Biol Inorg Chem Research Articles

Dissecting molecular details and functional effects of the high-affinity copper binding site in plasminogen activator Inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor for plasminogen activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). As a unique member in the serine protease inhibitor (serpin) family, PAI-1 is metastable and converts to an inactive, latent structure with a half-life of 1-2 hr under physiological conditions. Unusual effects of metals on the rate of the latency conversion are incompletely understood. Previous work has identified two residues near the N-terminus, H2 and H3, which reside in a high-affinity copper-binding site in PAI-1 [Bucci JC, McClintock CS, Chu Y, Ware GL, McConnell KD, Emerson JP, Peterson CB (2017) J Biol Inorg Chem 22:1123-1,135]. In this study, neighboring residues, H10, E81, and H364, were tested as possible sites that participate in Cu(II) coordination at the high-affinity site. Kinetic methods, gel sensitivity assays, and isothermal titration calorimetry (ITC) revealed that E81 and H364 have different roles in coordinating metal and mediating the stability of PAI-1. H364 provides a third histidine in the metal-coordination sphere with H2 and H3. In contrast, E81 does not appear to be required for metal ligation along with histidines; contacts made by the side-chain carboxylate upon metal binding are perturbed and, in turn, influence dynamic fluctuations within the region encompassing helices D, E, and F and the W86 loop that are important in the pathway for the PAI-1 latency conversion. This investigation underscores a prominent role of protein dynamics, noncovalent bonding networks and ligand binding in controlling the stability of the active form of PAI-1.

Selective, Catalytic Oxidations of C–H Bonds in Polyethylenes Produce Functional Materials with Enhanced Adhesion

Summary The synthesis of functional and processable polyethylenes from simple vinyl monomers with controlled molecular weights and architectures has been a grand challenge of polymer chemistry. Post-polymerization modification of the homopolymers of ethylene is attractive for this purpose but has been hampered by the lack of efficient methods for the selective functionalization of C–H bonds in polyolefins. We report the selective, catalytic oxidation of C–H bonds in commodity polyethylenes with varying molecular weights and architectures. Remarkably, the functionalized materials, even at low levels of functionalization, exhibit physical properties that are absent in unmodified polyolefins, such as strong adhesion and the ability to be painted with common waterborne latex paint. Such observations indicate that selectively modified polyethylenes as described here may help to transform existing commodity plastics into more valuable and potentially more sustainable materials.

Augmentation of cytochrome P450 monooxygenase catalysis on its interaction with NADPH-cytochrome P450 reductase FMN domain from Trichoderma brevicompactum.

Cytochrome P450s are involved in a variety of monooxygenation reactions that require electron transfer from one redox partner to the other. We have recently shown the catalytic mechanism of a cytochrome P450 monooxygenase like protein (encoded by tri11 gene) that catalyzes the hydroxylation of 12,13-epoxytrichothec-9-ene (EPT) to produce trichodermol in the trichothecene biosynthetic pathway of trichodermin and harzianum A in Trichoderma brevicompactum [J Biol Inorg Chem. 22(8):1197-1209. doi: https://doi.org/10.1007/s00775-017-1496-6]. In the present work we have analyzed the effects of interaction of CPR FMN domain, a redox partner of tri11 protein, on its catalysis. The analysis of protein-protein complex interface showed various important contacts between the two protein partners that may aid in the process of electron transfer. The redox partner binding with tri11 protein on proximal side elicited catalytically important changes on the oppositely situated distal side that may help in stabilizing the active site and may play positive roles during the catalysis.

Kinetic characterization of the inhibition of protein tyrosine phosphatase-1B by Vanadyl (VO2+) chelates.

Protein tyrosine phosphatases (PTPases) are a prominent focus of drug design studies because of their roles in homeostasis and disorders of metabolism. These studies have met with little success because (1) virtually all inhibitors hitherto exhibit only competitive behavior and (2) a consensus sequence H/V-C-X5-R-S/T characterizes the active sites of PTPases, leading to low specificity of active site directed inhibitors. With protein tyrosine phosphatase-1B (PTP1B) identifed as the target enzyme of the vanadyl (VO2+) chelate bis(acetylacetonato)oxidovanadium(IV) [VO(acac)2] in 3T3-L1 adipocytes [Ou et al. J Biol Inorg Chem 10: 874-886, 2005], we compared the inhibition of PTP1B by VO(acac)2 with other VO2+-chelates, namely, bis(2-ethyl-maltolato)oxidovanadium(IV) [VO(Et-malto)2] and bis(3-hydroxy-2-methyl-4(1H)pyridinonato)oxidovanadium(IV) [VO(mpp)2] under steady-state conditions, using the soluble portion of the recombinant human enzyme (residues 1-321). Our results differed from those of previous investigations because we compared inhibition in the presence of the nonspecific substrate p-nitrophenylphosphate and the phosphotyrosine-containing undecapeptide DADEpYLIPQQG mimicking residues 988-998 of the epidermal growth factor receptor, a relevant, natural substrate. While VO(Et-malto)2 acts only as a noncompetitive inhibitor in the presence of either subtrate, VO(acac)2 exhibits classical uncompetitive inhibition in the presence of DADEpYLIPQQG but only apparent competitive inhibition with p-nitrophenylphosphate as substrate. Because uncompetitive inhibitors are more potent pharmacologically than competitive inhibitors, structural characterization of the site of uncompetitive binding of VO(acac)2 may provide a new direction for design of inhibitors for therapeutic purposes. Our results suggest also that the true behavior of other inhibitors may have been masked when assayed with only p-nitrophenylphosphate as substrate.

Theoretical insights into [NiFe]-hydrogenases oxidation resulting in a slowly reactivating inactive state.

[NiFe]-hydrogenases catalyse the relevant H2→2H++2e- reaction. Aerobic oxidation or anaerobic oxidation of this enzyme yields two inactive states called Ni-A and Ni-B. These states differ for the reactivation kinetics which are slower for Ni-A than Ni-B. While there is a general consensus on the structure of Ni-B, the nature of Ni-A is still controversial. Indeed, several crystallographic structures assigned to the Ni-A state have been proposed, which, however, differ for the nature of the bridging ligand and for the presence of modified cysteine residues. The spectroscopic characterization of Ni-A has been of little help due to small differences of calculated spectroscopic parameters, which does not allow to discriminate among the various forms proposed for Ni-A. Here, we report a DFT investigation on the nature of the Ni-A state, based on systematic explorations of conformational and configurational space relying on accurate energy calculations, and on comparisons of theoretical geometries with the X-ray structures currently available. The results presented in this work show that, among all plausible isomers featuring various protonation patterns and oxygenic ligands, the one corresponding to the crystallographic structure recently reported by Volbeda et al. (J Biol Inorg Chem 20:11-22, 19)-featuring a bridging hydroxide ligand and the sulphur atom of Cys64 oxidized to bridging sulfenate-is the most stable. However, isomers with cysteine residues oxidized to terminal sulfenate are very close in energy, and modifications in the network of H-bond with neighbouring residues may alter the stability order of such species.

Divalent copper ion bound amyloid-β(40) and amyloid-β(42) alloforms are less preferred than divalent zinc ion bound amyloid-β(40) and amyloid-β(42) alloforms.

Divalent copper and zinc ions bind to the amyloid-β(40) and amyloid-β(42) alloforms and affect their structural stability as well as their chemical and physical properties. Current literature debates the impact of copper ions on amyloid-β alloforms. Recently, we reported the structural and thermodynamic properties of apo amyloid-β and divalent zinc ion bound amyloid-β alloforms (see, Wise-Scira et al. in J Biol Inorg Chem 17:927-938, 2012 and Coskuner et al. in ACS Chem Neurosci 4: 310-320, 2013). In our search for understanding the impacts of transition metal ions on disordered amyloid-β, we also developed and reported new potential functions using quantum mechanics, which are required for high-quality molecular dynamics simulations of divalent copper ion bound amyloid-β alloforms (see, Wise and Coskuner in J Comput Chem 35:1278-1289, 2014). The structures and thermodynamic properties of the divalent copper ion bound amyloid-β(40) and amyloid-β(42) alloforms in an aqueous medium are studied. The secondary and tertiary structures of divalent copper ion bound amyloid-β(40) and amyloid-β(42) along with their thermodynamic properties including enthalpy, entropy, Gibbs free energy and potential of mean force surface are investigated. Results are compared to those for apo amyloid-β and divalent zinc ion bound amyloid-β alloforms. Results demonstrate that copper binding to Aβ alloforms is thermodynamically less preferred rather than zinc binding. Less compact structures of copper ion bound amyloid-β alloforms possess reduced stability in comparison to zinc ion bound amyloid-β alloforms. Cu(II) binding impacts the thermodynamic properties, secondary and tertiary structural properties of Aβ40 and Aβ42.

Residual Erythropoiesis Protects Against Cardiac Iron Loading in Transfusion Dependent Thalassaemia (TDT) By Lowering Labile Plasma Iron (LPI) through Transient Apotransferrin Generation

▪IntroductionMyocardial iron loading frequently complicates TDT but factors affecting the highly variable risk are not fully understood and current available predictors do not explain this variability. Chronic blood transfusion in TDT, by correcting anemia, is life saving but also aims to reduce ineffective erythropoiesis (IE) and its complications. Such transfusion will also decrease utilisation of transferrin (Tf) iron by the erythron and increase Tf saturation (TfSat) (Porter et al, Brit J Haematol 2014). Here we examine for the first time, how variable levels of residual erythropoiesis in TDT (marked by soluble transferrin receptors, sTfR), and the consequent presence of ApoTf, affect myocardial iron loading. We then examine how the difference between the transfusional iron loading rate (ILR) and Tf iron utilisation (calculated from sTfR levels) impact on myocardial iron loading. We finally test the hypotheses generated by these findings, using in vitro models of cellular uptake of NTBI and its redox active component LPI, the main conduits of myocardial iron uptake, in the presence and absence of ApoTf.MethodsA cohort of 73 TDT patients on deferasirox (DFX) had cardiac MRI and blood biomarkers measured at baseline and at 1.5 years later, over which time the ILR was calculated. Patients were divided into those with cardiac iron (24 cases, cardiac T2*<20ms, R2* >50s-1) and those without (59 controls, T2*>20ms, R2* <50s-1): biomarkers of iron metabolism and routine biochemical variables were compared in both groups with data shown for the 1.5 year measurements. 48h washout from DFX was ensured prior to blood sampling for which patients gave written informed consent. NTBI, LPI, sTfR, TfSat were performed as in Porter et al 2014, mouse HL-1 cardiomyocytes grown as in Claycomb et al, PNAS 1998, Ferric citrate (Fe-cit) prepared as in Evans et al, J Biol Inorg Chem 2008, and total cellular iron was assayed by ferrozine as in Riemer et al Anal Biochem 2004.Results and DiscussionTf iron utilisation and myocardial iron in TDT. Of all biomarkers analysed, sTfR was most significantly associated with heart iron (p<0.001) being >3x higher in controls than cases. NTBI, LPI, TfSat and ILR were not significantly different in cases and controls, but LIC and ferritin were higher in cases (p=0.003 and 0.002). With sTfR expressed as Tf iron utilisation rate (or erythroid Tf uptake rate, ETUR) after Beguin et al, Blood 1993 and compared to ILR, myocardial iron was present only when the net ILR (ILR-ETUR) exceeded 0.21 mg/kg/d (p<0.001). Above and below this threshold LIC, TfSat and NTBI were similar (9.7 vs 10.1 mg/g dry weight, 96 vs 98%, 3 vs 2.6µM, p=ns), while LPI was 3x higher (0.35 vs 0.1 µM p=0.01). Thus LPI was high only in patients where the net ILR exceeded 0.21 whereas LIC, TfSat, NTBI showed no difference between patients with and without myocardial siderosis, even when adjusted for the net ILR (Figure 1).Effect of ApoTf and Fe:cit ratio on NTBI uptake into cultured cardiomyocytes. ApoTf or TfSat <100% inhibited NTBI uptake into HL-1 cells as well as LPI but did not increase iron release from cells preloaded with iron. Fe-cit, the main NTBI species, is less visible as LPI than NTA iron.Increasing excess of cit:Fe increases the proportion of iron visible to the LPI assay,increases myocardial iron uptake and iron binding to Tf. Previous work (Evans et al J Biol Inorg Chem 2008) showed that Fe:cit ratio does not affect the NTBI assay, by contrast we show it affects the LPI assay. The ratio affects uptake and because uptake may be redox dependent (Oudit et al, Nat Med 2003), it is very likely that a redox facile NTBI, i.e. LPI, is the key species taken up by the heart, further supported by Fe:cit ratios <1:100 (more citrate) accounting for the majority of uptake (EC90=1.09µM, Figure 2)ConclusionTaken together these data suggest that the risk of myocardial siderosis in TDT is critically dependent on the degree of residual erythropoiesis and the net balance between the transfusional iron delivery (ILR) and the iron utilisation rate by the erythron (ETUR). Myocardial siderosis occurs when the net ILR-ETUR exceeds 0.21 mg/kg/day, the same threshold at which LPI is increased. The likely mechanism is that higher ILR-ETUR values decrease TfSat<100% hence allowing transiently formed ApoTf to decrease LPI. This mechanism is supported by the finding that as little as 1µM ApoTf inhibits NTBI uptake into cardiomyocytes in culture while also decreasing LPI. [Display omitted] [Display omitted] DisclosuresPorter:Celgene: Consultancy; Shire: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding.

SAPP modulates iron efflux from brain microvascular endothelial cells by stabilizing the ferrous iron exporter ferroportin.

A sequence within the E2 domain of soluble amyloid precursor protein (sAPP) stimulates iron efflux. This activity has been attributed to a ferroxidase activity suggested for this motif. We demonstrate that the stimulation of efflux supported by this peptide and by sAPPα is due to their stabilization of the ferrous iron exporter, ferroportin (Fpn), in the plasma membrane of human brain microvascular endothelial cells (hBMVEC). The peptide does not bind ferric iron explaining why it does not and thermodynamically cannot promote ferrous iron autoxidation. This peptide specifically pulls Fpn down from the plasma membrane of hBMVEC; based on these results, FTP, for ferroportin-targeting peptide, correctly identifies the function of this peptide. The data suggest that in stabilizing Fpn via the targeting due to the FTP sequence, sAPP will increase the flux of iron into the cerebral interstitium. This inference correlates with the observation of significant iron deposition in the amyloid plaques characteristic of Alzheimer's disease.

Electron self-exchange and self-amplified posttranslational modification in the hemoglobins from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002

Many heme proteins undergo covalent attachment of the heme group to a protein side chain. Such posttranslational modifications alter the thermodynamic and chemical properties of the holoprotein. Their importance in biological processes makes them attractive targets for mechanistic studies. We have proposed a reductively driven mechanism for the covalent heme attachment in the monomeric hemoglobins produced by the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 (GlbN) (Nothnagel et al. in J Biol Inorg Chem 16:539-552, 2011). These GlbNs coordinate the heme iron with two axial histidines, a feature that distinguishes them from most hemoglobins and conditions their redox properties. Here, we uncovered evidence for an electron exchange chain reaction leading to complete heme modification upon substoichiometric reduction of GlbN prepared in the ferric state. The GlbN electron self-exchange rate constants measured by NMR spectroscopy were on the order of 10(2)-10(3) M(-1) s(-1) and were consistent with the proposed autocatalytic process. NMR data on ferrous and ferric Synechococcus GlbN in solution indicated little dependence of the structure on the redox state of the iron or cross-link status of the heme group. This allowed the determination of lower bounds to the cross-exchange rate constants according to Marcus theory. The observations illustrate the ability of bishistidine hemoglobins to undergo facile interprotein electron transfer and the chemical relevance of such transfer for covalent heme attachment.

Zinc is decreased in prostate cancer: an established relationship of prostate cancer!

This minireview is prompted by the recent report of Banas et al. (J Biol Inorg Chem 15:1147-1155, 2010), which purports to show and concludes that zinc levels are increased in prostate cancer. Such a conclusion conflicts with the overwhelming corroborating clinical and experimental evidence that has amassed from numerous reports over the past approximately 60 years; these consistently show that prostate zinc levels are decreased in the development and progression of prostate cancer. We submit that this is an established relationship in prostate cancer that must be considered and described in any studies that purport to identify results that are inconsistent with this established relationship. In support of this relationship, we provide a minireview of the information that has led to the establishment of this relationship. As with most established clinical relationships, exceptions and anomalies often exist. However, these must be described and explained in the context of the established relationship, and not in the context of refutation of the established relationship, at least not until sufficient corroborating evidence overwhelms the existing evidence. This provides a background to address and to critique the report of Banas et al. Of broader and more serious implications are the widespread recalcitrance and/or lack of knowledge within the clinical and biomedical research community for recognition that zinc decrease in prostate cancer is an established relationship. This leads to misinformation and misinterpretations regarding clinical, experimental, and epidemiological issues that do not serve the best interests of the scientific, medical, and public communities.

Redox and metal-regulated oligomeric state for human porphobilinogen synthase activation

The oligomeric state of human porphobilinogen synthase (PBGS) [EC.4.2.1.24] is homooctamer, which consists of conformationally heterogenous subunits in the tertiary structure under air-saturated conditions. When PBGS is activated by reducing agent with zinc ion, a reservoir zinc ion coordinated by Cys(223) is transferred in the active center to be coordinated by Cys(122), Cys(124), and Cys(132) (Sawada et al. in J Biol Inorg Chem 10:199-207, 2005). The latter zinc ion serves as an electrophilic catalysis. In this study, we investigated a conformational change associated with the PBGS activation by reducing agent and zinc ion using analytical ultracentrifugation, negative staining electron microscopy, native PAGE, and enzyme activity staining. The results are in good agreement with our notion that the main component of PBGS is octamer with a few percent of hexamer and that the octamer changes spatial subunit arrangement upon reduction and further addition of zinc ion, accompanying decrease in f/f (0). It is concluded that redox-regulated PBGS activation via cleavage of disulfide bonds among Cys(122), Cys(124), and Cys(132) and coordination with zinc ion is closely linked to change in the oligomeric state.

Spectroscopic and computational investigation of three Cys-to-Ser mutants of nickel superoxide dismutase: insight into the roles played by the Cys2 and Cys6 active-site residues.

Nickel-dependent superoxide dismutase (NiSOD) is a member of a class of metalloenzymes that protect aerobic organisms from the damaging superoxide radical (O(2) (.-)). A distinctive and fascinating feature of NiSOD is the presence of active-site nickel-thiolate interactions involving the Cys2 and Cys6 residues. Mutation of one or both Cys residues to Ser prevents catalysis of O(2) (.-), demonstrating that both residues are necessary to support proper enzymatic activity (Ryan et al., J Biol Inorg Chem, 2010). In this study, we have employed a combined spectroscopic and computational approach to characterize three Cys-to-Ser (Cys --> Ser) mutants (C2S, C6S, and C2S/C6S NiSOD). Similar electronic absorption and magnetic circular dichroism spectra are observed for these mutants, indicating that they possess nearly identical active-site geometric and electronic structures. These spectroscopic data also reveal that the Ni(2+) ion in each mutant adopts a high-spin (S = 1) configuration, characteristic of a five- or six-coordinate ligand environment, as opposed to the low-spin (S = 0) configuration observed for the four-coordinate Ni(2+) center in the native enzyme. An analysis of the electronic absorption and magnetic circular dichroism data within the framework of density functional theory computations performed on a series of five- and six-coordinate C2S/C6S NiSOD models reveals that the active site of each Cys --> Ser mutant possesses an essentially six-coordinate Ni(2+) center with a rather weak axial bonding interaction. Factors contributing to the lack of catalytic activity displayed by the Cys --> Ser NiSOD mutants are explored.

Expanding the Range of Redox Potentials of the 2Fe-2S Clusters of the Outer Mitochondrial Membrane Protein MitoNEET

MitoNEET is a recently discovered outer mitochondrial membrane protein that harbors a 2Fe-2S cluster bound to a unique 3Cys-1His coordination (1). We measured a pH-dependent redox potential with a value near 0 mV at pH 7.0 (Em,7). This value lies intermediate between most low potential 4Cys-coordinated ferredoxin-like (Fd) centers (∼-300 mV) and most high potential 2Cys-2His-coordinated Rieske centers (∼+300 mV) (2). Upon replacing the single His87 ligand with Cys, we obtained an Em,7 near −300 mV closer to that of Fd clusters (Figure). Upon replacing Lys55 located near His87 with Met, Em,7 increases to near +250 mV, closer to that of high potential Rieske clusters (Figure). This shows that there is a large interaction between Lys55 and His87 and that mitoNEET is robust to large changes in the Em,7 of its 2Fe-2S clusters. Thus, we have engineered stable mutant mitoNEET with Em,7 values over a range of almost 600 mV.(1) Paddock et al. (2007) Proc Natl. Acad. Sci USA 104, 14342-14347(2) Meyer (2008) J Biol Inorg Chem 13, 157-170Supported by NIH (GM41637, GM54038 and DK54441).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Scanning chimeragenesis: the approach used to change the substrate selectivity of fatty acid monooxygenase CYP102A1 to that of terpene ω-hydroxylase CYP4C7

CYP102A1 is a highly active, water-soluble, bacterial monooxygenase enzyme that contains both substrate-binding heme and diflavin reductase subunits, both in a single polypeptide. Recently we developed a procedure which uses the known structure of the substrate-bound heme domain of CYP102A1 and its sequence homology with a cytochrome P450 of unknown structure, both of which react with a common substrate but produce different products, to create recombinant enzymes which have substrate selectivity different from that of CYP102A1, and produce the product of the enzyme of unknown structure. Insect CYP4C7, a terpene hydroxylase from the cockroach, was chosen as the cytochrome P450 of unknown structure, and farnesol was chosen as the substrate. CYP102A1 oxidizes farnesol to three products (2,3-epoxyfarnesol, 10,11-epoxyfarnesol, and 9-hydroxyfarnesol), whereas CYP4C7 produces 12-hydroxyfarnesol as the major product. In earlier work it was found that the chimera C(78-82,F87L) showed a change in substrate selectivity from fatty acids to farnesol, and was approximately sixfold more active than wild-type CYP102A1 (Chen et al. in J Biol Inorg Chem 13:813-824, 2008), but neither it nor any other earlier chimera produced 12-hydroxyfarnesol. In this work we added amino acid residues 327-332, to create six new full-length, functional chimeric proteins. Four of these, the most active of which was C(78-82,F87L,328-330), produce 12-hydroxyfarnesol as the major product, with approximately twofold increase in turnover number as compared with wild-type CYP102A1 toward farnesol. Methylfarnesoate was metabolized to 12-hydroxymethylfarnesoate (70%) and 10,11-epoxymethylfarnesoate (juvenile hormone III) (30%). The latter is metabolized to 65% 12-hydroxy-10,11-epoxymethylfarnesoate and 35% 15-hydroxy-10,11-epoxymethylfarnesoate. Substitution of residues 328-330, APA, by VPL was crucial to accomplishing this change in product.

Redox Potential of the Outer-Mitochondrial Membrane 2Fe-2S Protein MitoNEET

MitoNEET was recently discovered as a binding target for the anti-diabetes drug pioglitazone (1). It harbors a pH-labile 2Fe-2S cluster coordinated by three cysteines and one histidine (His87) (2). We measured a pH-dependent redox potential of +35 mV (pH 7.5) that lies intermediate between most low potential 4Cys-coordinated ferredoxin-like centers (∼-300 mV) and most high potential 2Cys-2His-coordinated Rieske centers (∼+300 mV) (3). In addition, its redox potential was ∼40mV lower in the presence of phosphate ions. This can be explained by binding of a phosphate ion near the cluster as reported elsewhere (Homer, poster). The H87C mutant, which becomes 4 Cys coordinated, has a more negative reduction potential similar to a ferredoxin (∼ -200mV). Our results show that the redox potential is sensitive to the coordination of the cluster and that mitoNEET's unique coordination geometry is likely essential for its unknown redox function. (1) Colca et al. (2004) Am J Physiol Endocrinol Metab 286 E252-E260. (2) Paddock et al. (2007) Proc Natl. Acad. Sci USA 104, 14342-14347. (3) Meyer (2008) J Biol Inorg Chem 13, 157-170 Supported by NIH (GM41637, GM54038 and DK54441).View Large Image Figure ViewerDownload Hi-res image Download (PPT) View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Comparative Study of Chemical Approaches to the Solid-Phase Synthesis of a Tumor-Seeking α-MSH Analogue

The synthesis of a cyclic melanocortin analogue (H-pz-βAla-Nle-cyclo[Asp-His-DPhe-Arg-Trp-Lys]-NH2), where the Boc-protected derivative of a metal-chelating pyrazolyl ligand (pz) was inserted as N-terminal residue, was addressed by several different Fmoc/tBu and Boc/Bzl solid-phase strategies. On-resin cyclization was achieved immediately following incorporation of Asp, by condensation of the Asp side chain carboxyl with the Lys side chain primary amine after selective and simultaneous removal of side chain protecting groups. The success of the synthesis was highly dependent on the chemical strategy employed, with Boc/Bzl chemistry giving the best results. On the light of our findings, Fmoc/tBu strategies are not advantageous for the solid-phase synthesis of this particular type of lactam-bridged peptides. Last, but not least, the target peptide was recently found to have promising tumor-seeking properties (J Biol Inorg Chem 13:449–459, 2008).

Structural basis for VO2+-inhibition of nitrogenase activity: (B) pH-sensitive inner-sphere rearrangements in the 1H-environment of the metal coordination site of the nitrogenase Fe–protein identified by ENDOR spectroscopy

The nitrogenase Fe-protein is the specific ATP-activated electron donor to the active site-containing nitrogenase MoFe-protein. It has been previously demonstrated that different VO(2+)-nucleotide coordination environments exist for the Fe-protein that depend on pH and are distinguishable by EPR spectroscopy. After having studied the nitrogenase 31P and 23Na superhyperfine structure for this system by electron nuclear double resonance (ENDOR) spectroscopy (Petersen et al. 2008 in J Biol Inorg Chem. doi:10.1007/s00775-008-0360-0), we here report on the 1H-interactions with the nucleotide-bound metal center after substitution of the natural diamagnetic metal Mg2+ with paramagnetic oxo-vanadium(IV). ENDOR spectra show a number of resonances arising from interactions of the VO2+ ion with protons. In the presence of reduced Fe-protein and VO2+ ADP, at least three sets of nonexchangeable protons are detected. At low pH the superhyperfine couplings of most of these are consistent with proton interactions originating from the nucleotide. There is no indication of 1H-resonances that exchange in D2O at neutral pH and could be assigned to inner-sphere hydroxyl coordination. Exchangeable hydroxyl protons in the inner coordination sphere with reduced Fe-protein are only found in the low pH form; based on their hyperfine tensor components these have been assigned to an axially coordinated hydroxyl water molecule. The pH-dependent alterations of the proton couplings that exchange in D2O suggest that they are partially caused by a rearrangement in the local hydroxyl coordination environment of the metal center. These rearrangements especially affect the apical metal position, where an axially coordinated water present at low pH is absent at neutral pH. Oxidation of the Fe-protein induced substantial changes in the electron-nucleus interactions. This indicates that the oxidation state of the iron-sulfur cluster has an important effect on the metal coordination environment at the nucleotide binding site of the Fe-protein. The distinct VO(2+)-nucleotide coordination structures with ADP and ATP and the redox state of the [4Fe-4S] cluster imply that VO2+ has a critical influence on the switch regions of the regulatory protein, and, taken together, this provides a plausible explanation for the inhibitory action of VO2+.

Erratum to Oxoiron(IV) complexes of the tris(2-pyridylmethyl)amine ligand family: effect of pyridine α-substituents

Erratum to: J Biol Inorg Chem 11:272–276 DOI 10.1007/s00775-006-0089-6

Substrate binding to mononuclear metallo-beta-lactamase from Bacillus cereus.

Structure and dynamics of substrate binding (cefotaxime) to the catalytic pocket of the mononuclear zinc-beta-lactamase from Bacillus cereus are investigated by molecular dynamics simulations. The calculations, which are based on the hydrogen-bond pattern recently proposed by Dal Peraro et al. (J Biol Inorg Chem 2002; 7:704-712), are carried out for both the free and the complexed enzyme. In the resting state, active site pattern and temperature B-factors are in agreement with crystallographic data. In the complexed form, cefotaxime is accommodated into a stable orientation in the catalytic pocket within the nanosecond timescale, interacting with the enzyme zinc-bound hydroxide and the surrounding loops. The beta-lactam ring remains stable and very close to the hydroxide nucleophile agent, giving a stable representation of the productive enzyme-substrate complex.

Is Ag(I) an adequate probe for Cu(I) in structural copper-metallothionein studies? The binding features of Ag(I) to mammalian metallothionein 1.

The binding abilities of silver(I) to mammalian MT 1 have been studied and compared with those of copper(I), recently reported [Bofill et al. (2001) J Biol Inorg Chem 6:408-417], with the aim of analyzing the suitability of Ag(I) as a Cu(I) probe in Cu-MT studies. The Zn/Ag replacement in recombinant mouse Zn(7)-MT 1 and corresponding Zn(4)-alphaMT 1 and Zn(3)-betaMT 1 fragments, as well as the stepwise incorporation of Ag(I) to the corresponding apo-MTs, have been followed in parallel by various spectroscopic techniques including electronic absorption (UV-vis), circular dichroism (CD) and electrospray mass spectrometry coupled to capillary zone electrophoresis (CZE-ESI-MS). A comparative analysis of the sets of data obtained in the titration of Zn(7)-MT 1, Zn(4)-alphaMT 1 and Zn(3)-betaMT 1 with AgClO(4) at pH 7.5 and 2.5 has led to the reaction pathways followed during the incorporation of silver to these proteins under these specific conditions, disclosing unprecedented stoichiometries and structural features for the species formed. Thus, the Zn/Ag replacement in Zn(7)-MT 1 at pH 7.5 has revealed the subsequent formation of Ag(4)Zn(5)-MT, Ag(7)Zn(3)-MT, Ag(8)Zn(3)-MT, Ag(10)Zn(2)-MT, Ag(12)Zn(1)-MT, Ag(x)-MT, x=14-19, whose structure consists of two additive domains only if Zn(II) remains coordinated to the protein. A second structural role for Zn(II) has been deduced from the different folding found for the Ag(x)-MT species of the same stoichiometry formed at pH 7.5 or 2.5. Comparison of the binding features of Cu(I) and Ag(I) to the entire MT at pH 7.5 shows that, among all the micro(x)Zn(y)-MT (0<or= y<7) species found, only M(I)(4)Zn(5)-MT [(Zn(4))(alpha)(micro(4)Zn(1))(beta)] and M(I)(7)Zn(3)-MT [(micro(3)Zn(2))(alpha)(micro(4)Zn(1))(beta)], which form during the first stages of the Zn(II)/M(I) metal replacement, show comparable 3D structures; thus, they are the only species where Ag(I) ions can be predicted to be an adequate probe for Cu(I).