1,073 research outputs found
A Nonclassical Dihydrogen Adduct of S = Ā½ Fe(I)
We have exploited the capacity of the ā(SiP^(iPr)_3)Fe(I)ā scaffold to accommodate additional axial ligands and characterized the mononuclear S = 1/2 H_2 adduct complex (SiP^(iPr)_3)Fe^I(H_2). EPR and ENDOR data, in the context of X-ray structural results, revealed that this complex provides a highly unusual example of an open-shell metal complex that binds dihydrogen as a ligand. The H2 ligand at 2 K dynamically reorients within the ligand-binding pocket, tunneling among the energy minima created by strong interactions with the three FeāP bonds
Q-band electron nuclear double resonance (ENDOR) and X-band EPR of the sulfobetaine 12 heat-treated cytochrome c oxidase complex
Heat treatment of the bovine cytochrome c oxidase complex in the zwitterionic detergent sulfobetaine 12 (SB-12) results in loss of subunit III and the appearance of a type II copper center as characterized by electron paramagnetic resonance (EPR) spectroscopy. Previous authors (Nilsson, T., Copeland, R. A., Smith, P. A., and Chan, S. I. (1988) Biochemistry 27, 8254-8260) have interpreted this type II copper center as a modified version of the CuA site. By using electron nuclear double resonance spectroscopy, it is found that the CuA proton and nitrogen resonances remain present in the SB-12 heat-treated enzyme and that three new nitrogen resonances appear having hyperfine coupling constants consistent with histidine ligation. These hyperfine coupling constants correlate well with those recently found for the CuB histidines from the cytochrome aa3-600 quinol oxidase from Bacillus subtilis (Fann, Y. C., Ahmed, I., Blackburn, N. J., Boswell, J. S., Verkhovskaya, M. L., Hoffman, B. M., and Wikstrƶm, M. (1995) Biochemistry 34, 10245-10255). In addition, the total EPR-detectable copper concentration per enzyme molecule approximately doubles upon SB-12 heat treatment. Finally, the observed type II copper EPR spectrum is virtually indistinguishable from the EPR spectrum of CuB of the as-isolated cytochrome bo3 complex from Escherichia coli. These data indicate that the type II copper species that appears results from a breaking of the strong antiferromagnetic coupling of the heme a3-CuB binuclear center
Allosteric Control of O2 Reactivity in Rieske Oxygenases
Oxygen is Natureās perfect reagent. On one hand, it is potentially a very strong oxidant. On the other hand, this potential is caged because the two highest energy valence electrons of the O2 molecule are unpaired. As a result, O2 is relatively unreactive with most other molecules, as almost all of these have paired electrons. Consequently, by modulating the properties of the O2 valence electrons, Nature can generate a reactive species under controlled conditions, catalyzing difficult reactions while still rigorously enforcing specificity. Special sets of enzymes termed oxygenases and oxidases have evolved to perform this task
Advanced paramagnetic resonance spectroscopies of ironāsulfur proteins: Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM)
AbstractThe advanced electron paramagnetic resonance (EPR) techniques, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies, provide unique insights into the structure, coordination chemistry, and biochemical mechanism of nature's widely distributed ironāsulfur cluster (FeS) proteins. This review describes the ENDOR and ESEEM techniques and then provides a series of case studies on their application to a wide variety of FeS proteins including ferredoxins, nitrogenase, and radical SAM enzymes. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases
Composition and Structure of the Inorganic Core of Relaxed Intermediate
Activation of the diferrous center of the Ī²2 (R2) subunit of the class 1a Escherichia coli ribonucleotide reductases by reaction with O2 followed by one-electron reduction yields a spin-coupled, paramagnetic Fe(III)/Fe(IV) intermediate, denoted X, whose identity has been sought by multiple investigators for over a quarter of a century. To determine the composition and structure of X, the present study has applied 57Fe, 14,15N, 17O, and 1H electron nuclear double resonance (ENDOR) measurements combined with quantitative measurements of 17O and 1H electron paramagnetic resonance line-broadening studies to wild-type X, which is very short-lived, and to X prepared with the Y122F mutant, which has a lifetime of many seconds. Previous studies have established that over several seconds the as-formed X(Y122F) relaxes to an equilibrium structure. The present study focuses on the relaxed structure. It establishes that the inorganic core of relaxed X has the composition [(OHā)FeIIIāOāFeIV]: there is no second inorganic oxygenic bridge, neither oxo nor hydroxo. Geometric analysis of the 14N ENDOR data, together with recent extended X-ray absorption fine structure measurements of the FeāFe distance (Dassama, L. M.; et al. J. Am. Chem. Soc. 2013, 135, 16758), supports the view that X contains a ādiamond-coreā Fe(III)/Fe(IV) center, with the irons bridged by two ligands. One bridging ligand is the oxo bridge (OBr) derived from O2 gas. Given the absence of a second inorganic oxygenic bridge, the second bridging ligand must be protein derived, and is most plausibly assigned as a carboxyl oxygen from E238.United States. National Institutes of Health (GM 111097)United States. National Institutes of Health (GM 29595
Electron Transfer Precedes ATP Hydrolysis during Nitrogenase Catalysis
The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 sā1, 25 Ā°C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 sā1, 25 Ā°C), (ii) ATP hydrolysis (kATP = 70 sā1, 25 Ā°C), (iii) Phosphate release (kPi = 16 sā1, 25 Ā°C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 sā1, 25 Ā°C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and proteināprotein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein
Responses of Mn\u3csup\u3e2+\u3c/sup\u3e Speciation in \u3cem\u3eDeinococcus radiodurans\u3c/em\u3e and \u3cem\u3eEscherichia coli\u3c/em\u3e to Ī³-Radiation by Advanced Paramagnetic Resonance Methods
The remarkable ability of bacterium Deinococcus radiodurans to survive extreme doses of Ī³-rays (12,000 Gy), 20 times greater than Escherichia coli, is undiminished by loss of Mn-dependent superoxide dismutase (SodA). D. radiodurans radiation resistance is attributed to the accumulation of low-molecular-weight (LMW) āantioxidantā Mn2+āmetabolite complexes that protect essential enzymes from oxidative damage. However, in vivo information about such complexes within D. radiodurans cells is lacking, and the idea that they can supplant reactive-oxygen-species (ROS)āscavenging enzymes remains controversial. In this report, measurements by advanced paramagnetic resonance techniques [electron-spin-echo (ESE)-EPR/electron nuclear double resonance/ESE envelope modulation (ESEEM)] reveal differential details of the in vivo Mn2+ speciation in D. radiodurans and E. coli cells and their responses to 10 kGy Ī³-irradiation. The Mn2+ of D. radiodurans exists predominantly as LMW complexes with nitrogenous metabolites and orthophosphate, with negligible EPR signal from Mn2+ of SodA. Thus, the extreme radiation resistance of D. radiodurans cells cannot be attributed to SodA. Correspondingly, 10 kGy irradiation causes no change in D. radiodurans Mn2+ speciation, despite the paucity of holo-SodA. In contrast, the EPR signal of E. coli is dominated by signals from low-symmetry enzyme sites such as that of SodA, with a minority pool of LMW Mn2+ complexes that show negligible coordination by nitrogenous metabolites. Nonetheless, irradiation of E. coli majorly changes LMW Mn2+ speciation, with extensive binding of nitrogenous ligands created by irradiation. We infer that E. coli is highly susceptible to radiation-induced ROS because it lacks an adequate supply of LMW Mn antioxidants
Recommended from our members
Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases.
The clinical success of multitargeted kinase inhibitors has stimulated efforts to identify promiscuous drugs with optimal selectivity profiles. It remains unclear to what extent such drugs can be rationally designed, particularly for combinations of targets that are structurally divergent. Here we report the systematic discovery of molecules that potently inhibit both tyrosine kinases and phosphatidylinositol-3-OH kinases, two protein families that are among the most intensely pursued cancer drug targets. Through iterative chemical synthesis, X-ray crystallography and kinome-level biochemical profiling, we identified compounds that inhibit a spectrum of new target combinations in these two families. Crystal structures revealed that the dual selectivity of these molecules is controlled by a hydrophobic pocket conserved in both enzyme classes and accessible through a rotatable bond in the drug skeleton. We show that one compound, PP121, blocks the proliferation of tumor cells by direct inhibition of oncogenic tyrosine kinases and phosphatidylinositol-3-OH kinases. These molecules demonstrate the feasibility of accessing a chemical space that intersects two families of oncogenes
Recommended from our members
Fluorescent humanized anti-CEA antibody specifically labels metastatic pancreatic cancer in a patient-derived orthotopic xenograft (PDOX) mouse model.
Pancreatic cancer is a highly lethal disease in part due to incomplete tumor resection. Targeting by tumor-specific antibodies conjugated with a fluorescent label can result in selective labeling of cancer in vivo for surgical navigation. In the present study, we describe a patient-derived orthotopic xenograft model of pancreatic cancer that recapitulated the disease on a gross and microscopic level, along with physiologic clinical manifestations. We additionally show that the use of an anti-CEA antibody conjugated to the near-infrared (NIR) fluorescent dye, IRDye800CW, can selectively highlight the pancreatic cancer and its metastases in this model with a tumor-to-background ratio of 3.5 (SEM 0.9). The present results demonstrate the clinical potential of this labeling technique for fluorescence-guided surgery of pancreatic cancer
- ā¦