Conserved C-Terminal Tail Is Responsible for Membrane Localization and Function of Pseudomonas aeruginosa Hemerythrin.

Many bacteria have hemerythrin (Hr) proteins that bind O2, including Pseudomonas aeruginosa, in which microoxia-induced Hr (Mhr) provide fitness advantages under microoxic conditions. Mhr has a 23 amino-acid extension at its C-terminus relative to a well-characterized Hr from Methylococcus capsulatus, and similar extensions are also found in Hrs from other bacteria. The last 11 amino acids of this extended, C-terminal tail are highly conserved in gammaproteobacteria and predicted to form a helix with positively charged and hydrophobic faces. In cellular fractionation assays, wild-type (WT) Mhr was found in both membrane and cytosolic fractions, while a MhrW143* variant lacking the last 11 residues was largely in the cytosol and did not complement Mhr function in competition assays. MhrL112Y, a variant that has a much longer-lived O2-bound form, was fully functional and had a similar localization pattern to that of WT Mhr. Both MhrW143* and MhrL112Y had secondary structures, stabilities, and O2-binding kinetics similar to those of WT Mhr. Fluorescence studies revealed that the C-terminal tail, and particularly the fragment corresponding to its last 11 residues, was sufficient and necessary for association with lipid vesicles. Molecular dynamics simulations and subsequent cellular analysis of Mhr variants have demonstrated that conserved, positively charged residues in the tail are important for Mhr interactions with negatively charged membranes and the contribution of this protein to competitive fitness. Together, these data suggest that peripheral interactions of Mhr with membranes are guided by the C-terminal tail and are independent of O2-binding.

Associations of Single Versus Multiple Human Papillomavirus Infections With the Prevalence of Cervical Intraepithelial Neoplasia 2/3 and Squamous Cell Carcinoma Lesions: Human Papillomavirus Type-Specific Attribution.

The risk of developing cervical squamous lesions in women with multiple high-risk human papillomavirus (hrHPV) infections is uncertain. The aim of this retrospective study was to investigate the type-specific attribution and phylogenetic effects of single and multiple hrHPV subtypes in cervical squamous lesions. All cases with cervical histopathologic diagnosis and human papillomavirus (HPV) genotyping results in the 6 months preceding biopsy from October 2018 to December 2022 were studied and analyzed. Over the study period, 70,361 cases with histopathologic follow-up and prior HPV genotyping were identified. The hrHPV-positive rate was 55.6% (39,104/70,361), including single hrHPV detected in 27,182 (38.6%), 2 types of hrHPV detected in 8158 (11.6%), and 3 types of hrHPV detected in 2486 (3.5%). Among 16,457 cases with a histologically diagnosed squamous lesion (cervical intraepithelial neoplasia 1: 11411; cervical intraepithelial neoplasia 2/3: 4192; squamous cell carcinoma: 854 cases), the prevalence of single hrHPV infection increased, but the rate of multiple concomitant hrHPV infections showed negative association as the degree of squamous lesions increased. Among women with a single HPV16 infection, cervical intraepithelial neoplasia 2/3 and squamous cell carcinoma (CIN2+) diagnostic rate was 30.6%, and it increased to 47.6% when coinfected with HPV33 (P < .001) but significantly decreased when coinfected with all other hrHPV types (P < .05). By comparing CIN2+ diagnostic rates in 40 most common 2 types of hrHPV infections with related single hrHPV infection, CIN2+ rates were decreased in 12 combinations (30.0%), equivalent in 26 combinations (65.0%), and increased in 2 combinations (5.0%). The cases with 3 types of HPV infections reduced the risk for CIN2+ compared with related single HPV infections. HPV16+52+53, HPV16+52+68, HPV16+52+51, HPV16+39+52, and HPV16+58+53 significantly decreased the risk of CIN2+ compared with HPV16 single infection (P < .05). This study demonstrates that multiple hrHPV infections are not associated with cumulatively higher risk for CIN2+ development, suggesting that oncogenic progression of multiple hrHPV-associated cervical squamous lesions is neither synergistic nor a cumulative effect at the phylogenetic level, possibly a way of competitive interference.

Feasibility of Using Magnetic Resonance Spectroscopy Test Biomarkers to Diagnose Alzheimer's Disease: Systematic Evaluation and Meta-Analysis.

BACKGROUND: Alzheimer's disease (AD) is the leading cause of dementia, resulting in impairments in memory, cognition, decision-making, and social skills. Thus, accurate preclinical diagnosis of Alzheimer's disease is paramount. The identification of biomarkers for Alzheimer's disease through magnetic resonance spectroscopy (MRS) represents a novel adjunctive diagnostic approach. OBJECTIVE: This study conducted a meta-analysis of the diagnostic results of this technology to explore its feasibility and accuracy. METHODS: PubMed, Cochrane Library, EMBASE, and Web of Science databases were searched without restrictions, with the search period extending up to July 31, 2022. The search strategy employed a combination of subject headings and keywords. All retrieved documents underwent screening by two researchers, who selected them for meta-analysis. The included literature was analyzed using Review Manager 5.4 software, with corresponding bias maps, forest plots, and summary receiver operating characteristic (SROC) curves generated and analyzed. RESULTS: A total of 344 articles were retrieved initially, with 11 articles meeting the criteria for inclusion in the analysis. The analysis encompassed data from approximately 1766 patients. In the forest plot, both sensitivity (95% CI) and specificity (95% CI) approached 1. Examining the true positive rate, false positive rate, true negative rate, and false negative rate, all studies on the summary receiver operating characteristic (SROC) curve clustered in the upper left quadrant, suggesting a very high accuracy of biomarkers detected by MRS for diagnosing Alzheimer's disease. CONCLUSION: The detection of biomarkers by MRS demonstrates feasibility and high accuracy in diagnosing AD. This technology holds promise for widespread adoption in the clinical diagnosis of AD in the future.

Prevalence and Associations of Beta2-Microglobulin Mutations in MSI-H/dMMR Cancers.

Microsatellite instability (MSI) has emerged as an important predictor of sensitivity for immunotherapy-based strategies. ß-2-Microglobulin (B2M) contains microsatellites within the coding regions and is prone to somatic changes in MSI/mismatch repair deficiency (MSI/dMMR) tumors. To delineate prevalence and associations of B2M mutations in MSI-H/dMMR cancers, we investigated the mutational profile of B2M and clinical and pathological features in gastric cancer (GC), colorectal cancer (CRC), and endometrial cancer (EC) with a high incidence of microsatellite instability-high (MSI-H)/dMMR. Formalin-fixed paraffin-embedded (FFPE) tumor tissues along with matched normal tissues were collected from 108 MSI/dMMR patients with GC, CRC, and EC. Genomic profiling of tissue and blood samples were assessed next-generation sequencing (NGS). Immunohistochemistry (IHC) was used to examine the presence or absence of B2M protein. Alternations in the exonic microsatellite regions of B2M were observed at various but high frequencies (57.5% in CRC, 23.9% in GC, and 13.6% in EC) and in different forms. NGS assay revealed that genes involved in chromatin regulation, the PI3K pathway, the WNT pathway, and mismatch repair were extensively altered in the MSI-H cohort. Signature 6 and 26, 2 of 4 mutational signatures associated with defective DNA mismatch repair, featured with high numbers of small insertion/deletions (INDEL) dominated in all 3 types of cancer. Alternations in the exonic microsatellite regions of B2M were observed at various but high frequencies (57.5% in CRC, 23.9% in GC, and 13.6% in EC) and in different forms. Tumor mutational burden (TMB) was significantly higher in the patients carrying MSI-H/dMMR tumors with B2M mutation than that in patients with wild-type B2M (P = .026).The frame shift alteration occurring at the exonic microsatellite sties caused loss of function of B2M gene. In addition, a case with CRC carrying indels in B2M gene resisted the ICI treatment was reported. In conclusion, patients carrying MSI-H/dMMR tumors with B2M mutation showed significantly higher TMB. Prescription of ICIs should be thoroughly evaluated for these patients.

The epigenetic factor CHD4 contributes to metastasis by regulating the EZH2/ß-catenin axis and acts as a therapeutic target in ovarian cancer.

BACKGROUND: The overall survival rate of patients with advanced ovarian cancer (OC) has remained static for several decades. Advanced ovarian cancer is known for its poor prognosis due to extensive metastasis. Epigenetic alterations contribute to tumour progression and therefore are of interest for potential therapeutic strategies. METHODS: Following our previous study, we identified that CHD4, a chromatin remodelling factor, plays a strong role in ovarian cancer cell metastasis. We investigated the clinical significance of CHD4 through TCGA and GEO database analyses and explored the effect of CHD4 expression modulation and romidepsin treatment on the biological behaviour of ovarian cancer through CCK-8 and transwell assays. Bioluminescence imaging of tumours in xenografted mice was applied to determine the therapeutic effect of romidepsin. GSEA and western blotting were used to screen the regulatory mechanism of CHD4. RESULTS: In ovarian cancer patient specimens, high CHD4 expression was associated with a poor prognosis. Loss of function of CHD4 in ovarian cancer cells induced suppression of migration and invasion. Mechanistically, CHD4 knockdown suppressed the expression of EZH2 and the nuclear accumulation of ß-catenin. CHD4 also suppressed the metastasis of ovarian cancer cells and prevented disease progression in a mouse model. To inhibit the functions of CHD4 that are mediated by histone deacetylase, we evaluated the effect of the HDAC1/2 selective inhibitor romidepsin. Our findings indicated that treatment with romidepsin suppressed the progression of metastases in vitro and in vivo. CONCLUSIONS: Collectively, our results uncovered an oncogenic function of CHD4 in ovarian cancer and provide a rationale for clinical trials of romidepsin in ovarian cancer patients.

Urban residents' self-rescue in response to public health emergencies in China: a qualitative study.

BACKGROUND: The abject uncertainty and unpredictability of public health emergencies have plagued various countries. Global health governance and international communities are facing long-term and arduous challenges. The self-rescue ability of individuals in a public emergency may be the most powerful trait to improve the survival rate outside the hospital. The study explores the cognitive ability and attitudes of urban residents in China towards self-rescue in response to public health emergencies. It provides appropriate evidence for improving the self-rescue ability of urban residents in China. METHODS: Sixteen urban residents were selected using the purposive sampling method for semi-structured interviews. Theme analysis was used to collate and analyse the interview data. RESULTS: Two themes and five sub-themes were analysed. The two themes included cognition and attitude of Chinese urban residents for self-rescue in an emergency. Urban residents believed that their knowledge and skills for self-rescue in an emergency were low. The ability for emergency self-rescue is affected by multiple factors, with relatively limited options for improvement. Nonetheless, the respondents expressed a desire to accept interventions under psychological crisis and a strong willingness to acquire knowledge and skills required for emergency self-rescue. CONCLUSION: This study investigated the perceptions and attitudes of Chinese urban residents towards emergency self-rescue. The results support enhanced ability of urban residents to respond to public health emergencies, thereby diminishing the negative outcomes. The findings suggest the need for strategies to address the factors affecting emergency self-rescue.

Pseudomonas aeruginosa lasR mutant fitness in microoxia is supported by an Anr-regulated oxygen-binding hemerythrin.

Pseudomonas aeruginosa strains with loss-of-function mutations in the transcription factor LasR are frequently encountered in the clinic and the environment. Among the characteristics common to LasR-defective (LasR-) strains is increased activity of the transcription factor Anr, relative to their LasR+ counterparts, in low-oxygen conditions. One of the Anr-regulated genes found to be highly induced in LasR- strains was PA14_42860 (PA1673), which we named mhr for microoxic hemerythrin. Purified P. aeruginosa Mhr protein contained the predicted di-iron center and bound molecular oxygen with an apparent Kd of ∼1 µM. Both Anr and Mhr were necessary for fitness in lasR+ and lasR mutant strains in colony biofilms grown in microoxic conditions, and the effects were more striking in the lasR mutant. Among genes in the Anr regulon, mhr was most closely coregulated with the Anr-controlled high-affinity cytochrome c oxidase genes. In the absence of high-affinity cytochrome c oxidases, deletion of mhr no longer caused a fitness disadvantage, suggesting that Mhr works in concert with microoxic respiration. We demonstrate that Anr and Mhr contribute to LasR- strain fitness even in biofilms grown in normoxic conditions. Furthermore, metabolomics data indicate that, in a lasR mutant, expression of Anr-regulated mhr leads to differences in metabolism in cells grown on lysogeny broth or artificial sputum medium. We propose that increased Anr activity leads to higher levels of the oxygen-binding protein Mhr, which confers an advantage to lasR mutants in microoxic conditions.

Exploring the pharmacological mechanisms of Shuanghuanglian against T-cell acute lymphoblastic leukaemia through network pharmacology combined with molecular docking and experimental validation.

CONTEXT: Due to the poor prognosis of T-cell acute lymphoblastic leukaemia (T-ALL), there is an urgent need to identify safer and more cost-effective drugs. OBJECTIVE: This study evaluated the antitumour activity of Shuanghuanglian (SHL) on T-ALL cells and elucidated the mechanism. MATERIALS AND METHODS: Jurkat and Molt4 cells were treated with SHL (0.1, 0.2 and 0.4 mg/mL) for 24 and 48 h. The controls were treated with RPMI 1640 containing 10% foetal bovine serum. Cell viability was evaluated through Cell Counting Kit-8 assay. Patterns of death and signalling pathway alterations caused by SHL were identified by network pharmacology combined with GO enrichment analysis and then were verified by Hoechst 33342 staining, Annexin V-FITC/PI staining and Western blotting. Interactions of the active ingredients with targets were analysed by molecular docking. RESULTS: The IC50 values of SHL in Jurkat and Molt4 cells were 0.30 ± 0.10 and 0.48 ± 0.07 mg/mL, respectively, at 24 h and 0.27 ± 0.05 and 0.30 ± 0.03 mg/mL at 48 h. In T-ALL, 117 target genes of SHL were mainly enriched in the apoptosis and NOTCH signalling pathways. SHL induced apoptosis was confirmed by Hoechst 33342 staining and flow cytometry. The protein levels of cleaved caspase-7 and cleaved PARP were significantly increased but those of cleaved NOTCH1 and MYC were reduced. The active ingredients of SHL can interact with γ-secretase.Discussion and conclusions: SHL induces apoptosis in T-ALL cells via the NOTCH1-MYC pathway and may be a potential drug for the treatment of T-ALL.

Influence of the Interdomain Interface on Structural and Redox Properties of Multiheme Proteins.

Multiheme proteins are important in energy conversion and biogeochemical cycles of nitrogen and sulfur. A diheme cytochrome c4 (c4) was used as a model to elucidate roles of the interdomain interface on properties of iron centers in its hemes A and B. Isolated monoheme domains c4-A and c4-B, together with the full-length diheme c4 and its Met-to-His ligand variants, were characterized by a variety of spectroscopic and stability measurements. In both isolated domains, the heme iron is Met/His-ligated at pH 5.0, as in the full-length c4, but becomes His/His-ligated in c4-B at higher pH. Intradomain contacts in c4-A are minimally affected by the separation of c4-A and c4-B domains, and isolated c4-A is folded. In contrast, the isolated c4-B is partially unfolded, and the interface with c4-A guides folding of this domain. The c4-A and c4-B domains have the propensity to interact even without the polypeptide linker. Thermodynamic cycles have revealed properties of monomeric folded isolated domains, suggesting that ferrous (FeII), but not ferric (FeIII) c4-A and c4-B, is stabilized by the interface. This study illustrates the effects of the interface on tuning structural and redox properties of multiheme proteins and enriches our understanding of redox-dependent complexation.

Comparing Properties of Common Bioinorganic Ligands with Switchable Variants of Cytochrome c.

Ligand substitution at the metal center is common in catalysis and signal transduction of metalloproteins. Understanding the effects of particular ligands, as well as the polypeptide surrounding, is critical for uncovering mechanisms of these biological processes and exploiting them in the design of bioinspired catalysts and molecular devices. A series of switchable K79G/M80X/F82C (X = Met, His, or Lys) variants of cytochrome (cyt) c was employed to directly compare the stability of differently ligated proteins and activation barriers for Met, His, and Lys replacement at the ferric heme iron. Studies of these variants and their nonswitchable counterparts K79G/M80X have revealed stability trends Met < Lys < His and Lys < His < Met for the protein FeIII-X and FeII-X species, respectively. The differences in the hydrogen-bonding interactions in folded proteins and in solvation of unbound X in the unfolded proteins explain these trends. Calculations of free energy of ligand dissociation in small heme model complexes reveal that the ease of the FeIII-X bond breaking increases in the series amine < imidazole < thioether, mirroring trends in hardness of these ligands. Experimental rate constants for X dissociation in differently ligated cyt c variants are consistent with this sequence, but the differences between Met and His dissociation rates are attenuated because the former process is limited by the heme crevice opening. Analyses of activation parameters and comparisons to those for the Lys-to-Met ligand switch in the alkaline transition suggest that ligand dissociation is entropically driven in all the variants and accompanied by Lys protonation at neutral pH. The described thiolate redox-linked switches have offered a wealth of new information about interactions of different protein-derived ligands with the heme iron in cyt c model proteins, and we anticipate that the strategy of employing these switches could benefit studies of other redox metalloproteins and model complexes.

Autophagy inhibitors increase the susceptibility of KRAS-mutant human colorectal cancer cells to a combined treatment of 2-deoxy-D-glucose and lovastatin.

RAS-driven colorectal cancer relies on glucose metabolism to support uncontrolled growth. However, monotherapy with glycolysis inhibitors like 2-deoxy-D-glucose causes limited effectiveness. Recent studies suggest that anti-tumor effects of glycolysis inhibition could be improved by combination treatment with inhibitors of oxidative phosphorylation. In this study we investigated the effect of a combination of 2-deoxy-D-glucose with lovastatin (a known inhibitor of mevalonate pathway and oxidative phosphorylation) on growth of KRAS-mutant human colorectal cancer cell lines HCT116 and LoVo. A combination of lovastatin (>3.75 µM) and 2-deoxy-D-glucose (>1.25 mM) synergistically reduced cell viability, arrested cells in the G2/M phase, and induced apoptosis. The combined treatment also reduced cellular oxygen consumption and extracellular acidification rate, resulting in decreased production of ATP and lower steady-state ATP levels. Energy depletion markedly activated AMPK, inhibited mTOR and RAS signaling pathways, eventually inducing autophagy, the cellular pro-survival process under metabolic stress, whereas inhibition of autophagy by chloroquine (6.25 µM) enhanced the cytotoxic effect of the combination of lovastatin and 2-deoxy-D-glucose. These in vitro experiment results were reproduced in a nude mouse xenograft model of HCT116 cells. Our findings suggest that concurrently targeting glycolysis, oxidative phosphorylation, and autophagy may be a promising regimen for the management of RAS-driven colorectal cancers.

Clinical analysis of uterine intravenous leiomyomatosis: A retrospective study of 260 cases.

METHODS: We collected the clinical data of 260 patients admitted to the hospital from April 2003 to September 2019 with pathologically confirmed intravenous leiomyomatosis (IVL) and followed up with these patients regularly. Univariate and multivariate logistic regression analyses were carried out on the relevant recurrence factors. RESULTS: A total of 166 patients were regularly followed up, the median follow-up time was 36 (range 2-168) months, 14 (5.4%) patients eventually relapsed, and the median recurrence time was 8.5 (range 2-42) months. The univariate analysis showed that age (p = 0.003) and surgical type (p < 0.001) were associated with recurrence, and multivariate regression analysis demonstrated that surgical type was the only factor associated with recurrence (p < 0.001, OR 20.01). CONCLUSIONS: The use of gonadotrophin releasing hormone agonist (GnRHa) cannot reduce the postsurgical recurrence rate of patients with UIVL. Compared to total hysterectomy and bilateral salpingo-oophorectomy (TH-BSO), total hysterectomy (TH) does not increase the odds of recurrence, but the chance of recurrence with tumorectomy (TE) is 20 times higher than that of TH-BSO.

Highly Stable Vanadium Redox-Flow Battery Assisted by Redox-Mediated Catalysis.

With good operation flexibility and scalability, vanadium redox-flow batteries (VRBs) stand out from various electrochemical energy storage (EES) technologies. However, traditional electrodes in VRBs, such as carbon and graphite felt with low electrochemical activities, impede the interfacial charge transfer processes and generate considerable overpotential loss, which significantly decrease the energy and voltage efficiencies of VRBs. Herein, by using a facile electrodeposition technique, Prussian blue/carbon felt (PB/CF) composite electrodes with high electrochemical activity for VRBs are successfully fabricated. The PB/CF electrode exhibits excellent electrochemical activity toward VO2+ /VO2 + redox couple in VRB with an average cell voltage efficiency (VE) of 90% and an energy efficiency (EE) of 88% at 100 mA cm-2 . In addition, due to the uniformly distributed PB particles that are strongly bound to the surface of carbon fibers in CF, VRBs with the PB/CF electrodes show much better long-term stabilities compared with the pristine CF-based battery due to the redox-mediated catalysis. A VRB stack consisting of three single cells (16 cm2 ) is also constructed to assess the reliability of the redox-mediated PB/CF electrodes for large-scale application. The facile technique for the high-performance electrode with redox-mediated reaction is expected to shed new light on commercial electrode design for VRBs.

Mechanistic Studies of Proton-Coupled Electron Transfer in a Calorimetry Cell.

Mechanistic studies of proton-coupled electron-transfer (PCET) reactions in proteins are complicated by the challenge of following proton transfer (PT) in these large molecules. Herein we describe the use of isothermal titration calorimetry (ITC) to establish proton involvement in protein redox reactions and the identity of PT sites. We validate this approach with three variants of a heme protein cytochrome c (cyt c) and show that the method yields a wealth of thermodynamic information that is important for characterizing PCET reactions, including reduction potentials, redox-dependent p Ka values, and reaction enthalpies for both electron-transfer (ET) and PT steps. We anticipate that this facile and label-free ITC approach will find widespread applications in studies of other redox proteins and enhance our knowledge of PCET reaction mechanisms.

Effect of Molecular Conformation Restriction on the Photophysical Properties of N

Using naphthalimide (NI), complexes (Pt-PhNI and Pt-PhMeNI) based on the N^N platinum(II) bis(phenylacetylide) coordination framework were prepared, in which there are two close-lying triplet states, i.e., the metal-to-ligand-charge-transfer (3MLCT) and the NI localized emissive state (3LE). Pt-PhNI has better electronic communication between the Pt coordination center and the NI moiety, whereas in Pt-PhMeNI, they are more isolated by orthogonal geometry. For Pt-PhMeNI, the S0 → 1MLCT and S0 → 1LE absorption bands are separated by 5655 cm-1, while they are more overlapped in Pt-PhNI. The 3MLCT → S0 and 3LE → S0 dual phosphorescence emissions were observed for both Pt-PhNI (in toluene) and Pt-PhMeNI (in benzonitrile). The molecular conformation tunes the 3MLCT/3LE state population ratio, and the orthogonal geometry makes the 3LE state in Pt-PhMeNI basically a dark state (in toluene). Switching of the relative energy levels of the 3MLCT/3LE states by variation of the solvent polarity and temperature was achieved. For Pt-PhMeNI, the energy level of 3MLCT state is higher in a polar solvent; thus, the 3MLCT emission decreases, while the phosphorescence lifetime is prolonged from 9.5 µs (in toluene) to 58 µs (in benzonitrile) because of the different equilibria with the nonemissive 3LE state. Conversely, increasing the temperature enhances the upward transition from the nonemissive 3LE state to the emissive 3MLCT state; as such, the phosphorescence of Pt-PhMeNI was intensified at higher temperature (which is unusual), and the phosphorescence lifetime decreased from 58 µs (298 K) to ca. 5 µs (348 K). The ultrafast intersystem crossing (ca. 0.5 ps) and intramolecular triplet-triplet energy transfer (3-11 ps) were studied by femtosecond transient absorption spectroscopy. These results are useful for an in-depth understanding of the photophysics of multichromophore transition-metal complexes and for the design of external stimuli-responsive sensing materials, for instance, temperature or microenvironment sensing materials.

The K79G Mutation Reshapes the Heme Crevice and Alters Redox Properties of Cytochrome c.

The two roles of cytochrome c (cyt c), in oxidative phosphorylation and apoptosis, critically depend on redox properties of its heme iron center. The K79G mutant has served as a parent protein for a series of mutants of yeast iso-1 cyt c. The mutation preserves the Met80 coordination to the heme iron, as found in WT* (K72A/C102S), and many spectroscopic properties of K79G and WT* are indistinguishable. The K79G mutation does not alter the global stability, fold, rate of Met80 dissociation, or thermodynamics of the alkaline transition (p Ka) of the protein. However, the reduction potential of the heme iron decreases; further, the p KH of the trigger group and the rate of the Met-to-Lys ligand exchange associated with the alkaline transition decrease, suggesting changes in the environment of the heme. The rates of electron self-exchange and bimolecular electron transfer (ET) with positively charged inorganic complexes increase, as does the intrinsic peroxidase activity. Analysis of the reaction rates suggests that there is increased accessibility of the heme edge in K79G and supports the importance of the Lys79 site for bimolecular ET reactions of cyt c, including those with some of its native redox partners. Structural modeling rationalizes the observed effects to arise from changes in the volume of the heme pocket and solvent accessibility of the heme group. Kinetic and structural analyses of WT* characterize the properties of the heme crevice of this commonly employed reference variant. This study highlights the important role of Lys79 for defining functional redox properties of cyt c.

Ligation and Reactivity of Methionine-Oxidized Cytochrome c.

Met80, one of the heme iron ligands in cytochrome c (cyt c), is readily oxidized to Met sulfoxide (Met-SO) by several biologically relevant oxidants. The modification has been suggested to affect both the electron-transfer (ET) and apoptotic functions of this metalloprotein. The coordination of the heme iron in Met-oxidized cyt c (Met-SO cyt c) is critical for both of these functions but has remained poorly defined. We present electronic absorption, NMR, and EPR spectroscopic investigations as well as kinetic studies and mutational analyses to identify the heme iron ligands in yeast iso-1 Met-SO cyt c. Similar to the alkaline form of native cyt c, Lys73 and Lys79 ligate to the ferric heme iron in the Met80-oxidized protein, but this coordination takes place at much lower pH. The ferrous heme iron is ligated by Met-SO, implying the redox-linked ligand switch in the modified protein. Binding studies with the model peptide microperoxidase-8 provide a rationale for alterations in ligation and for the role of the polypeptide packing in native and Met-SO cyt c. Imidazole binding experiments have revealed that Lys dissociation from the ferric heme in K73A/K79G/M80K (M80K#) and Met-SO is more than 3 orders of magnitude slower than the opening of the heme pocket that limits Met80 replacement in native cyt c. The Lys-to-Met-SO ligand substitution gates ET of ferric Met-SO cyt c with Co(terpy)22+. Owing to the slow Lys dissociation step, ET reaction is slow but possible, which is not the case for nonswitchable M80A and M80K#. Acidic conditions cause Lys replacement by a water ligand in Met-SO cyt c (p Ka = 6.3 ± 0.1), increasing the intrinsic peroxidase activity of the protein. This pH-driven ligand switch may be a mechanism to boost peroxidase function of cyt c specifically in apoptotic cells.

Redox-dependent stability, protonation, and reactivity of cysteine-bound heme proteins.

Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to Cys binding studies with the minimalist heme peptide microperoxidase-8, demonstrate that the protein scaffold and solvent interactions play important roles in stabilizing a particular Cys-heme coordination. The increased stability of ferric thiolate compared with ferrous thiol arises mainly from entropic factors. This robust cyt c model system provides access to all four forms of Cys-bound heme, including the ferric thiol. Protein motions control the rates of heme redox reactions, and these effects are amplified at low pH, where the proteins are less stable. Thermodynamic signatures and redox reactivity of the model Cys-bound hemes highlight the critical role of the protein scaffold and its dynamics in modulating redox-linked transitions between thiols and thiolates.

The triplet excited state of Bodipy: formation, modulation and application.

Boron dipyrromethene (Bodipy) is one of the most extensively investigated organic chromophores. Most of the investigations are focused on the singlet excited state of Bodipy, such as fluorescence. In stark contrast, the study of the triplet excited state of Bodipy is limited, but it is an emerging area, since the triplet state of Bodipy is tremendously important for several areas, such as the fundamental photochemistry study, photodynamic therapy (PDT), photocatalysis and triplet-triplet annihilation (TTA) upconversion. The recent developments in the study of the production, modulation and application of the triplet excited state of Bodipy are discussed in this review article. The formation of the triplet state of Bodipy upon photoexcitation, via the well known approach such as the heavy atom effect (including I, Br, Ru, Ir, etc.), and the new methods, such as using a spin converter (e.g. C60), charge recombination, exciton coupling and the doubly substituted excited state, are summarized. All the Bodipy-based triplet photosensitizers show strong absorption of visible or near IR light and the long-lived triplet excited state, which are important for the application of the triplet excited state in PDT or photocatalysis. Moreover, the methods for switching (or modulation) of the triplet excited state of Bodipy were discussed, such as those based on the photo-induced electron transfer (PET), by controlling the competing Förster-resonance-energy-transfer (FRET), or the intermolecular charge transfer (ICT). Controlling the triplet excited state will give functional molecules such as activatable PDT reagents or molecular devices. It is worth noting that switching of the singlet excited state and the triplet state of Bodipy may follow different principles. Application of the triplet excited state of Bodipy in PDT, hydrogen (H2) production, photoredox catalytic organic reactions and TTA upconversion were discussed. The challenges and the opportunities in these areas were briefly discussed.