Cysteine Radical Research Articles

Computational insights into chemoselectivity of Trans-4-Hydroxy-L-Proline dehydratase HypD

The dehydration of trans-4-hydroxy-L-proline (t4L-HP) yielding 1-pyrroline-5-carboxylate in certain gut bacteria requires the glycyl radical enzyme HypD, while isomerisation of trans-4-hydroxy-D-proline (t4D-HP) generating 2-amino-4-ketopentanoate involves a homologous enzyme HplG. Although the former enzyme exhibits high chemoselectivity when catalysing the dehydration and is regarded as a promising target for treating bacteria-caused gut diseases, its catalytic mechanism remains elusive. Herein, we deeply investigated the two reaction pathways by carrying out theoretical studies at the QM/MM level. Our calculations show that, in HypD, a catalytic Cys434 radical facilitates the Cδ–H activation in an initially deprotonated zwitterionic t4L-HP, which is followed by a heterolytic cleavage of t4L-HP’s Cγ–O bond with a water formed being assisted by the conserved Asp279. In the isomerisation pathway, the Cys434 radical would abstract the hydrogen atom from Cγ of zwitterionic t4L-HP, and then Asp279 cleaves the Cδ–N bond by abstracting a proton from the hydroxyl group of t4L-HP. The two reaction paths are calculated to have barriers of 18.0 and 22.8 kcal/mol, respectively, which well explain HypD’s chemoselectivity. In the mechanisms suggested by our study, Asp279 plays a role as “proton shuttle” and regulates the protonation state of t4L-HP in the selective hydrogen atom abstraction invoked by the cysteine radical. Our findings provide an insight of universal significance into radical-based enzymatic catalysis and expand the understanding of how nature wisely modulates catalytic reactivity and selectivity by switching the substrate’s protonation state.

Cysteine Radical and Glutamate Collaboratively Enable C-H Bond Activation and C-N Bond Cleavage in a Glycyl Radical Enzyme HplG.

Hydroxyprolines are abundant in nature and widely utilized by many living organisms. Isomerization of trans-4-hydroxy-d-proline (t4D-HP) to generate 2-amino-4-ketopentanoate has been found to need a glycyl radical enzyme HplG, which catalyzes the cleavage of the C-N bond, while dehydration of trans-4-hydroxy-l-proline involves a homologous enzyme of HplG. Herein, molecular dynamics simulations and quantum mechanics/molecular mechanics (QM/MM) calculations are employed to understand the reaction mechanism of HplG. Two possible reaction pathways of HplG have been explored to decipher the origin of its chemoselectivity. The QM/MM calculations reveal that the isomerization proceeds via an initial hydrogen shift from the Cγ site of t4D-HP to a catalytic cysteine radical, followed by cleavage of the Cδ-N bond in t4D-HP to form a radical intermediate that captures a hydrogen atom from the cysteine. Activation of the Cδ-H bond in t4D-HP to bring about dehydration of t4D-HP possesses an extremely high energy barrier, thus rendering the dehydration pathway implausible in HplG. On the basis of the current calculations, conserved residue Glu429 plays a pivotal role in the isomerization pathway: the hydrogen bonding between it and t4D-HP weakens the hydroxyalkyl Cγ-Hγ bond, and it acts as a proton acceptor to trigger the cleavage of the C-N bond in t4D-HP. Our current QM/MM calculations rationalize the origin of the experimentally observed chemoselectivity of HplG and propose an H-bond-assisted bond activation strategy in radical-containing enzymes. These findings have general implications on radical-mediated enzymatic catalysis and expand our understanding of how nature wisely and selectively activates the C-H bond to modulate catalytic selectivity.

Direct effects of low-energy electrons on including sulfur bonds in proteins: a second-order Møller–Plesset perturbation (MP2) theory approach

In an attempt to describe how low-energy electrons (LEEs) damage the polypeptide chain at disulfide bridges, ab initio electronic structure estimates on LEE interactions with cysteine-cysteine (Cys-Cys) disulfide bond model have been performed. Here, the fundamental mechanisms in LEE impression on S-S and C-S bond ruptures in the Cys-Cys model have been discussed. The electronic energy was calculated using the MP2 method with a Hartree–Fock exchange during the SCF and the Møller–Plesset correlation energy correction on the converged HF orbitals with 6-311++G(d,p) atomic orbital basis set. Further, six more sets of diffuse s and p functions with extra basis on the sulfur and relevant carbon atoms were used to describe the added electron to located away as much as possible from the nuclei in anions. The bonds rupture mechanisms involve the primary placement of LEEs to the π* orbital of the model to construct the shape-resonance state following by an adiabatic or nonadiabatic electron migration to either S-S or C-S bond σ* orbital. The formed radical anion undergoes S-S or C-S bonds cleavage by energy barriers of ca. 5.68 and 9.19 kcal/mol, respectively, to produce either (2-amino-2-carboxyethyl) sulfanyl (cysteine radical), aziridine-2-carboxylic acid or mercapto-L-cysteine lesions. In SMD solvent, calculations suggest electronically stable of the formed π* and σ* states by solvation, something that induces either S-S or C-S bond break even when the electron energy is near zero. The required barrier energy of only 0 to < 0.4 eV indicates a high kinetic favorable fragmentation for involved sulfur polypeptides with LEEs. Communicated by Ramaswamy H. Sarma

Probing the Cys-Tyr Cofactor Biogenesis in Cysteine Dioxygenase by the Genetic Incorporation of Fluorotyrosine.

Cysteine dioxygenase (CDO) is a nonheme iron enzyme that adds two oxygen atoms from dioxygen to the sulfur atom of l-cysteine. Adjacent to the iron site of mammalian CDO, there is a post-translationally generated Cys-Tyr cofactor, whose presence substantially enhances the oxygenase activity. The formation of the Cys-Tyr cofactor in CDO is an autocatalytic process, and it is challenging to study by traditional techniques because the cross-linking reaction is a side, uncoupled, single-turnover oxidation buried among multiple turnovers of l-cysteine oxygenation. Here, we take advantage of our recent success in obtaining a purely uncross-linked human CDO due to site-specific incorporation of 3,5-difluoro-l-tyrosine (F2-Tyr) at the cross-linking site through the genetic code expansion strategy. Using EPR spectroscopy, we show that nitric oxide (•NO), an oxygen surrogate, similarly binds to uncross-linked F2-Tyr157 CDO as in wild-type human CDO. We determined X-ray crystal structures of uncross-linked F2-Tyr157 CDO and mature wild-type CDO in complex with both l-cysteine and •NO. These structural data reveal that the active site cysteine (Cys93 in the human enzyme), rather than the generally expected tyrosine (i.e., Tyr157), is well-aligned to be oxidized should the normal oxidation reaction uncouple. This structure-based understanding is further supported by a computational study with models built on the uncross-linked ternary complex structure. Together, these results strongly suggest that the first target to oxidize during the iron-assisted Cys-Tyr cofactor biogenesis is Cys93. Based on these data, a plausible reaction mechanism implementing a cysteine radical involved in the cross-link formation is proposed.

Adsorption‐induced Symmetry Distortions in W@Au12 Nanoclusters, Leading to Enhanced Hyperpolarizabilities

AbstractWe study how the adsorption of molecules on the stable gold icosahedral W@Au12 nanocluster affects its structure, specifically the inversion symmetry, in relation to the hyperpolarizability of the cluster‐molecule complex. As adsorbed molecules, we selected the achiral physisorbed H2S and chemisorbed HS radical, the chiral physisorbed cysteine and the chemisorbed cysteine radical. We show that adsorption leads to deformation of W@Au12 that removes the inversion symmetry of the cluster itself. Employing the continuous symmetry measure (CSM) methodology we are able to quantify the distortions, relative to the undisturbed cluster. We then evaluate the hyperpolarizabilities which emerge due to the lack of centrosymmetry. We find that large hyperpolarizabilities, by far exceeding those of the organic molecules, emerge as a result of the complexation of these two different species. This result is of relevance to studies of nonlinear optics, where methods of enhancing that property in molecules of choice are desired.

Cysteine Radical/Metal Ion Adducts: A Gas-Phase Structural Elucidation and Reactivity Study.

The formation and investigation of sulfur-based cysteine radicals cationized by a group 1A metal ion or Ag+ in the gas phase are reported. Gas-phase ion-molecule reactions (IMR) and infrared multiple-photon dissociation (IRMPD) spectroscopy revealed that the Li+ , Na+ , and K+ adducts of the cysteine radical remain S-based radicals as initially formed. Theoretical calculations for the three alkali metal ions found that the lowest-energy isomers are Cα -based radicals, but they are not observed experimentally owing to the barriers associated with the hydrogen-atom transfer. A mechanism for the S-to-Cα radical rearrangement in the metal ion complexes was proposed, and the relative energies of the associated energy barriers were found to be Li+ >Na+ >K+ at all levels of theory. Relative to the B3LYP functional, other levels of calculation gave significantly higher barriers (by 35-40 kJ mol-1 at MP2 and 44-47 kJ mol-1 at the CCSD level) using the same basis set. Unlike the alkali metal adducts, the cysteine radical/Ag+ complex rearranged from the S-based radical to an unreactive species as indicated by IMRs and IRMPD spectroscopy. This is consistent with the Ag+ /cysteine radical complex having a lower S-to-Cα radical conversion barrier, as predicted by the MP2 and CCSD levels of theory.

Gas-phase tyrosine-to-cysteine radical migration in model systems.

Radical migration, both intramolecular and intermolecular, from the tyrosine phenoxyl radical Tyr(O(∙)) to the cysteine radical Cys(S(∙)) in model peptide systems was observed in the gas phase. Ion-molecule reactions (IMRs) between the radical cation of homotyrosine and propyl thiol resulted in a fast hydrogen atom transfer. In addition, radical cations of the peptide LysTyrCys were formed via two different methods, affording regiospecific production of Tyr(O(∙)) or Cys(S(∙)) radicals. Collision-induced dissociation of these isomeric species displayed evidence of radical migration from the oxygen to sulfur, but not for the reverse process. This was supported by theoretical calculations, which showed the Cys(S(∙)) radical slightly lower in energy than the Tyr(O(∙)) isomer. IMRs of the LysTyrCys radical cation with allyl iodide further confirmed these findings. A mechanism for radical migration involving a proton shuttle by the C-terminal carboxylic group is proposed.

Radical Propagation in E. coli Class Ia Ribonucleotide Reductase Initiated by a 2,3,5‐trifluorotyrosyl Radical Cofactor

Escherichia coli class Ia ribonucleotide reductase (RNR) catalyzes the formation of all four deoxynucleoside 5′‐diphosphates from their corresponding nucleoside 5′‐diphosphates. Class Ia RNRs are composed of two homodimeric subunits, α2 and β2, that form the active complex. A stable tyrosyl radical (Y122•) in the β2 subunit generates a transient cysteine radical (C439•) located 35 Å away in the α2 subunit by a mechanism that is proposed to involve the concerted movement of protons and electrons (proton‐coupled electron transfer or PCET) through several aromatic amino acid residues (Y122 ‐ [W48] ‐ Y356 in β2 to Y731 ‐ Y730 ‐ C439 in α2). 2,3,5‐trifluorotyrosine (2,3,5‐F3Y) has been site‐specifically inserted at Y122 using a polyspecific aminoacyl tRNA synthetase to modulate the driving force of radical propagation. The diferric‐2,3,5‐F3Y• radical cofactor self assembles in vitro producing 0.8‐1.0 F3Y•/β2. The reaction of Y122(2,3,5)F3Y‐β2, α2, substrate and effector generates 0.3‐0.5 equiv. of a putative Y356• that is kinetically and chemically competent in nucleotide reduction. At 5°C, complete re‐oxidation of 2,3,5‐F3Y by Y356• is recorded and represents our first observation of the disappearance and reappearance of the stable radical at position 122. Altering the driving force for radical propagation by simply 10 mV partially lifts the protein conformational gate that governs radical transport allowing mechanistic insight into the details of PCET.

Quantum-chemical Study on the Thermodynamical Aspect of Competitive Inhibition of Ribonucleotide Reductase by trans-Resveratrol, trans- Piceatannol and Hydroxyurea

Ribonucleotide reductase is an enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides. We present for the first time a quantum-chemical study of the thermodynamics of reactions of naturally occurring potent inhibitors (trans-resveratrol, trans-piceatannol and hydroxyurea) of ribonucleotide reductase with tyrosine radical and cysteine radical, scavenging of which is crucial for the inhibition of the enzyme. Density functional theory has been applied to compute the Gibbs free enthalpy changes for these reactions in the gas phase and in the presence of water medium. Various reaction pathways have been analyzed. The results obtained prove that trans-resveratrol 4’-OH group is mainly responsible for effective reaction with these enzymatic free radicals. In water medium, the reactions studied are characterized by more negative values of the Gibbs free enthalpy changes than in vacuum. It was found that transresveratrol and trans-piceatannol may be efficient inhibitors of the enzyme (trans-piceatannol is more efficient than transresveratrol). Because inhibition of ribonucleotide reductase is essential for blockage of the cancer development pathways, the polyphenols studied may block diverse processes (including cancerogenesis) by inhibition of free radical reaction steps that occur during the catalytic action of this enzyme. Keywords: Enzyme inhibition, ribonucleotide reductase, trans-resveratrol, trans-piceatannol.

A 2.8 Å Fe–Fe Separation in the Fe2III/IV Intermediate, X, from Escherichia coli Ribonucleotide Reductase

A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe2(III/III)/tyrosyl radical cofactor in its β subunit to oxidize a cysteine residue ~35 Å away in its α subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the Escherichia coli RNR-β cofactor, reaction of the protein's Fe2(II/II) complex with O2 results in accumulation of an Fe2(III/IV) cluster, termed X, which oxidizes the adjacent tyrosine (Y122) to the radical (Y122(•)) as the cluster is converted to the μ-oxo-Fe2(III/III) product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, X has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a Fe-Fe separation (d(Fe-Fe)) of 2.5 Å, which was interpreted to imply the presence of three single-atom bridges (O(2-), HO(-), and/or μ-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have d(Fe-Fe) ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of X. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O2 in situ from chlorite using the enzyme chlorite dismutase to prepare X at ~2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured d(Fe-Fe) = 2.78 Å is fully consistent with computational models containing a (μ-oxo)2-Fe2(III/IV) core. Correction of the d(Fe-Fe) brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which X generates Y122(•).

Structure and Reactivity of Homocysteine Radical Cation in the Gas Phase Studied by Ion–Molecule Reactions and Infrared Multiple Photon Dissociation

The reactivity of the cysteine (Cys) and homocysteine (Hcy) radical cation was studied using ion-molecule reactions. The radical cations were generated via collision-induced dissociation (CID) of their S-nitrosylated precursors. Cleavage of the S-NO bond led to the formation of the radical initially positioned on the sulfur atom. The reactions of the radical cations with dimethyl disulfide revealed that the cysteine radical cation reacts more quickly than the homocysteine radical cation. Infrared multiple photon dissociation (IRMPD) spectroscopy and density functional theory (DFT) calculations were used to determine the structure of the homocysteine radical cation, which was compared to the previously published structure of the cysteine radical cation (Sinha et al. Phys. Chem. Chem. Phys. 2010, 12, 9794-9800). IRMPD spectroscopy and DFT calculations revealed that this difference in radical reactivity was not a result of a radical rearrangement for the homocysteine radical cation but rather that the reactivity was modulated by stronger hydrogen bonding.

Freezing-thawing induces alterations in histone H1-DNA binding and the breaking of protein-DNA disulfide bonds in boar sperm

The main aim of this work is to gain insight into the mechanisms by which freezing-thawing alters the nucleoprotein structure of boar sperm. For this purpose, the freezing-thawing-related changes of structure and location of histones-DNA domains in the boar sperm head were analyzed through Western blot and immunocytochemistry. Afterwards, it was analyzed whether freezing-thawing induced changes in tyrosine phosphorylation levels of both protamine 1 and histone H1, through Western blot analyses in samples previously subjected to immunoprecipitation. This analysis was completed with the determination of the changes induced by freezing-thawing on the overall levels of sperm-head disulfide bonds through analysis of free-cysteine radicals levels. Freezing-thawing induced significant changes in the histones-DNA structures, which were manifested in the appearance of a freezing-thawing-linked histone H1-DNA aggregate of about a 35-kDa band and in the spreading of histone H1-positive markings from the caudal area of the sperm head to more cranial zones. Freezing-thawing did not have any significant effect on the tyrosine phosphorylation levels of either protamine 1 or histone H1. However, thawed samples showed a significant (P < 0.05) increase in the free cysteine radical content (from 3.1 ± 0.5nmol/μg protein in fresh samples to 6.7 ± 0.8 nmol/μg protein). In summary, our results suggest that freezing-thawing causes significant alterations in the nucleoprotein structure of boar sperm head by mechanism/s linked with the rupture of disulfide bonds among the DNA. These mechanisms seem to be unspecific, affecting both the protamines-DNA unions and the histones-DNA bonds in a similar way. Furthermore, results suggest that the boar-sperm nuclear structure is heterogeneous suggesting the existence of a zonated pattern, differing in their total DNA density and the compactness of the precise nucleoprotein structures present in each zone.

Structure and Reactivity of the N-Acetyl-Cysteine Radical Cation and Anion: Does Radical Migration Occur?

The structure and reactivity of the N-acetyl-cysteine radical cation and anion were studied using ion-molecule reactions, infrared multi-photon dissociation (IRMPD) spectroscopy, and density functional theory (DFT) calculations. The radical cation was generated by first nitrosylating the thiol of N-acetyl-cysteine followed by the homolytic cleavage of the S-NO bond in the gas phase. IRMPD spectroscopy coupled with DFT calculations revealed that for the radical cation the radical migrates from its initial position on the sulfur atom to the α-carbon position, which is 2.5 kJ mol(-1) lower in energy. The radical migration was confirmed by time-resolved ion-molecule reactions. These results are in contrast with our previous study on cysteine methyl ester radical cation (Osburn et al., Chem. Eur. J. 2011, 17, 873-879) and the study by Sinha et al. for cysteine radical cation (Phys. Chem. Chem. Phys. 2010, 12, 9794-9800) where the radical was found to stay on the sulfur atom as formed. A similar approach allowed us to form a hydrogen-deficient radical anion of N-acetyl-cysteine, (M - 2H)( •- ). IRMPD studies and ion-molecule reactions performed on the radical anion showed that the radical remains on the sulfur, which is the initial and more stable (by 63.6 kJ mol(-1)) position, and does not rearrange.

Chasing the ubiquitous RET proto-oncogene in South African MEN2 families - implications for the surgeon

The RET proto-oncogene (REarranged during Transfection; RET) plays an important role in the causation of many thyroid tumours. Germline RET proto-oncogene missense mutations have been clearly linked to medullary thyroid carcinoma (MTC) and the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN2A, MEN2B). We investigated a cohort of MEN2-related patients referred to Tygerberg Hospital, W Cape (2003-2009). The study cohort was divided into three groups based on pathology (viz. MEN/MTC, phaeochromocytoma, and a miscellaneous group of MEN pathologies). Families with identified high-risk factors were recalled. Serum calcitonin levels were monitored where indicated. DNA was extracted from whole blood by standard techniques and polymerase chain reaction (PCR) products screened for RET gene variations by heteroduplex single-strand duplication techniques (heteroduplex single-strand conformation polymorphism analysis) being validated with automated sequencing techniques showing conformational variants in acrylamide gel. We screened 40 persons, male/female ratio 1:1.5. Three ethnic groups were represented (white (12), black (11) and mixed race (17)). Nine were index MTC cases, 5 phaeochromocytoma, 3 Hirschsprung's disease-MEN associations and 2 miscellaneous (1 neuroblastoma, 1 intestinal neuronal dysplasia), while 1 fell into the MEN2B category. The remaining 19 were unaffected relatives screened for carrier status, among whom afamilial recurrence was observed in 7. On genetic testing, an RET point mutation at the high-risk 634 cysteine allele was identified in 11 cases. A further cysteine radical mutation at the 620 position was related to MEN2 in 3 families plus 1 other family referred from elsewhere. Other less-recognised gene variations were detected throughout the RET gene in 70% of cases and included the 691 position on codon 11 (11 cases); the 432 position (4 cases, 1 homozygous) intronic mutations on exon 4 (1 case); and an IVS19-37G/C and a D1017N variation in exon 19 in 2 MEN families. Fifteen MTC patients have had thyroidectomies, of which 2 were prophylactic (C-cell hyperplasia; early occult MTC). A further 3 are awaiting prophylactic surgery. RET gene mutation carries a risk of MEN2 and MTC in all ethnic groups in South Africa. Prophylactic surgery may prevent MTC, so genetic screening is important to identify and treat high-risk patients.

Cysteine radical cation: A distonic structure probed by gas phase IR spectroscopy

The interest in the radical cations of amino acids is twofold. On the one hand, these species are relevant in enzymatic catalysis and in oxidative damage of proteins. On the other hand, as constituents of peptides and proteins, they aid the mass spectrometric characterization of these biomolecules, yielding diagnostic fragmentation patterns and providing complementary information with respect to the one obtained from even electron ions. The cysteine radical cation has been obtained by S-NO bond cleavage of protonated S-nitrosocysteine and thoroughly characterized by IRMPD spectroscopy, both in the 1000-2000 cm(-1) range (the highly structurally diagnostic, so-called 'fingerprint' range) and in the 2900-3700 cm(-1) spectral range, encompassing O-H and N-H stretching vibrations. In this way the distonic structure in which the charge is on the NH(3) group and the spin is on the sulfur atom is unambiguously demonstrated. This tautomer is a local minimum on the potential energy surface, at 29.7 kJ mol(-1) with respect to the most stable tautomer, a captodative structure allowing extensive delocalization of charge and spin.

Clinical and genetic differences in total colonic aganglionosis in Hirschsprung's disease

Although apparently the same condition as Hirschsprung's disease (HSCR), total colonic aganglionosis (TCA) patients (2%-14% congenital aganglionosis) display clinical, histopathologic, and genetic differences that may account for altered clinical presentations. Patients and methods Clinical, radiologic, and histologic features of 22 TCA patients of 114 HSCR cases (including 16 kindreds) were retrospectively evaluated by chart review. With ethical permission, DNA mutation analysis of the RET and EDNRB genes was carried out. Polymerase Chain Reaction (PCR) products were screened for genetic variation of by Hetroduplex Single Strand conformation polymorphism (HEX/SSCP) analysis and compared with 60 normal population control samples (20/ethnic groups). The SSCP variants were validated with automated sequencing techniques showing conformational variants in acrylamide gel. Results Of the 22 patients, 12 (55%) presented within the first 28 days of extrauterine life, but 10 presented later with 3 (14%) presenting more than 6 months of age. The TCA patients evaluated differed clinically, radiologically, and histologically, and misdiagnosis occurred in 23% (5/22). Seven patients (32%) were familial—the remainder being nonrelated. Histologic features varied, and difficulties in diagnosis occurred in 5 (24%), with unclear histologic condition delaying diagnosis in one and a mistaken aganglionic level, requiring repeat surgery in two. RET variations were detected in 82% (18/22)of TCA as opposed to 33% short segment (S-HSCR) with multiple genetic RET variations in 5 (28%). Genetic variations included exon 2 SNPs but less than in S-HSCR. One had an isolated RET A4 variation with no other abnormalities. Intronic RET variations occurred in intron 6 (2 patients) (IVS6+56delG) and intron 16 (2 patients) (IVS16-38delG). A cysteine radical mutation ( C620R) (2 patients) was related to Multiple Endocrine Neoplasia Type 2 (MEN2) in the family. In contrast to S-HSCR, genetic variations in TCA aggregated to the important tyrosine kinase (intracellular) region in 5 patients suggesting a possible pathogenetic link. EDNRB variations occurred in 7 patients (32%) all within exon 4 of the gene. Conclusions Total colonic aganglionosis differs clinically from other HSCR phenotypes and may lead to misdiagnosis. Potential disease-related RET gene mutations include exon 17-21 genetic variations that suggest the possibility of disrupted downstream signaling pathways from vital gene recruitment sites as possible TCA contributing factors.

Gas-Phase Fragmentation of Long-Lived Cysteine Radical Cations Formed Via NO Loss from Protonated S-Nitrosocysteine

In this work, we describe two different methods for generating protonated S-nitrosocysteine in the gas phase. The first method involves a gas-phase reaction of protonated cysteine with t-butylnitrite, while the second method uses a solution-based transnitrosylation reaction of cysteine with S-nitrosoglutathione followed by transfer of the resulting S-nitrosocysteine into the gas phase by electrospray ionization mass spectrometry (ESI-MS). Independent of the way it was formed, protonated S-nitrosocysteine readily fragments via bond homolysis to form a long-lived radical cation of cysteine (Cys(*+)), which fragments under collision-induced dissociation (CID) conditions via losses in the following relative abundance order: *COOH CH(2)S >> *CH(2)SH approximately = H(2)S. Deuterium labeling experiments were performed to study the mechanisms leading to these pathways. DFT calculations were also used to probe aspects of the fragmentation of protonated S-nitrosocysteine and the radical cation of cysteine. NO loss is found to be the lowest energy channel for the former ion, while the initially formed distonic Cys(*+) with a sulfur radical site undergoes proton and/or H atom transfer reactions that precede the losses of CH(2)S, *COOH, *CH(2)SH, and H(2)S.

Branched Activation- and Catalysis-Specific Pathways for Electron Relay to the Manganese/Iron Cofactor in Ribonucleotide Reductase from Chlamydia trachomatis

A conventional class I (subclass a or b) ribonucleotide reductase (RNR) employs a tyrosyl radical (Y (*)) in its R2 subunit for reversible generation of a 3'-hydrogen-abstracting cysteine radical in its R1 subunit by proton-coupled electron transfer (PCET) through a network of aromatic amino acids spanning the two subunits. The class Ic RNR from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor (specifically, the Mn (IV) ion) in place of the Y (*) for radical initiation. Ct R2 is activated when its Mn (II)/Fe (II) form reacts with O 2 to generate a Mn (IV)/Fe (IV) intermediate, which decays by reduction of the Fe (IV) site to the active Mn (IV)/Fe (III) state. Here we show that the reduction step in this sequence is mediated by residue Y222. Substitution of Y222 with F retards the intrinsic decay of the Mn (IV)/Fe (IV) intermediate by approximately 10-fold and diminishes the ability of ascorbate to accelerate the decay by approximately 65-fold but has no detectable effect on the catalytic activity of the Mn (IV)/Fe (III)-R2 product. By contrast, substitution of Y338, the cognate of the subunit interfacial R2 residue in the R1 <--> R2 PCET pathway of the conventional class I RNRs [Y356 in Escherichia coli ( Ec) R2], has almost no effect on decay of the Mn (IV)/Fe (IV) intermediate but abolishes catalytic activity. Substitution of W51, the Ct R2 cognate of the cofactor-proximal R1 <--> R2 PCET pathway residue in the conventional class I RNRs (W48 in Ec R2), both retards reduction of the Mn (IV)/Fe (IV) intermediate and abolishes catalytic activity. These observations imply that Ct R2 has evolved branched pathways for electron relay to the cofactor during activation and catalysis. Other R2s predicted also to employ the Mn/Fe cofactor have Y or W (also competent for electron relay) aligning with Y222 of Ct R2. By contrast, many R2s known or expected to use the conventional Y (*)-based system have redox-inactive L or F residues at this position. Thus, the presence of branched activation- and catalysis-specific electron relay pathways may be functionally important uniquely in the Mn/Fe-dependent class Ic R2s.

The cysteine radical cation: structures and fragmentation pathways

A theoretical study on the structures, relative energies, isomerization reactions and fragmentation pathways of the cysteine radical cation, [NH(2)CH(CH(2)SH)COOH].+, is reported. Hybrid density functional theory (B3LYP) has been used in conjunction with the 6-311++G(d,p) basis set. The isomer at the global minimum, Captodative-1, has the structure NH(2)C.(CH(2)SH)C(OH)(2)+; the stability of this ion is attributed to the captodative effect in which the NH(2) functions as a powerful pi-electron donor and C(OH)(2)+ as a powerful pi-electron acceptor. Ion Distonic-S-1, H(3)N(+)CH(CH(2)S.)COOH, in which the radical is formally situated on the S atom, is higher in enthalpy (DeltaH degrees (0)) than Captodative-1 by 6.1 kcal mol(-1), but is lower in enthalpy than another isomer Distonic-C-1, H(3)N(+)C.(CH(2)SH)COOH, by 8.2 kcal mol(-1). Isomerization of the canonical radical cation of cysteine, [H(2)NCH(CH(2)SH)COOH].+, (Canonical-1), to Captodative-1 has an enthalpy of activation of 25.8 kcal mol(-1), while the barrier against isomerization of Canonical-1 to Distonic-S-1 is only 9.6 kcal mol(-1). Two additional transient tautomers, one with the radical located at C(alpha) and the charge on SH(2), and the other a carboxy radical with the charge on NH(3), are reported. Plausible fragmentation pathways (losses of small molecules, CO(2), CH(2)S, H(2)S and NH(3), and neutral radicals COOH. , HSCH(2). and NH(2).) from Canonical-1 are examined.

Structure and fragmentation of glycine, alanine, serine and cysteine radical cations. A theoretical study

The structure of glycine, alanine, serine and cysteine radical cations, as well as their C α–R fragmentations have been studied at the B3LYP/6-31++G(d,p) level of theory. For all systems the loss of COOH is found to be the most favourable process. However, when R 1 size increases, fragmentations that lead to R 1 or R 1 + start become competitive. The energy of a C α–R fragmentation can be related to the ionization potential of the two generated fragments, in such a way that the preferred process is the one that leaves the positive charge in the fragment with a lower ionization potential.