This intermediate corresponds to that obtained during the reaction of laccase [Sundaram, U. M., Zhang, H. H., Hedman, B., Hodgson, K. O., and Solomon, E. I. (1997) J. Am. Chem. Soc. 119, 12525-12540; Huang, H., Zoppellaro, G., and Sakurai, T. (1999) J. Biol. Chem. 274, 32718-32724]. According to a three-dimensional model of bilirubin oxidase, Asp105 is positioned near the trinuclear center. Asp105Glu and Asp105Ala exhibited 46 and 7.5% bilirubin oxidase activity compared to the wild-type enzyme, respectively, indicating that Asp105 conserved in all multi-copper oxidases donates a proton to reaction intermediates I and II. In addition, this amino acid might be involved in the formation of the trinuclear center and in the binding of dioxygen based on the difficulties in incorporating four Cu ions in Asp105Ala and Asp105Asn and their reactions with dioxygen.

Heme/non-heme diiron(II) complexes and O2, CO, and NO adducts as reduced and substrate-bound models for the active site of bacterial nitric oxide reductase.

As a first generation model for the reactive reduced active-site form of bacterial nitric oxide reductase, a heme/non-heme diiron(II) complex [(6L)Fe(II)...Fe(II)-(Cl)]+ (2) {where 6L = partially fluorinated tetraphenylporphyrin with a tethered tetradentate TMPA chelate; TMPA = tris(2-pyridyl)amine} was generated by reduction of the corresponding mu-oxo diferric compound [(6L)Fe(III)-O-Fe(III)-Cl]+ (1). Coordination chemistry models for reactions of reduced NOR with O2, CO, and NO were also developed. With O2 and CO, adducts are formed, [(6L)Fe(III)(O2-))(thf)...Fe(II)-Cl]B(C6F5)4 (2a x O2) {lambda(max) 418 (Soret), 536 nm; nu(O-O) = 1176 cm(-1), nu(Fe-O) = 574 cm(-1) and [(6L)Fe(II)(CO)(thf)Fe(II)-Cl]B(C6F5)4 (2a x CO) {nu(CO) 1969 cm(-1)}, respectively. Reaction of purified nitric oxide with 2 leads to the dinitrosyl complex [(6L)Fe(NO)Fe(NO)-Cl]B(C6F5)4 (2a x (NO)2) with nu(NO) absorptions at 1798 cm(-1) (non-heme Fe-NO) and 1689 cm(-1) (heme-NO).

Heme-copper/dioxygen adduct formation relevant to cytochrome c oxidase: spectroscopic characterization of [(6L)FeIII-(O2(2-))-CuII]+.

In the further development and understanding of heme-copper dioxygen reactivity relevant to cytochrome c oxidase O(2)-reduction chemistry, we describe a high-spin, five-coordinate dioxygen (peroxo) adduct of an iron(II)-copper(I) complex, [((6)L)Fe(II)Cu(I)](BArF(20)) (1), where (6)L is a tetraarylporphyrinate with a tethered tris(2-pyridylmethyl)amine chelate for copper. Reaction of 1 with O(2) in MeCN affords a remarkably stable [t(1/2) (rt; MeCN) approximately 60 min] adduct, [((6)L)Fe(III)-(O(2) (2-))-Cu(II)](+) (2) [EPR silent; lambda(max)=418 (Soret), 561 nm], formulated as a peroxo complex based on manometry (1:O(2)=1:1; spectrophotometric titration, -40 degrees C, MeCN), mass spectrometry {MALDI-TOF-MS: (16)O(2), m/z 1191 ([((6)L)Fe(III)-((16)O(2) (2-))-Cu(II)](+)); (18)O(2), m/z 1195}, and resonance Raman spectroscopy (nu((O-O))=788 cm(-1); Delta(16)O(2)/(18)O(2)=44 cm(-1); Delta(16)O(2)/(16/18)O(2)=22 cm(-1)). (1)H and (2)H NMR spectroscopy (-40 degrees C, MeCN) reveals that 2 is the first heme-copper peroxo complex which is high-spin, with downfield-shifted pyrrole resonances (delta(pyrrole)=75 ppm, s, br) and upfield shifted peaks at delta= -22, -35, and -40 ppm, similar to the pattern observed for the mu-oxo complex [((6)L)Fe(III)-O-Cu(II)](BAr(F)) (3) (known S=2 system, antiferromagnetically coupled high-spin Fe(III) and Cu(II)). The corresponding magnetic moment measurement (Evans method, CD(3)CN, -40 degrees C) also confirms the S=2 spin state, with mu(B)=4.9. Structural insights were obtained from X-ray absorption spectroscopy, showing Fe-O (1.83 A) and Cu-O (1.882 A) bonds, and an Fe...Cu distance of 3.35(2) A, suggestive of a mu-1,2-peroxo ligand present in 2. The reaction of 2 with cobaltocene gives 3, differing from the observed full reduction seen with other heme-Cu peroxo complexes. Finally, thermal decomposition of 2 yields 3, with concomitant release of 0.5 mol O(2) per mol 2, as confirmed quantitatively by an alkaline pyrogallol dioxygen scavenging solution.

Synthesis and spectroscopy of micro-oxo (O(2)(-))-bridged heme/non-heme diiron complexes: models for the active site of nitric oxide reductase.

In this paper, we describe the synthesis and study of a series of heme/non-heme Fe-O-Fe' complexes supported by a porphyrin and the tripodal nitrogen ligand TMPA [TMPA = tris(2-pyridylmethyl)amine]. The complete synthesis of [((6)L)Fe-O-Fe(X)](+) (1) (X = OMe(-) or Cl(-), 69:31 ratio), where (6)L is the dianion of 5-(o-O-[(N,N-bis(2-pyridylmethyl)-2-(6-methoxyl)pyridinemethanamine)phenyl]-10,15,20-tris(2,6-difluorophenyl)porphine, is reported. The crystal structure for 1.PF(6) reveals an intramolecular heme/non-heme diferric complex bridged by an Fe-O-Fe' moiety; 90 degree angle (Fe-O-Fe') = 166.7(3) degrees, and d(Fe.Fe') = 3.556 A. Crystal data for C(70)H(57)ClF(12)Fe(2)N(8)O(3)P (1.PF(6)): triclinic, Ponemacr;, a = 13.185(3) A, b = 14.590 (3) A, c = 16.885(4) A, alpha = 104.219(4) degrees, beta = 91.572(4) degrees, gamma = 107.907(4) degrees, V = 2977.3(11) A(3), Z = 2, T = 150(2) K. Complex 1 (where X = Cl(-)) is further characterized by UV-vis (lambda(max) = 328, 416 (Soret), 569 nm), (1)H NMR (delta 27-24 [TMPA -CH(2)-], 16.1 [pyrrole-H], 15.2-10.5 [PY-3H, PY-5H], 7.9-7.2 [m- and p-phenyl-H], 6.9-5.8 [PY-4H] ppm), resonance Raman (nu(as)(Fe-O-Fe') 844 cm(-)(1)), and Mössbauer (delta(Fe) = 0.47, 0.41 mm/s; deltaE(A) = 1.59, 0.55 mm/s; 80 K) spectroscopies, MALDI-TOF mass spectrometry (m/z 1202), and SQUID susceptometry (J = - 114.82 cm(-)(1), S = 0). We have also synthesized a series of 3-, 4-, and 5-methyl-substituted as well as selectively deuterated TMPA(Fe') complexes and condensed these with the hydroxo complex (F(8))FeOH or (F(8)-d(8))FeOH to yield "untethered" Fe-O-Fe' analogues. Along with selective deuteration of the methylene hydrogens in TMPA, complete (1)H NMR spectroscopic assignments for 1 have been accomplished. The magnetic properties of several of the untethered complexes and a comparison to those of 1 are also presented. Complex 1 and related species represent good structural and spectroscopic models for the heme/non-heme diiron active site in the enzyme nitric oxide reductase.

Coupled oxidation vs heme oxygenation: insights from axial ligand mutants of mitochondrial cytochrome b5.

Mutation of His-39, one of the axial ligands in rat outer mitochondrial membrane cytochrome b(5) (OM cyt b(5)), to Val produces a mutant (H39V) capable of carrying out the oxidation of heme to biliverdin when incubated with hydrazine and O(2). The reaction proceeds via the formation of an oxyferrous complex (Fe(II)(-)O(2)) that is reduced by hydrazine to a ferric hydroperoxide (Fe(III)(-)OOH) species. The latter adds a hydroxyl group to the porphyrin to form meso-hydroxyheme. The observation that catalase does not inhibit the oxidation of the heme in the H39V mutant is consistent with the formation of a coordinated hydroperoxide (Fe(III)(-)OOH), which in heme oxygenase is the precursor of meso-hydroxyheme. By comparison, mutation of His-63, the other axial ligand in OM cyt b(5), to Val results in a mutant (H63V) capable of oxidizing heme to verdoheme in the absence of catalase. However, the oxidation of heme by H63V is completely inhibited by catalase. Furthermore, whereas the incubation of Fe(III)(-)H63V with H(2)O(2) leads to the nonspecific degradation of heme, the incubation of Fe(II)(-)H63V with H(2)O(2) results in the formation of meso-hydroxyheme, which upon exposure to O(2) is rapidly converted to verdoheme. These findings revealed that although meso-hydroxyheme is formed during the degradation of heme by the enzyme heme oxygenase or by the process of coupled oxidation of model hemes and hemoproteins not involved in heme catabolism, the corresponding mechanisms by which meso-hydroxyheme is generated are different. In the coupled oxidation process O(2) is reduced to noncoordinated H(2)O(2), which reacts with Fe(II)-heme to form meso-hydroxyheme. In the heme oxygenation reaction a coordinated O(2) molecule (Fe(II)(-)O(2)) is reduced to a coordinated peroxide molecule (Fe(III)(-)OOH), which oxidizes heme to meso-hydroxyheme.