Exposure of Bacillus subtilis to silver inhibits activity of cytochrome c oxidase in vivo via interaction with SCO, the CuA assembly protein.

Silver has long been used as an antimicrobial agent in general and medicinal use. Here, we observe that exposure of the Gram-positive, endospore-forming bacterium Bacillus subtilis to Ag(i) effects growth in a biphasic manner. In the first phase at Ag(i) concentrations below 50 µM B. subtilis growth is not affected, but activity of the respiratory enzyme cytochrome c oxidase is disrupted completely. Between 50 to 100 µM Ag(i) B. subtilis growth is drastically diminished and completely absent above 100 µM Ag(i). Synthesis of cytochrome c oxidase, or SCO proteins, have been shown to play a role in assembly of the CuA center of cytochrome c oxidase and we suppose that the effects observed here of silver on Bacillus subtilis in culture may be explained at least in part by the interaction of Bacillus SCO (BsSCO) with Ag(i). We find that Ag(i) forms a high affinity complex with BsSCO in vitro that blocks SCO's interaction with copper indicating competition between the metals for binding BsSCO. The interaction of BsSCO with Ag(i) exhibits multiple phases and is more complex than that observed for the high-affinity, 1 : 1 copper complex with BsSCO. We propose that the initial response of B. subtilis cultures is due to high affinity binding of Ag(i) to BsSCO that blocks the functionality of BsSCO required for assembly of cytochrome c oxidase. Our results provide evidence of a specific effect of silver on Bacillus subtilis cells and implies that SCO proteins play a role in sensitivity to Ag(i).

Using Tryptophan Mutants To Probe the Structural and Functional Status of BsSCO, a Copper Binding, Cytochrome c Oxidase Assembly Protein from Bacillus subtilis.

The synthesis of cytochrome c oxidase protein from Bacillus subtilis (i.e., BsSCO) binds copper with picomolar affinity, which increases the protein's melting temperature (i.e., TM) by 20 °C. Here two native tryptophans (i.e., W36 and W101) are identified as major contributors to BsSCO's structural form, and their contributions to the stability, intrinsic fluorescence, and copper binding properties of BsSCO are explored. Single mutations of tryptophan to phenylalanine decrease the TM by 10 °C and the folding free energy by 3-4 kcal/mol. A more severe change to alanine (i.e., W36A BsSCO) decreases the TM by 20 °C and the stability by 9 kcal/mol. However, these mutants bind copper with high affinity and assemble cytochrome c oxidase in vivo. Replacing phenylalanine at a position near (∼5 Å) the copper binding site with tryptophan (i.e., F42W) increases the TM of apo-BsSCO by 3 °C but diminishes the effect of copper binding. When both native tryptophans are changed to alanine, apo-BsSCO is unfolded in vitro and is not functional in cytochrome c oxidase assembly in vivo. A double-mutant of BsSCO in which W36A is combined with F42W exhibits a form of metastability. Apo-W36A/F42W BsSCO melts at 37 °C, which upon binding of copper shifts to 65 °C. B. subtilis expressing W36A/F42W BsSCO and grown at 37 °C does not assemble cytochrome c oxidase. However, when these cells are cooled to 25 °C, cytochrome c oxidase activity is recovered. Our results illustrate the subtle relationship between the structural stability and functional properties of BsSCO in the assembly of cytochrome c oxidase.

Subthalamic oscillations and phase amplitude coupling are greater in the more affected hemisphere in Parkinson's disease.

OBJECTIVE: Determine the incidence of resting state oscillations in alpha/beta, high frequency (HFO) bands, and their phase amplitude coupling (PAC) in a large cohort in Parkinson's disease (PD). METHODS: Intra-operative local field potentials (LFPs) from subthalamic nucleus (STN) were recorded from 100 PD subjects, data from 74 subjects were included in the analysis. RESULTS: Alpha/beta oscillations were evident in >99%, HFO in 87% and PAC in 98% of cases. Alpha/beta oscillations (P<0.01) and PAC were stronger in the more affected (MA) hemisphere (P=0.03). Alpha/beta oscillations were primarily found in 13-20Hz (low beta). Beta and HFO frequencies with the greatest coupling, were positively correlated (P=0.001). Tremor attenuated alpha (P=0.002) and beta band oscillations (P<0.001). CONCLUSIONS: STN alpha/beta band oscillations and PAC were evident in ⩾98% cases and were greater in MA hemisphere. Resting tremor attenuated underlying alpha/beta band oscillations. SIGNIFICANCE: Beta band LFP power may be used to drive adaptive deep brain stimulation (aDBS), augmented by a kinematic classifier in tremor dominant PD.

Beta oscillations in freely moving Parkinson's subjects are attenuated during deep brain stimulation.

BACKGROUND: Investigations into the effect of deep brain stimulation (DBS) on subthalamic (STN) beta (13-30 Hz) oscillations have been performed in the perioperative period with the subject tethered to equipment. Using an embedded sensing neurostimulator, this study investigated whether beta power was similar in different resting postures and during forward walking in freely moving subjects with Parkinson's disease (PD) and whether STN DBS attenuated beta power in a voltage-dependent manner. METHODS: Subthalamic local field potentials were recorded from the DBS lead, using a sensing neurostimulator (Activa(®) PC+S, Medtronic, Inc., Food and Drug Administration- Investigational Device Exemption (IDE)-, institutional review board-approved) from 15 PD subjects (30 STNs) off medication during lying, sitting, and standing, during forward walking, and during randomized periods of 140 Hz DBS at 0 V, 1 V, and 2.5/3 V. Continuous video, limb angular velocity, and forearm electromyography recordings were synchronized with neural recordings. Data were parsed to avoid any movement or electrical artifact during resting states. RESULTS: Beta power was similar during lying, sitting, and standing (P = 0.077, n = 28) and during forward walking compared with the averaged resting state (P = 0.466, n = 24), although akinetic rigid PD subjects tended to exhibit decreased beta power when walking. Deep brain stimulation at 3 V and at 1 V attenuated beta power compared with 0 V (P < 0.003, n = 14), and this was voltage dependent (P < 0.001). CONCLUSIONS: Beta power was conserved during resting and forward walking states and was attenuated in a voltage-dependent manner during 140-Hz DBS. Phenotype may be an important consideration if this is used for closed-loop DBS.

Sixty hertz neurostimulation amplifies subthalamic neural synchrony in Parkinson's disease.

High frequency subthalamic nucleus (STN) deep brain stimulation (DBS) improves the cardinal motor signs of Parkinson's disease (PD) and attenuates STN alpha/beta band neural synchrony in a voltage-dependent manner. While there is a growing interest in the behavioral effects of lower frequency (60 Hz) DBS, little is known about its effect on STN neural synchrony. Here we demonstrate for the first time that during intra-operative 60 Hz STN DBS, one or more bands of resting state neural synchrony were amplified in the STN in PD. We recorded intra-operative STN resting state local field potentials (LFPs) from twenty-eight STNs in seventeen PD subjects after placement of the DBS lead (model 3389, Medtronic, Inc.) before and during three randomized neurostimulation sets (130 Hz/1.35V, 130 Hz/2V, 60 Hz/2V). During 130 Hz/2V DBS, baseline (no DBS) STN alpha (8-12 Hz) and beta (13-35 Hz) band power decreased (N=14, P < 0.001 for both), whereas during 60 Hz/2V DBS, alpha band and peak frequency power increased (P = 0.012, P = 0.007, respectively). The effect of 60 Hz/2V DBS opposed that of power-equivalent (130 Hz/1.35V) DBS (alpha: P < 0.001, beta: P = 0.006). These results show that intra-operative 60 Hz STN DBS amplified whereas 130 Hz STN DBS attenuated resting state neural synchrony in PD; the effects were frequency-specific. We demonstrate that neurostimulation may be useful as a tool to selectively modulate resting state resonant bands of neural synchrony and to investigate its influence on motor and non-motor behaviors in PD and other neuropsychiatric diseases.

The affinity of yeast and bacterial SCO proteins for CU(I) and CU(II). A capture and release strategy for copper transfer.

SCO (Synthesis of Cytochrome c Oxidase) proteins are present in prokaryotic and eukaryotic cells, and are often required for efficient synthesis of the respiratory enzyme cytochrome c oxidase. The Bacillus subtilis version of SCO (i.e., BsSCO) has much greater affinity for Cu(II) than it does for Cu(I) (Davidson and Hill, 2009), and this has been contrasted to mitochondrial SCO proteins that are characterized as being specific for Cu(I) (Nittis, George and Winge, 2001). This differential affinity has been proposed to reflect the different physiological environments in which these two members of the SCO protein family reside. In this study the affinity of mitochondrial SCO1 from yeast is compared directly to that of BsSCO in vitro. We find that the yeast SCO1 protein has similar preference for Cu(II) over Cu(I), as does BsSCO. We propose a mechanism for SCO function which would involve high-affinity binding to capture Cu(II), and relatively weak binding of Cu(I) to facilitate copper transfer.

Reactivity of ligand-swapped mutants of the SCO protein from Bacillus subtilis. Isomers of the CCH metal binding motif.

The Synthesis of Cytochrome Oxidase protein, or SCO protein, is required for the assembly of cytochrome c oxidase in many mitochondrial and bacterial respiratory chains. SCOs have been proposed to deliver copper to the CuA site of cytochrome c oxidase. We have reported that Bacillus subtilis SCO (i.e., BsSCO) binds Cu(II) with high-affinity via a two-step process mediated by three conserved residues (i.e., two cysteines and one histidine, or the CCH motif). A remarkable feature in the reaction of reduced (i.e., di-thiol) BsSCO with copper is that it does not generate any of the disulfide form of BsSCO. This molecular aversion is proposed to be a consequence of a binding mechanism in which the initial copper complex of BsSCO does not involve cysteine, but instead involves nitrogen ligands. We test this proposal here by constructing two isomers of BsSCO in which the conserved copper binding residues (i.e., the CCH-motif) are retained, but their positions are altered. In these variants the two cysteines are exchanged with histidine, and both react transiently with copper (II) with distinct kinetic profiles. The reaction generates Cu(I) and the protein is oxidized to its disulfide form. EPR analysis supports a copper binding model in which cysteine, which is at the "histidine position" in the mutant, is part of an initial encounter complex with copper. When cysteine is the initial ligating residue an oxidation reaction ensues. In contrast initial binding to native BsSCO uses nitrogen-based ligands, and thereby avoids the opportunity for thiol oxidation.

Resting beta hypersynchrony in secondary dystonia and its suppression during pallidal deep brain stimulation in DYT3+ Lubag dystonia.

OBJECTIVES: 1) To characterize patterns of globus pallidus interna neural synchrony in patients with secondary dystonia; 2) to determine whether neural hypersynchrony in the globus pallidus externa (GPe) and interna (GPi) is attenuated during high frequency deep brain stimulation (HF DBS) in a patient with DYT3+ dystonia and in a patient with secondary dystonia due to childhood encephalitis. MATERIALS AND METHODS: We recorded local field potentials from the DBS lead in the GPi of four patients (seven hemispheres) with secondary dystonia and from one patient (two hemispheres) with primary DYT3+ dystonia. In two patients, we also recorded pallidal local field potentials during the administration of 10 sec epochs of HF DBS. RESULTS: Power spectral densities during rest demonstrated visible peaks in the beta band in seven out of nine cases. In DYT3+ dystonia, power in the alpha and beta bands, but not theta band, was attenuated during HF DBS in the GPe and in GPi, and attenuation was most prominent in the high beta band. This patient demonstrated an early and maintained improvement in dystonia. There was no beta peak and the power spectrum was not attenuated during HF DBS in a patient with secondary dystonia due to childhood encephalitis. CONCLUSIONS: These results suggest that beta hypersynchrony, demonstrated now in both primary and secondary dystonia, may play a pathophysiological role in pathological hyperkinesis. Further investigation is needed in a larger cohort of well-characterized primary and secondary dystonia patients.

Differential affinity of BsSCO for Cu(II) and Cu(I) suggests a redox role in copper transfer to the Cu(A) center of cytochrome c oxidase.

SCO (synthesis of cytochrome c oxidase) proteins are involved in the assembly of the respiratory chain enzyme cytochrome c oxidase acting to assist in the assembly of the Cu(A) center contained within subunit II of the oxidase complex. The Cu(A) center receives electrons from the reductive substrate ferrocytochrome c, and passes them on to the cytochrome a center. Cytochrome a feeds electrons to the oxygen reaction site composed of cytochrome a(3) and Cu(B). Cu(A) consists of two copper ions positioned within bonding distance and ligated by two histidine side chains, one methionine, a backbone carbonyl and two bridging cysteine residues. The complex structure and redox capacity of Cu(A) present a potential assembly challenge. SCO proteins are members of the thioredoxin family which led to the early suggestion of a disulfide exchange function for SCO in Cu(A) assembly, whereas the copper binding capacity of the Bacillus subtilis version of SCO (i.e., BsSCO) suggests a direct role for SCO proteins in copper transfer. We have characterized redox and copper exchange properties of apo- and metalated-BsSCO. The release of copper (II) from its complex with BsSCO is best achieved by reducing it to Cu(I). We propose a mechanism involving both disulfide and copper exchange between BsSCO and the apo-Cu(A) site. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Copper binding traps the folded state of the SCO protein from Bacillus subtilis.

The SCO protein from the aerobic bacterium Bacillus subtilis (BsSCO) is involved in the assembly of the cytochrome c oxidase complex, and specifically with the Cu(A) center. BsSCO has been proposed to play various roles in Cu(A) assembly including, the direct delivery of copper ions to the Cu(A) site, and/or maintaining the appropriate redox state of the cysteine ligands during formation of Cu(A). BsSCO binds copper in both Cu(II) and Cu(I) redox states, but has a million-fold higher affinity for Cu(II). As a prerequisite to kinetic studies, we measured equilibrium stability of oxidized, reduced and Cu(II)-bound BsSCO by chemical and thermal induced denaturation. Oxidized and reduced apo-BsSCO exhibit two-state behavior in both chemical- and thermal-induced unfolding. However, the Cu(II) complex of BsSCO is stable in up to nine molar urea. Thermal or guanidinium-induced unfolding of BsSCO-Cu(II) ensues only as the Cu(II) species is lost. The effect of copper (II) on the folding of BsSCO is complicated by a rapid redox reaction between copper and reduced, denatured BsSCO. When denatured apo-BsSCO is refolded in the presence of copper (II) some of the population is recovered as the BsSCO-Cu(II) complex and some is oxidized indicating that refolding and oxidation are competing processes. The proposed functional roles for BsSCO in vivo require that its cysteine residues are reduced and the presence of copper during folding may be detrimental to BsSCO attaining its functional state.

Avoiding premature oxidation during the binding of Cu(II) to a dithiolate site in BsSCO. A rapid freeze-quench EPR study.

The Bacillus subtilis version of SCO1 (BsSCO) is required for assembly of Cu(A) in cytochrome c oxidase and may function in thiol-disulfide exchange and/or copper delivery. BsSCO binds Cu(II) with ligation by two cysteines, one histidine and one water. However, copper is a catalyst of cysteine oxidation and BsSCO must avoid this reaction to remain functional. Time resolved, rapid freeze-quench (RFQ) electron paramagnetic resonance of apo-BsSCO reacting with Cu(II) reveals an initial Cu(II) species with two equatorially coordinated nitrogen atoms, but no sulfur. We propose that BsSCO evolves from this initial sulfur free coordination of Cu(II) to the final dithiolate species via a change in conformation, and that the initial binding by nitrogen is a means for BsSCO to avoid premature thiol oxidation.

Maximal subthalamic beta hypersynchrony of the local field potential in Parkinson's disease is located in the central region of the nucleus.

A pathological marker of Parkinson's disease is the existence of abnormal synchrony of neuronal activity within the beta frequency range (13-35 Hz) in the subthalamic nucleus (STN). Recent studies examining the topography of this rhythm have located beta hypersynchrony in the most dorsal part of the STN. In contrast, this study of the topography of the local field potential beta oscillations in 18 STNs with a 1 mm spatial resolution revealed that the point of maximal beta hypersynchrony was located at 53 ± 24% of the trajectory span from the dorsal to the ventral borders of the STN (corresponding to a 3.0 ± 1.6 mm depth for a 5.9 ± 0.75 mm STN span). This suggests that maximal beta hypersynchrony is located in the central region of the nucleus and that further investigation should be done before using STN spectral profiles as an indicator for guiding placement of deep brain stimulation leads.

Bilateral symmetry and coherence of subthalamic nuclei beta band activity in Parkinson's disease.

Abnormal synchronization of neuronal activity in the basal ganglia has been associated with the dysfunction of sensorimotor circuits in Parkinson's disease (PD). In particular, oscillations at frequencies within the beta range (13-35 Hz) are specifically modulated by dopaminergic medication and are correlated with the clinical state of the subjects. While these oscillations have been shown to be coherent ipsilaterally within the basal ganglia and between the basal ganglia nuclei and the ipsilateral motor cortex in PD, the bilateral extent of their coherence has never been characterized. Here we demonstrate for the first time that the beta band oscillations recorded in the local field potential of the subthalamic nuclei (STN), while appearing different across subjects, are occurring at the same frequencies bilaterally (p<0.001) and are coherent between the two STNs of individual PD subjects (11/12 cases, p<0.05). These findings suggest the existence of a bilateral network controlling the beta band activity in the basal ganglia in PD.

Probing the kinetics and thermodynamics of copper(II) binding to Bacillus subtilis Sco, a protein involved in the assembly of the Cu(A) center of cytochrome c oxidase.

BsSco is a member of the Sco protein family involved in the assembly of the Cu(A) center within cytochrome c oxidase. BsSco forms a complex with Cu(II) that has properties consistent with dithiolate ligation. Stopped-flow UV-visible absorbance and fluorescence coupled with multiwavelength analysis reveal biphasic binding kinetics between BsSco and Cu(II). An initial species appears with absorbance centered at 382 nm at a copper concentration-dependent rate (2.9 x 10(4) M(-1) s(-1)). The initial species decays at a first-order rate (1.5 s(-1)) to the equilibrium form with a maximum at 352 nm. Formation of the BsSco-Cu(II) complex is accompanied by quenching of protein fluorescence. The copper concentration-dependent phase gives 70% of the total quenching, while the final 30% develops during the second phase of the absorbance change. The pH dependence of copper binding shows that the copper-dependent rate increases by 50-fold as the pH decreases from 8.5 to 5.5 with an apparent pK(a) of 6.7. The slower phase rate is independent of pH. Comparison of circular dichroism spectra between apo-BsSco and the BsSco-Cu(II) complex reveals a small change in the UV region consistent with a subtle conformational change upon copper binding. There is formation of a distinctive visible CD spectrum in the BsSco-Cu(II) complex. A model is presented in which the kinetic and thermodynamic stability of the BsSco-Cu(II) complex results from a two-step mechanism. Release of copper would be facilitated in the intermediate form of BsSco, and attaining such a low-Cu(II) affinity state may be important for BsSco's function in Cu(A) assembly.

Stability of oxidized, reduced and copper bound forms of Bacillus subtilis Sco.

Sco is an accessory protein required for assembly of the Cu(A) center of cytochrome c oxidase. Functions proposed for Sco include as a copper chaperone and as a thiol-disulfide exchange protein. Differential scanning calorimetry (DSC) is used here to assess the interaction between the Bacillis subtilis version of Sco (BsSco) and Cu(II). When BsSco binds Cu(II) its melting temperature increases by 23 degrees C, which corresponds to an equilibrium dissociation constant of 3.50 pM. In contrast BsSco exhibits a much weaker affinity for Cu(I) (K(D)=10 microM). BsSco-Cu(II) is stable over days indicating an extremely slow dissociation for BsSco-Cu(II). However, at high ionic strength in the presence of excess copper, BsSco-Cu(II) returns to its oxidized, disulfide-bonded state and loses its copper binding capacity with a half time of 100 s. DSC of BsSco at high ionic strength indicates an increase in stability of metal free, reduced BsSco combined with a small destabilization of BsSco-Cu(II). It is proposed that BsSco undergoes an ionic strength induced conformational change that promotes electron transfer from the thiol groups on BsSco to Cu(II) to effect copper release. Such a redox transformation could be an important aspect of the copper transfer role proposed for BsSco in Cu(A) assembly.

The STN beta-band profile in Parkinson's disease is stationary and shows prolonged attenuation after deep brain stimulation.

Producing accurate movements may rely on the functional independence of sensorimotor circuits within basal ganglia nuclei. In parkinsonism there is abnormal synchrony of electrical activity within these circuits that results in a loss of independence across motor channels. Local field potential (LFP) recordings reflect the summation of local electrical fields and an increase in LFP power reflects increased synchrony in local neuronal networks. We recorded LFPs from the subthalamic nucleus (STN) deep brain stimulation (DBS) lead in the operating room in 22 cases from 16 subjects with Parkinson's disease (PD) who were off medication. There was elevated LFP power at beta frequencies (13-35 Hz) at rest. The LFP spectral profile was consistent across several periods of rest that were separated by movement and/or DBS, and appeared to be a relatively stationary phenomenon. The spectral profile and frequencies of the beta-band peak(s) varied among subjects but were similar between the right and left STNs within certain individuals. These results suggest that the LFP spectrum at rest may characterize a "signature" rhythm for an individual with PD. Beta-band power was attenuated after intra-operative STN DBS (p<0.05). The attenuation lasted for 10 s after short periods (30 s) and for up to 50 s after longer periods (5 min) of DBS. The finding that longer periods of DBS attenuated beta power for a longer time suggests that there may be long-acting functional changes to networks in the STN in PD after chronic DBS.

Fluoroscopic registration and localization for image-guided cranial neurosurgical procedures: a feasibility study.

BACKGROUND/AIMS: Frameless image-guided neurosurgical techniques can achieve high degrees of accuracy when skull-implanted fiducials are used for registration. However, fiducial placement is invasive and uncomfortable for patients. Development of a noninvasive registration method for accurate image-guided functional neurosurgery such as deep brain stimulator placement would therefore be highly desirable. We performed an initial series of experiments using a commercially available fluoroscopic registration package to assess the feasibility of this approach for image-guided functional neurosurgery. We also evaluated the accuracy of landmark placement in the fluoroscopic images using the navigational capability of the software. METHODS: A fluoroscopic target was created by etching a hexagonal pattern of 1-mm diameter holes on a copper-clad board (0.0254-mm copper cladding on fiberglass). The target was then mounted in a plastic phantom skull, oriented in a mid-sagittal plane. Five implantable fiducial markers were screwed into the phantom in positions which approximated those commonly used clinically. 1.25-mm CT slices were obtained, uploaded to a Stealthstation neuronavigational system and were displayed using the Fluoromerge software package. Lateral and AP images were generated with 2 approximately orthogonal views of the phantom. Registration was carried out both fluoroscopically and using the implanted fiducials. Targets were localized using both methods and the localization errors recorded. RESULTS: Localization error was less than 1 mm using fiducial-based registration, and between 0.8 and 2.9 mm using fluoroscopic registration. Error varied depending on location within the volume of the phantom. CONCLUSION: Initial experiments show that fluoroscopic registration is feasible for the performance of frameless functional neurosurgical procedures, although accuracy is still insufficient. Intraoperative verification of lead location was also shown to be feasible in one case.

Direct mitochondrial dysfunction precedes reactive oxygen species production in amiodarone-induced toxicity in human peripheral lung epithelial HPL1A cells.

Amiodarone (AM), a drug used in the treatment of cardiac dysrrhythmias, can produce severe pulmonary adverse effects, including fibrosis. Although the pathogenesis of AM-induced pulmonary toxicity (AIPT) is not clearly understood, several hypotheses have been advanced, including increased inflammatory mediator release, mitochondrial dysfunction, and free-radical formation. The hypothesis that AM induces formation of reactive oxygen species (ROS) was tested in an in vitro model relevant for AIPT. Human peripheral lung epithelial HPL1A cells, as surrogates for target cells in AIPT, were susceptible to the toxicity of AM and N-desethylamiodarone (DEA), a major AM metabolite. Longer incubations (> or =6 h) of HPL1A cells with 100 microM AM significantly increased ROS formation. In contrast, shorter incubations (2 h) of HPL1A cells with AM resulted in mitochondrial dysfunction and cytoplasmic cytochrome c translocation. Preexposure of HPL1A cells to ubiquinone and alpha-tocopherol was more effective than that with Trolox C or 5,5-dimethylpyrolidine N-oxide (DMPO) at preventing AM cytotoxicity. These data suggest that mitochondrial dysfunction, rather than ROS overproduction, represents an early event in AM-induced toxicity in peripheral lung epithelial cells that may be relevant for triggering AIPT, and antioxidants that target mitochondria may potentially have beneficial effects in AIPT.

Aryl radical involvement in amiodarone-induced pulmonary toxicity: investigation of protection by spin-trapping nitrones.

Amiodarone (AM), an antidysrrhythmic drug, can produce serious adverse effects, including potentially fatal AM-induced pulmonary toxicity (AIPT). AM-induced cytotoxicity and pulmonary fibrosis are well recognized, but poorly understood mechanistically. The hypothesis of aryl radical involvement in AM toxicity was tested in non-biological and biological systems. Photolysis of anaerobic aqueous solutions of AM, or N-desethylamiodarone (DEA) resulted in the formation of an aryl radical, as determined by spin-trapping and electron paramagnetic resonance (EPR) spectroscopy experiments. The non-iodinated AM analogue, didesiodoamiodarone (DDIA), did not form aryl radicals under identical conditions. The toxic susceptibility of human lung epithelioid HPL1A cells to AM, DEA, and DDIA showed time- and concentration-dependence. DEA had a more rapid and potent toxic effect (LC(50)=8 microM) than AM (LC(50)=146 microM), whereas DDIA cytotoxicity was intermediate (LC(50)=26 microM) suggesting a minor contribution of the iodine atoms. Incubation of human lung epithelial cells with the spin-trapping nitrones alpha-phenyl-N-t-butylnitrone (PBN, 10 mM) or alpha-(4-pyridyl N-oxide)-N-t-butylnitrone (POBN, 5.0 mM) did not significantly protect against AM, DEA, or DDIA cytotoxicity. Intratracheal administration of AM to hamsters produced pulmonary fibrosis at day 21, which was not prevented by 4 days of treatment with 150 mg/kg/day PBN or 164 mg/kg/day POBN. However, the body weight loss in AM-treated animals was counteracted by PBN. These results suggest that, although AM can generate an aryl radical photochemically, its in vivo formation may not be a major contributor to AM toxicity, and that spin-trapping reagents do not halt the onset of AM toxicity.

Formamide probes a role for water in the catalytic cycle of cytochrome c oxidase.

Formamide is a slow-onset inhibitor of mitochondrial cytochrome c oxidase that is proposed to act by blocking water movement through the protein. In the presence of formamide the redox level of mitochondrial cytochrome c oxidase evolves over the steady state as the apparent electron transfer rate from cytochrome a to cytochrome a(3) slows. At maximal inhibition cytochrome a and cytochrome c are fully reduced, whereas cytochrome a(3) and Cu(B) remain fully oxidized consistent with the idea that formamide interferes with electron transfer between cytochrome a and the oxygen reaction site. However, transient kinetic studies show that intrinsic rates of electron transfer are unchanged in the formamide-inhibited enzyme. Formamide inhibition is demonstrated for another member of the heme-oxidase family, cytochrome c oxidase from Bacillus subtilis, but the onset of inhibition is much quicker than for mitochondrial oxidase. If formamide inhibition arises from a steric blockade of water exchange during catalysis then water exchange in the smaller bacterial oxidase is more open. Subunit III removal from the mitochondrial oxidase hastens the onset of formamide inhibition suggesting a role for subunit III in controlling water exchange during the cytochrome c oxidase reaction.