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.

The LCLS variable-energy hard X-ray single-shot spectrometer.

The engineering design, implementation, operation and performance of the new variable-energy hard X-ray single-shot spectrometer (HXSSS) for the LCLS free-electron laser (FEL) are reported. The HXSSS system is based on a cylindrically bent Si thin crystal for dispersing the incident polychromatic FEL beam. A spatially resolved detector system consisting of a Ce:YAG X-ray scintillator screen, an optical imaging system and a low-noise pixelated optical camera is used to record the spectrograph. The HXSSS provides single-shot spectrum measurements for users whose experiments depend critically on the knowledge of the self-amplified spontaneous emission FEL spectrum. It also helps accelerator physicists for the continuing studies and optimization of self-seeding, various improved mechanisms for lasing mechanisms, and FEL performance improvements. The designed operating energy range of the HXSSS is from 4 to 20 keV, with the spectral range of order larger than 2% and a spectral resolution of 2 × 10(-5) or better. Those performance goals have all been achieved during the commissioning of the HXSSS.

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.

Pulse energy measurement at the SXR instrument.

A gas monitor detector was implemented and characterized at the Soft X-ray Research (SXR) instrument to measure the average, absolute and pulse-resolved photon flux of the LCLS beam in the energy range between 280 and 2000 eV. The detector is placed after the monochromator and addresses the need to provide reliable absolute pulse energy as well as pulse-resolved measurements for the various experiments at this instrument. This detector provides a reliable non-invasive measurement for determining flux levels on the samples in the downstream experimental chamber and for optimizing signal levels of secondary detectors and for the essential need of data normalization. The design, integration into the instrument and operation are described, and examples of its performance are given.

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.

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.

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.

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.

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.

Recovery of Cronobacter sakazakii from environmental surface swabbing materials using a 5-h enrichment procedure.

This study aimed to reduce the time taken to detect low numbers of Cronobacter sakazakii inoculated onto environmental swabs (100, 10 or 1 cfu per swab) using a simple plating procedure for application in a dairy testing laboratory. Three types of environmental swabs (Biolab FlexiSwab, gauze swabs and the Whatman SwabCheck Polywipe sponge) were inoculated with either Cron. sakazakii in single culture or Cron. sakazakii together with Citrobacter freundii. A 5-h enrichment procedure of swabs in Cronobacter enrichment broth at 37 degrees C prior to plating was then compared with no enrichment or 24-h enrichment. The 5-h enrichment procedure was as efficient at detecting Cron. sakazakii on environmental swabs at low cell concentrations (100 cfu per swab), and in pure culture or in competition with other coliforms (Citrobacter), as pre-enrichment for 24 h. This protocol was also successful in detecting 10 cfu per swab 80% of the time. The results also indicated that the type of swab selected for use in environmental safety programmes is influential on the outcome, with the FlexiSwab and gauze swabs being the most efficient swabbing materials evaluated in this study.

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.

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.

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.

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).

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.

The potential of electricity transmission corridors in forested areas as bumblebee habitat.

Declines in pollinator abundance and diversity are not only a conservation issue, but also a threat to crop pollination. Maintained infrastructure corridors, such as those containing electricity transmission lines, are potentially important wild pollinator habitat. However, there is a lack of evidence comparing the abundance and diversity of wild pollinators in transmission corridors with other important pollinator habitats. We compared the diversity of a key pollinator group, bumblebees (Bombus spp.), between transmission corridors and the surrounding semi-natural and managed habitat types at 10 sites across Sweden's Uppland region. Our results show that transmission corridors have no impact on bumblebee diversity in the surrounding area. However, transmission corridors and other maintained habitats such as roadsides have a level of bumblebee abundance and diversity comparable to semi-natural grasslands and host species that are important for conservation and ecosystem service provision. Under the current management regime, transmission corridors already provide valuable bumblebee habitat, but given that host plant density is the main determinant of bumblebee abundance, these areas could potentially be enhanced by establishing and maintaining key host plants. We show that in northern temperate regions the maintenance of transmission corridors has the potential to contribute to bumblebee conservation and the ecosystem services they provide.

Intermediate forms of cytochrome oxidase observed in transient kinetic experiments and those visited in the catalytic cycle.

The cytochrome oxidase family of heme-copper oxidases has been the subject of intense kinetic and mechanistic enquiry. Much of this work has focussed on transient kinetic studies of the partial reactions of the enzyme with the goal being to build a kinetic model describing the catalytic cycle that the enzyme undergoes to direct the oxidation of substrate, reduction of oxygen and vectorial proton transfer. A key aspect of such a model is to define the structures of each of the intermediate forms the enzyme takes up as it traverses the catalytic cycle. One complication that has been prevalent with mitochondrial cytochrome c oxidase is the existence of structural variants of the enzyme, as isolated, that may not be participants in catalysis. Studies of structurally simpler procaryotic members of the family may offer new insight on the intermediates of catalysis. In this paper transient-state and steady-state kinetic studies of cytochrome aa(3)-600 from Bacillus subtilis are integrated into a model of the catalytic cycle. This model specifies that the P intermediate accumulates in the steady-state and it is proposed that the step following its formation is limited by proton uptake.

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.