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John T Groves

Publications and source records attributed to John T Groves.

At least 19 recordsLinked to original sources
PubMed2006

The peroxynitrite decomposition catalyst FP15 improves ageing-associated cardiac and vascular dysfunction.

Overproduction of oxidants and free radicals in ageing tissues induces nitro-oxidative stress, which has recently been implicated in the pathogenesis of cardiovascular dysfunction associated with ageing. Peroxynitrite, a strong cytotoxic oxidant damages proteins and DNA and activates several pathways causing tissue injury, including the peroxynitrite-poly(ADP-ribose) polymerase (PARP) pathway. In this study, we investigated the effectiveness of the peroxynitrite decomposition catalyst FP15 on ageing-associated cardiac and vascular dysfunction. Young and ageing rats were treated with vehicle or FP15 intraperitoneally. Using a microtip Millar pressure catheter we performed left ventricular blood pressure analysis to assess systolic and diastolic function. Endothelium-dependent and -independent vasorelaxation of isolated aortic rings were investigated by using acetylcholine and sodium nitroprusside. Ageing animals showed a marked reduction of systolic and diastolic cardiac function and loss of endothelium-dependent relaxant responsiveness of aortic rings. FP15-treatment significantly improved cardiac performance and endothelial function. Immunohistochemical staining confirmed that FP15 effectively reduced nitrosative stress and prevented the activation of PARP in the aortic wall of ageing rats. Our results demonstrate the importance of endogenous peroxynitrite-overproduction in the pathogenesis of ageing-associated cardiovascular dysfunction. Pharmacological decomposition of peroxynitrite by FP15 may represent a novel therapeutic utility to improve cardiac and vascular dysfunction associated with ageing.

Acetylcholine↗
PubMed2006

Fast catalytic hydroxylation of hydrocarbons with ruthenium porphyrins.

Ruthenium porphyrin complexes such as carbonylruthenium(II) tetrakispentafluorophenylporphyrin [Ru(II)(TPFPP)(CO)] were found to be efficient catalysts for the hydroxylation of alkanes in the presence of 2,6-dichloropyridine N-oxide as the oxidant under mild, nonacidic conditions. Up to 14 800 turnovers (TO) and rates of 800 TO/min were obtained for the hydroxylation of adamantane. The hydroxylation of cis-decalin afforded cis-9-decalol and cis-decalin-9,10-diol, exclusively, thus, excluding a long-lived radicals mechanism. The kinetics of product evolution in a typical catalytic oxygenation showed an initial induction period followed by a fast, apparently zero-order phase with maximum rates and high efficiencies. Deuterium isotope effects (kH/kD) in the range of 4.2-6.4 were found for the hydroxylation of alkanes. A Hammett treatment of the data for the oxidation of para-substituted toluene derivatives showed a linear correlation with a highly negative rho+ value of -2.0. On the basis of kinetic and spectroscopic evidence, Ru(VI)(TPFPP)(O)2, Ru(II)(TPFPP)(CO), and Ru(IV)(TPFPP)Cl2 observed during catalysis were ruled out as candidates for the active catalyst responsible for the high efficiencies and turnover rates in the oxidation reactions. The fastest rates of adamantane hydroxylation with 2,6-dichloropyridine N-oxide were achieved by the reductive activation of Ru(IV)(TPFPP)Cl2 with a zinc amalgam. This redox activation is consistent with the formation of an active Ru(III) intermediate in situ by a one-electron reduction of the Ru(IV) porphyrin. EPR spectra characteristic of Ru(III) have been observed upon the reduction of Ru(IV)(TPFPP)Cl2 with a zinc amalgam. In the adamantane oxidation mediated with Ru(III)(TPFPP)(OEt), it was found that, during this process, the Ru(III) porphyrin was gradually converted to a dioxoRu(VI) porphyrin. Concomitant with this conversion, the reaction rates decreased. Catalyst activation was also stimulated by autoxidation of the solvent CH2Cl2. On the basis of these data, a mechanism is proposed that incorporates a "fast" cycle involving metastable Ru(III) and oxoRu(V) intermediates and a "slow" oxidation cycle, mediated by oxoRu(IV) and trans-dioxoRu(VI) porphyrin complexes.

Adamantane↗
PubMed2006

Electronic structure and reactivity of isomeric oxo-Mn(V) porphyrins: effects of spin-state crossing and pKa modulation.

The reactivity of the isomeric oxo-Mn(V)-2-tetra-N-methylpyridyl (2-TMPyP) and oxo-Mn(V)-4-tetra-N-methylpyridyl (4-TMPyP) porphyrins has been investigated by a combined experimental and theoretical approach based on density functional theory. The unusual higher reactivity of the more electron-rich 4-TMPyP species appears to be related to both the higher basicity of its oxo ligand, compared to that of the 2-TMPyP isomer, and the smaller low-spin-high-spin promotion energy of 4-TMPyP, compared to that of 2-TMPyP, because of the stabilization of the A2u orbital in the latter isomer. Therefore, in a two-state energy profile involving crossing of the initial singlet and final quintet potential energy surfaces, the 4-TMPyP isomer should be kinetically favored. The calculated differences in the singlet-quintet gaps for the 2-TMPyP and 4-TMPyP systems compare well with the measured differences in the activation energies for two isomeric porphyrins. Both effects, proton affinity and electron-promotion energy, contribute to reduce the reactivity of the more electrophilic oxidant when electron-withdrawing groups are closer to the active site, contrary to the usual expectations based on simple chemical reactivity correlations. These theoretical results are in accord with new experimental data showing O=Mn(V)-O-H pK(a)s of 7.5 and 8.6 for the isomeric 2-TMPyP and 4-TMPyP systems, respectively.

Computer Simulation↗
PubMed2006

High-valent iron in chemical and biological oxidations.

Various aspects of the reactivity of iron(IV) in chemical and biological systems are reviewed. Accumulated evidence shows that the ferryl species [Fe(IV)O](2+) can be formed under a variety of conditions including those related to the ferrous ion-hydrogen peroxide system known as Fenton's reagent. Early evidence that such a species could hydroxylate typical aliphatic C-H bonds included regioselectivities and stereospecificities for cyclohexanol hydroxylation that could not be accounted for by a freely diffusing hydroxyl radical. Iron(IV) porphyrin complexes are also found in the catalytic cycles of cytochrome P450 and chloroperoxidase. Model oxo-iron(IV) porphyrin complexes have shown reactivity similar to the proposed enzymatic intermediates. Mechanistic studies using mechanistically diagnostic substrates have implicated a radical rebound scenario for aliphatic hydroxylation by cytochrome P450. Likewise, several non-heme diiron hydroxylases, AlkB (Omega-hydroxylase), sMMO (soluble methane monooxygenase), XylM (xylene monooxygenase) and T4moH (toluene monooxygenase) all show clear indications of radical rearranged products indicating that the oxygen rebound pathway is a ubiquitous mechanism for hydrocarbon oxygenation by both heme and non-heme iron enzymes.

Catalysis↗
PubMed2006

Dynamics of carbon monoxide binding to cystathionine beta-synthase.

Cystathionine beta-synthase (CBS) condenses homocysteine, a toxic metabolite, with serine in a pyridoxal phosphate-dependent reaction. It also contains a heme cofactor to which carbon monoxide (CO) or nitric oxide can bind, resulting in enzyme inhibition. To understand the mechanism of this regulation, we have investigated the equilibria and kinetics of CO binding to the highly active catalytic core of CBS, which is dimeric. CBS exhibits strong anticooperativity in CO binding with successive association constants of 0.24 and 0.02 microm(-1). Stopped flow measurements reveal slow CO association (0.0166 s(-1)) limited by dissociation of the endogenous ligand, Cys-52. Rebinding of CO and of Cys-52 following CO photodissociation were independently monitored via time-resolved resonance Raman spectroscopy. The Cys-52 rebinding rate, 4000 s(-1), is essentially unchanged between pH 7.6 and 10.5, indicating that the pK(a) of Cys-52 is shifted below pH 7.6. This effect is attributed to the nearby Arg-266 residue, which is proposed to form a salt bridge with the dissociated Cys-52, thereby inhibiting its protonation and slowing rebinding to the Fe. This salt bridge suggests a pathway for enzyme inactivation upon CO binding, because Arg-266 is located on a helix that connects the heme and pyridoxal phosphate cofactor domains.

Binding Sites↗
PubMed2006

Enzymatic tailoring of enterobactin alters membrane partitioning and iron acquisition.

Enterobactin (Ent), a prototypic bacterial siderophore, is modified by both the C-glucosyltransferase IroB and the macrolactone hydrolase IroE in pathogenic bacteria that contain the iroA cluster. To investigate the possible effects of glucosylation and macrolactone hydrolysis on the physical properties of Ent, the membrane affinities and iron acquisition rates of Ent and Ent-derived siderophores were measured. The data obtained indicate that Ent has a high membrane affinity (K(x) = 1.5 x 10(4)) similar to that of ferric acinetoferrin, an amphiphile containing two eight-carbon hydrophobic chains. Glucosylation and macrolactone hydrolysis decrease the membrane affinity of Ent by 5-25-fold. Furthermore, in the presence of phospholipid vesicles, the iron acquisition rate is significantly increased by glucosylation and macrolactone hydrolysis, due to the resultant decrease in membrane sequestration of the siderophore. These results suggest that IroB and IroE enhance the ability of Ent-producing pathogens to acquire iron in membrane-rich microenvironments.

Bacteria↗
PubMed2005

Synthesis and structural modeling of the amphiphilic siderophore rhizobactin-1021 and its analogs.

We describe two convenient syntheses of rhizobactin-1021 (Rz), a citrate-based siderophore amphiphile produced by the nitrogen-fixing root symbiont Rhizobium meliloti-1021, and several analogs. Our approach features a singly amidated, tert-butyl-protected citrate intermediate that easily affords a variety of Rz analogs in the late stages of the synthesis. Structural modeling and the monolayer behavior of Rz and its metal complexes are consistent with a structural reorganization upon Rz-mediated iron chelation.

Alkenes↗
PubMed2005

Mycobactin-mediated iron acquisition within macrophages.

Restricting the availability of iron is an important strategy for defense against bacterial infection. Mycobacterium tuberculosis survives within the phagosomes of macrophages; consequently, iron acquisition is particularly difficult for M. tuberculosis, because the phagosomal membrane is an additional barrier for its iron access. However, little is known about the iron transport and acquisition pathways adapted by this microbe in vivo. Extracellular iron sources are usually mobilized by hydrophilic siderophores. Here, we describe direct evidence that mycobactins, the lipophilic siderophores of mycobacteria, efficiently extract intracellular macrophage iron. The metal-free siderophore is diffusely associated with the macrophage membrane, ready for iron chelation. Notably, the mycobactin-metal complex accumulates with high selectivity in macrophage lipid droplets, intracellular domains for lipid storage and sorting. In our experiments, these mycobactin-targeted lipid droplets were found in direct contact with phagosomes, poised for iron delivery. The existence of this previously undescribed iron acquisition pathway indicates that mycobacteria have taken advantage of endogenous macrophage mechanisms for iron mobilization and lipid sorting for iron acquisition during infection. The pathway could represent a new target for the control of mycobacterial infection.

Iron↗
PubMed2005

Membrane dynamics of the amphiphilic siderophore, acinetoferrin.

Acinetobacter haemolyticus is an antibiotic resistant, pathogenic bacterium responsible for an increasing number of hospital infections. Acinetoferrin (Af), the amphiphilic siderophore isolated from this organism, contains two unusual trans-2-octenoyl hydrocarbon chains reminiscent of a phospholipid structural motif. Here, we have investigated the membrane affinity of Af and its iron complex, Fe-Af, using small and large unilamellar phospholipid vesicles (SUV and LUV) as model membranes. Af shows a high membrane affinity with a partition coefficient, K(x)= 6.8 x 10(5). Membrane partitioning and trans-membrane flip-flop of Fe-Af have also been studied via fluorescence quenching of specifically labeled vesicle leaflets and (1)H NMR line-broadening techniques. Fe-Af is found to rapidly redistribute between lipid and aqueous phases with dissociation/partitioning rates of k(off) = 29 s(-1) and k(on) = 2.4 x 10(4) M(-1) s(-1), respectively. Upon binding iron, the membrane affinity of Af is reduced 30-fold to K'(x) = 2.2 x 10(4) for Fe-Af. In addition, trans-membrane flip-flop of Fe-Af occurs with a rate constant, k(p) = 1.2 x 10(-3) s(-1), with egg-PC LUV and a half-life time around 10 min with DMPC SUV. These properties are due to the phospholipid-like conformation of Af and the more extended conformation of Fe-Af that is enforced by iron binding. Remarkable similarities and differences between Af and another amphiphilic siderophore, marinobactin E, are discussed. The potential biological implications of Af and Fe-Af are also addressed. Our approaches using inner- and outer-leaflet-labeled fluorescent vesicles and (1)H NMR line-broadening techniques to discern Af-mediated membrane partitioning and trans-membrane diffusion are amenable to similar studies for other paramagnetic amphiphiles.

Acinetobacter↗
PubMed2005

Reaction mechanisms of non-heme diiron hydroxylases characterized in whole cells.

Whole cells expressing the non-heme diiron hydroxylases AlkB and toluene 4-monooxygenase (T4MO) were used to probe enzyme reaction mechanisms. AlkB catalyzes the hydroxylation of the radical clock substrates bicyclo[4.1.0]heptane (norcarane), spirooctane and 1,1-diethylcyclopropane, and does not catalyze the hydroxylation of the radical clocks 1,1-dimethylcyclopropane or 1,1,2,2-tetramethylcyclopropane. The hydroxylation of norcarane yields a distribution of products consistent with an "oxygen-rebound" mechanism for the enzyme in both the wild type Pseudomonas putida GPo1 and AlkB from P. putida GPo1 expressed in Escherichia coli. Evidence for the presence of a substrate-based radical during the reaction mechanism is clear. With norcarane, the lifetime of that radical varies with experimental conditions. Experiments with higher substrate concentrations yield a shorter radical lifetime (approximately 1 ns), while experiments with lower substrate concentrations yield a longer radical lifetime (approximately 19 ns). Consistent results were obtained using either wild type or AlkB-equipped host organisms using either "resting cell" or "growing cell" approaches. T4MO expressed in E. coli also catalyzes the hydroxylation of norcarane with a radical lifetime of approximately 0.07 ns. No radical lifetime dependence on substrate concentration was seen. Results from experiments with diethylcyclopropane, spirooctane, dimethylcyclopropane, and diethylcyclopropane are consistent with a restricted active site for AlkB.

Cytochrome P-450 CYP4A↗
PubMed2004

Remarkable aliphatic hydroxylation by the diiron enzyme toluene 4-monooxygenase in reactions with radical or cation diagnostic probes norcarane, 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane.

Toluene 4-monooxygenase (T4MO) catalyzes the hydroxylation of toluene to yield 96% p-cresol. This diiron enzyme complex was used to oxidize norcarane (bicyclo[4.1.0]heptane), 1,1-dimethylcyclopropane, and 1,1-diethylcyclopropane, substrate analogues that can undergo diagnostic reactions upon the production of transient radical or cationic intermediates. Norcarane closely matches the shape and volume of the natural substrate toluene. Reaction of isoforms of the hydroxylase component of T4MO (T4moH) with different regiospecificities for toluene hydroxylation (k(cat) approximately 1.9-2.3 s(-)(1) and coupling efficiency approximately 81-96%) revealed similar catalytic parameters for norcarane oxidation (k(cat) approximately 0.3-0.5 s(-)(1) and coupling efficiency approximately 72%). The products included variable amounts of the un-rearranged isomeric norcaranols and cyclohex-2-enyl methanol, a product attributed to rearrangement of a radical oxidation intermediate. A ring-expansion product derived from the norcaranyl C-2 cation, cyclohept-3-enol, was not produced by either the natural enzyme or any of the T4moH isoforms tested. Comparative studies of 1,1-dimethylcyclopropane and 1,1-diethylcyclopropane, diagnostic substrates with differences in size and with approximately 50-fold slower k(cat) values, gave products consistent with both radical rearrangement and cation ring expansion. Examination of the isotopic enrichment of the incorporated O-atoms for all products revealed high-fidelity incorporation of an O-atom from O(2) in the un-rearranged and radical-rearranged products, while the O-atom found in the cation ring-expansion products was predominantly obtained by reaction with H(2)O. The results show a divergence of radical and cation pathways for T4moH-mediated hydroxylation that can be dissected by diagnostic substrate probe rearrangements and by changes in the source of oxygen used for substrate oxygenation.

Cations↗
PubMed2004

Role for nitrosative stress in diabetic neuropathy: evidence from studies with a peroxynitrite decomposition catalyst.

Nitrosative stress, that is, enhanced peroxynitrite formation, has been documented in both experimental and clinical diabetic neuropathy (DN), but its pathogenetic role remains unexplored. This study evaluated the role for nitrosative stress in two animal models of type 1 diabetes: streptozotocin-diabetic mice and diabetic NOD mice. Control (C) and streptozotocin-diabetic (D) mice were treated with and without the potent peroxynitrite decomposition catalyst FP15 (5 mg kg(-1) d(-1)) for 1 wk after 8 wk without treatment. Sciatic nerve nitrotyrosine (a marker of peroxynitrite-induced injury) and poly(ADP-ribose) immunoreactivities were present in D and absent in C and D+FP15. FP15 treatment corrected sciatic motor and hind-limb digital sensory nerve conduction deficits and sciatic nerve energy state in D, without affecting those variables in C. Nerve glucose and sorbitol pathway intermediate concentrations were similarly elevated in D and D+FP15 vs C. In diabetic NOD mice, a 7-day treatment with either 1 or 3 mg kg(-1) d(-1) FP15 reversed increased tail-flick latency (a sign of reduced pain sensitivity); the effect of the higher dose was significant as early as 3 days after beginning of the treatment. In conclusion, nitrosative stress plays a major role in DN in, at least, type 1 diabetes. This provides the rationale for development of agents counteracting peroxynitrite formation and promoting peroxynitrite decomposition, and their evaluation in DN.

Animals↗
PubMed2004

Synthesis, structure, and molecular dynamics of gallium complexes of schizokinen and the amphiphilic siderophore acinetoferrin.

A new general synthesis of the citrate-based siderophores acinetoferrin (Af) and schizokinen (Sz) and their analogues is described. The molecular structure of gallium schizokinen, GaSz, was determined by combined (1)H NMR, Hartree-Fock ab initio calculations, DFT, and empirical modeling of vicinal proton NMR spin-spin couplings. The metal-coordination geometry of GaSz was determined from NOE contacts to be cis-cis with respect to the two chelating hydroxamates. One diaminopropane adopts a single chairlike conformation while another is a mixture of two ring pucker arrangements. Both amide hydrogens are internally hydrogen bonded to metal-ligating oxygen atoms. The acyl methyl groups are directed away from each other with an average planar angle of ca. 130 degrees. The kinetics of GaSz racemization were followed by selective, double spin-echo inversion-recovery (1)H NMR spectroscopy over the temperature range of 10-45 degrees C. The racemization proceeds by a multistep mechanism that is proton independent between pD 5 and 12 (k(0) = 1.47 (0.15 s(-1))) and acid catalyzed below pD 4 (k(1) = 2.25 (0.15) x 10(4) M(-1) s(-1)). The activation parameters found for the two sequential steps of the proton independent pathway were DeltaH(++) = 25 +/- 3 kcal M(-1), DeltaS(++) = 25 +/- 7 cal M(-1) K(-1) and DeltaH(++) = 17.1 +/- 0.2 kcal M(-1), DeltaS(++) = 0.3 +/- 2.7 cal M(-1) K(-1). The first step of the proton-independent mechanism was assigned to the dissociation of the carboxyl group. The second step was assigned to complex racemization. The proton-assisted step was assigned to a complete dissociation of the alpha-hydroxy carboxyl group at pD < 4. The ab initio modeling of gallium acinetoferrin, GaAf, and analogues derived from the structure of GaSz has shown that the pendant trans-octenoyl fragments are oriented in opposite directions with the average planar angle of ca. 130 degrees. This arrangement prevents GaAf from adopting a phospholipid-like structural motif. Significantly, iron siderophore complex FeAf was found to be disruptive to phospholipid vesicles and is considerably more hydrophilic than Af, with an eight-fold smaller partition coefficient.

Acinetobacter↗
PubMed2004

Dynamics of carbon monoxide binding to CooA.

CooA is a dimeric CO-sensing heme protein from Rhodospirillum rubrum. The heme iron in reduced CooA is six-coordinate; the axial ligands are His-77 and Pro-2. CO displaces Pro-2 and induces a conformation change that allows CooA to bind DNA and activate transcription of coo genes. Equilibrium CO binding is cooperative, with a Hill coefficient of n = 1.4, P(50) = 2.2 microm, and estimated Adair constants K(1) = 0.16 and K(2) = 1.3 microm(-1). The rates of CO binding and release are both strongly biphasic, with roughly equal amplitudes for the fast and slow phases. The association rates show a hyperbolic dependence on [CO], consistent with Pro-2 dissociation being rate-limiting. The kinetic characteristics of the transiently formed five-coordinate heme are probed via flash photolysis. These observations are integrated into a kinetic model, in which CO binding to one subunit decreases the rate of Pro-2 rebinding in the second, leading to a net increase in affinity for the second CO. The CO adduct exists in slowly interconverting "open" and "closed" forms. This interconversion probably involves the large-scale motions required to bring the DNA-binding domains into proper orientation. The combination of low CO affinity, slow CO binding, and slow conformational transitions ensures that activation of CooA only occurs at high (micromolar) and sustained (> or =1 min) levels of CO. When micromolar levels do occur, positive cooperativity allows efficient activation over a narrow range of CO concentrations.

Amino Acid Sequence↗