Fully activated structure of the sterol-bound Smoothened GPCR-Gi protein complex.

Smoothened (SMO) is an oncoprotein and signal transducer in the Hedgehog signaling pathway that regulates cellular differentiation and embryogenesis. As a member of the Frizzled (Class F) family of G protein-coupled receptors (GPCRs), SMO biochemically and functionally interacts with Gi family proteins. However, key molecular features of fully activated, G protein-coupled SMO remain elusive. We present the atomistic structure of activated human SMO complexed with the heterotrimeric Gi protein and two sterol ligands, equilibrated at 310 K in a full lipid bilayer at physiological salt concentration and pH. In contrast to previous experimental structures, our equilibrated SMO complex exhibits complete breaking of the pi-cation interaction between R4516.32 and W5357.55, a hallmark of Class F receptor activation. The Gi protein couples to SMO at seven strong anchor points similar to those in Class A GPCRs: intracellular loop 1, intracellular loop 2, transmembrane helix 6, and helix 8. On the path to full activation, we find that the extracellular cysteine-rich domain (CRD) undergoes a dramatic tilt, following a trajectory suggested by positions of the CRD in active and inactive experimental SMO structures. Strikingly, a sterol ligand bound to a shallow transmembrane domain (TMD) site in the initial structure migrates to a deep TMD pocket found exclusively in activator-bound SMO complexes. Thus, our results indicate that SMO interacts with Gi prior to full activation to break the molecular lock, form anchors with Gi subunits, tilt the CRD, and facilitate migration of a sterol ligand in the TMD to an activated position.

Structure and Molecular Mechanism of Signaling for the Glucagon-like Peptide-1 Receptor Bound to Gs Protein and Exendin-P5 Biased Agonist.

The glucagon-like peptide-1 receptor (GLP-1R) is a key regulator of blood glucose and a prime target for the treatment of type II diabetes and obesity with multiple public drugs. Here we present a comprehensive computational analysis of the interactions of the activated GLP-1R-Gs signaling complex with a G protein biased agonist, Exendin P5 (ExP5), which possesses a unique N-terminal sequence responsible for the signal bias. Using a refined all-atom model of the ExP5-GLP-1R-Gs complex in molecular dynamics (MD) simulations, we propose a novel mechanism of conformation transduction in which the unique interaction network of ExP5 N-terminus propagates the binding signal across an array of conserved residues at the transmembrane domain to enhance Gs protein coupling at the cytoplasmic end of the receptor. Our simulations reveal previously unobserved interactions important for activation by ExP5 toward GDP-GTP signaling, providing new insights into the mechanism of class B G protein-coupled receptor (GPCR) signaling. These findings offer a framework for the structure-based design of more effective therapeutics.

Use of right orbitofrontal repetitive transcranial magnetic stimulation (rTMS) augmentation for treatment-refractory obsessive-compulsive disorder with comorbid major depressive disorder.

We examined the safety and efficacy of repetitive Transcranial Magnetic Stimulation (rTMS) of the right orbitofrontal cortex (OFC) in patients with refractory obsessive-compulsive disorder (OCD) and comorbid Major Depressive Disorder. All participants (n = 26) received excitatory stimulation of the left dorsolateral prefrontal cortex followed by inhibitory stimulation of bilateral supplementary motor area for 10 sessions. In 18 patients with poor early OCD response, treatment was augmented with OFC inhibitory stimulation after the tenth treatment session. Augmentation with OFC stimulation was well-tolerated, and associated with further alleviation of both OCD and depression symptoms, particularly in individuals with more severe illnesses.

Full-Genome Sequences of Two Newcastle Disease Virus Strains Isolated in West Java, Indonesia.

The full-genome sequences of strains chicken/Indonesia/Cilebut/010WJ/2015 and chicken/Indonesia/ITA/012WJ/1951, isolated in West Java, Indonesia, in 2015 and 1951, respectively, were examined. Chicken/Indonesia/Cilebut/010WJ/2015 (genotype VII) caused a 2015 disease outbreak in Indonesia, and chicken/Indonesia/ITA/012WJ/1951 (genotype VI) is used as a standard strain for challenge in Newcastle disease virus (NDV) vaccine trials.

A simple laboratory algorithm for diagnosis of melioidosis in resource-constrained areas: a study from north-central Vietnam.

OBJECTIVES: Melioidosis may be endemic in many tropical developing countries, but diagnosis of the disease is currently unreliable in resource-limited areas. We aimed to validate a simple and cheap laboratory algorithm for the identification of Burkholderia pseudomallei from clinical specimens in parts of Vietnam where the disease has not previously been reported. METHODS: In June 2015, we conducted training courses at five general hospitals in north-central provinces in order to raise awareness of the disease and to introduce a simple and cheap laboratory identification algorithm for B. pseudomallei including the three-antibiotic disc test. RESULTS: Until the end of the year (7 months later), 94 suspected B. pseudomallei strains resistant to gentamicin and colistin but sensitive to amoxicillin/clavulanic acid were detected in clinical specimens from 70 patients. All strains were further confirmed as B. pseudomallei by using a specific TTSS1 real-time PCR assay and recA sequencing analysis. Among positive blood cultures, positive rates with B. pseudomallei ranged from 3.4% (5/147) to 10.2% (32/312) in the various clinics. A total of 82.8% (58/70) patients were bacteraemic, with a mortality of 50% (18/36) among patients with known outcome. No death occurred in nonbacteraemic patients. CONCLUSIONS: Our results demonstrate that the introduction of a simple and easy-to-perform laboratory algorithm for the identification of B. pseudomallei from clinical samples, together with clinical awareness raising, can lead to the diagnosis of a significant number of melioidosis cases in resource-limited clinical laboratories which previously did not identify the pathogen.

Intimate partner violence education for medical students in the USA, Vietnam and China.

OBJECTIVES: While intimate partner violence (IPV) is a global concern for women's health, there are few comparative studies of IPV training in medical schools. The aim of this study was to investigate medical students' knowledge of, and training in, IPV in the USA, Vietnam and China. STUDY DESIGN: Cross-national, cross-sectional study. METHODS: US (n = 60), Vietnamese (n = 232) and Chinese (n = 174) medical students participated in a cross-sectional self-administered survey that included demographic characteristics; opinions, training and knowledge regarding IPV against women; and personal experience with IPV victims. RESULTS: Attitudes, knowledge and training about IPV among medical students varied between the three countries. US participants reported higher levels of knowledge of IPV, were more likely to believe that IPV was a serious problem, and were more likely to consider IPV to be a healthcare problem compared with Vietnamese and Chinese participants. Chinese participants, in particular, did not appear to appreciate the importance of addressing IPV. Differences were found between the Vietnamese and Chinese students. CONCLUSIONS: While most medical schools in the USA include IPV training within their core medical curricula, education throughout medical school seems to be necessary to improve medical education regarding treatment of patients with a history of IPV. Vietnamese and Chinese medical schools should consider including IPV education in the training of their future physicians to improve the health of women who have experienced IPV. Practical opportunities for medical students to interact with women who have experienced IPV are essential to develop effective IPV education.

The vascular niche: home for normal and malignant hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are uniquely capable of self-renewal and provision of all of the mature elements of the blood and immune system throughout the lifetime of an individual. HSC self-renewal is regulated by both intrinsic mechanisms and extrinsic signals mediated via specialized microenvironments or 'niches' wherein HSCs reside. HSCs have been shown to reside in close association with bone marrow (BM) osteoblasts in the endosteal niche and also in proximity to BM sinusoidal vessels. An unresolved question surrounds whether the endosteal and vascular niches provide synchronous or redundant regulation of HSC fate or whether these niches provide wholly unique regulatory functions. Furthermore, while some aspects of the mechanisms through which osteoblasts regulate HSC fate have been defined, the mechanisms through which the vascular niche regulates HSC fate remain obscure. Here, we summarize the anatomic and functional basis supporting the concept of an HSC vascular niche as well as the precise function of endothelial cells, perivascular cells and stromal cells within the niche in regulating HSC fate. Lastly, we will highlight the role of the vascular niche in regulating leukemic stem cell fate in vivo.

Two types of recombination hotspots in bacteriophage T4: one requires DNA damage and a replication origin and the other does not.

Recombination hotspots have previously been discovered in bacteriophage T4 by two different approaches, marker rescue recombination from heavily damaged phage genomes and recombination during co-infection by two undamaged phage genomes. The phage replication origin ori(34) is located in a region that has a hotspot in both assays. To determine the relationship between the origin and the two kinds of hotspots, we generated phage carrying point mutations that should inactivate ori(34) but not affect the gene 34 reading frame (within which ori(34) is located). The mutations eliminated the function of the origin, as judged by both autonomous replication of plasmids during T4 infection and two-dimensional gel analysis of phage genomic replication intermediates. As expected from past studies, the ori(34) mutations also eliminated the hotspot for marker rescue recombination from UV-irradiated genomes. However, the origin mutations had no effect on the recombination hotspot that is observed with co-infecting undamaged phage genomes, demonstrating that some DNA sequence other than the origin is responsible for inflated recombination between undamaged genomes. The hotspots for marker rescue recombination may result from a replication fork restart process that acts upon origin-initiated replication forks that become blocked at nearby DNA damage. The two-dimensional gel analysis also revealed phage T4 replication intermediates not previously detected by this method, including origin theta forms.

Non-kramers ENDOR and ESEEM of the S = 2 ferrous ion of [Fe(II)EDTA](2-).

We report here the first non-Kramers (NK) ESEEM and ENDOR study of a mononuclear NK center, presenting extensive parallel-mode ESEEM and ENDOR measurements on the S(t) = 2 ferrous center of [Fe(II)ethylenediamine-N,N,N',N'-tetraacetato](2-); [Fe(II)EDTA)](2-). The results disclose an anomalous equivalence of the experimental patterns produced by the two techniques. A simple theoretical treatment of the frequency-domain patterns expected for NK-ESEEM and NK-ENDOR rationalizes this correspondence and further suggests that the very observation of NK-ENDOR is the result of an unprecedentedly large hyperfine enhancement effect. The mixed nitrogen-carboxylato oxygen coordination of [Fe(II)EDTA](2-) models that of the protein-bound diiron centers, although with a higher coordination number. Analysis of the NK-ESEEM measurements yields the quadrupole parameters for the (14)N ligands of [Fe(II)EDTA](2-), K = 1.16(1) MHz, 0

Effects of ACEA-1328, a NMDA receptor/glycine site antagonist, on U50,488H-induced antinociception and tolerance.

Previously, we have shown that inhibition of the glycine site associated with the N-methyl-D-aspartate (NMDA) receptor is another viable approach to blocking morphine tolerance. In the present study, we sought to investigate the involvement of the NMDA receptor/glycine site in kappa-opioid receptor-mediated antinociception and tolerance in CD-1 mice. In antinociception studies, mice were injected with 5-nitro-6,7-dimethyl-1,4-dihydro-2, 3-quinoxalinedione (ACEA-1328), a systemically bioavailable NMDA receptor/glycine site antagonist, or the vehicle (Bis-Tris, 0.2 M) and then immediately with trans-(+/-)-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid e methanesulfonate (U50,488H), a kappa-opioid receptor agonist. Thirty minutes later, mice were tested for changes in nociceptive responses in the tail flick assay. ACEA-1328, per se, prolonged tail flick latencies with an ED(50) of approximately 50 mg/kg. Concurrent administration of ACEA-1328, at doses that did not produce antinociception, with U50,488H increased the potency of U50,488H in a dose-dependent manner. In tolerance studies, mice were treated, either once a day for 9 days or twice daily for 4 days, with the vehicle or ACEA-1328. Immediately after the initial injection, mice then received an injection of saline or U50,488H. On the test day, mice were injected with U50,488H alone and tested for antinociception 30 min later. Chronic treatment with U50,488H by either method produced tolerance. Unlike the acute effect of the drug, chronic treatment with ACEA-1328 decreased the antinociceptive potency of U50,488H. Taken together, the data suggest that acute and chronic administration of ACEA-1328 differentially affected the antinociceptive effect of U50,488H. Furthermore, the decreased in the potency of U50,488H induced by chronic treatment with ACEA-1328 also confounded the interpretation of the tolerance data.

ACEA-1328, a NMDA receptor/glycine site antagonist, acutely potentiates antinociception and chronically attenuates tolerance induced by morphine.

The effect of ACEA-1328, a competitive and systemically bioavailable NMDA receptor/glycine site antagonist, was studied on morphine-induced antinociception and tolerance in CD-1 mice using the tail flick test. To study the effect of acute administration of ACEA-1328 on morphine-induced antinociception, mice were injected with either ACEA-1328 (1, 5, and 10 mg kg(-1)) or Bis-Tris (0.2 m) immediately followed by an injection of morphine and tested for antinociception 30 min later. ACEA-1328 significantly increased the antinociceptive potency of morphine. To study the effect of chronic administration of ACEA-1328 on morphine-induced antinociception and tolerance, mice were treated, either once per day for 9 days or twice daily for 4 days, with ACEA-1328 or with the vehicle. Mice were then, within 1 min, injected daily with either morphine or saline. On the day of the test, mice were injected with only morphine and tested for antinociception 30 min later. In comparison to the acute effect of ACEA-1328, chronic treatment with the NMDA receptor/glycine site antagonist did not affect the antinociceptive potency of morphine. Chronic treatment with morphine, by both methods, produced a significant degree of tolerance. Concurrent administration of ACEA-1328 with the opioid analgesic completely blocked morphine tolerance. Our results demonstrate that acute, but not chronic, treatment with ACEA-1328 increased the antinociceptive potency of morphine. Furthermore, co-administration of the NMDA receptor antagonist with morphine abolished the development of tolerance. Overall, the data support a growing body of evidence showing that activation of the NMDA receptor plays a functional role in opioid-induced antinociception and tolerance.

General analysis of (14)N (I = 1) electron spin echo envelope modulation.

The analysis methods described to date for (14)N electron spin echo envelope modulation (ESEEM) mostly deal with isotropic g- and (14)N hyperfine coupling tensors. However, many cases of rhombic tensors are encountered. In the present report we present general equations for analyzing orientation-selective ESEEM and illustrate their use. (i) We present general equations for the nuclear interactions in an electron spin system where the EPR signal arises from an isolated Kramers doublet, then give the nuclear (electron-nuclear double resonance) frequencies for I = 1 associated with such a system. (ii) These are incorporated into equations for single-crystal ESEEM amplitudes, which in turn are incorporated into general equations for the orientation-selective ESEEM that arises when the EPR envelope of a frozen-solution (powder) sample is determined by g anisotropy. (iii) This development is first used in the simplest limit of an isotropic g-tensor and leads to a more general picture of the response of the I = 1 modulation amplitude to variations in the nuclear hyperfine and quadrupole coupling constants, relative to the nuclear Zeeman interaction, than had been presented previously. We find that strong modulation occurs not only in the well-known regime where the "exact/near cancellation" condition (A/2 approximately nu(N)) is satisfied, but also when the nuclear hyperfine interaction is much larger than the nuclear Zeeman interaction (A/nu(N) > 3) with A/K = 4 approximately 5. (iv) We then describe the orientation-selective (14)N ESEEM frequency-domain patterns (g vs frequency) in the presence of anisotropic (rhombic) hyperfine and electron Zeeman interactions for both coaxial and noncoaxial cases. We derive analytical solutions when the g-, hyperfine, and nuclear quadrupole tensors are coaxial. (v) The method is applied to the ESEEM of the nitrogenase MoFe protein (Av1) to determine the full hyperfine and nuclear quadrupole tensors of (14)N nuclei interacting with the S = 32 FeMo-cofactor (Fe(7)S(8)Mo: homocitrate).

Changing patterns of drug use among US military recruits before and after enlistment.

OBJECTIVES: The US armed forces adopted "zero tolerance" policies concerning illicit drug use in 1980 and later developed policies to discourage tobacco and alcohol abuse. This article examines drug use among young active-duty recruits both before and after enlistment, compared with nonmilitary age-mates, and documents historical shifts in such drug use across 2 decades. METHODS: Analyses employed longitudinal panel data from 20 nationally representative samples of high school seniors (cohorts of 1976-1995), each surveyed just before graduation and again within 2 years. Separate analyses for men (n = 12,082) and women (n = 15,345) contrasted those who entered military service, college, and civilian employment. RESULTS: Illicit drug use declined more among young military recruits than among their civilian counterparts. Analyses of male recruits at multiple time periods showed (1) declines in the prevalence of marijuana use and cocaine use after the initiation of routine military drug testing and (2) lower proportions of smokers of half a pack or more of cigarettes per day who entered service after the initiation of tobacco bans during basic training. CONCLUSIONS: Recent military drug policies appear to deter illicit drug use among enlistees and discourage some smokers from enlisting.

ACEA-1328, an NMDA receptor antagonist, increases the potency of morphine and U50,488H in the tail flick test in mice.

The effect of 5-nitro-6,7-dimethyl-1,4-dihydro-2,3-quinoxalinedione (ACEA-1328), a competitive and systemically bioavailable NMDA receptor/glycine site antagonist, was examined on opioid-induced antinociception in the tail flick test. Swiss Webster mice were injected with ACEA-1328 either alone or in combination with morphine or (+/-)-trans-U-50488 methanesulfonate (U50,488H), a mu- and a kappa-opioid receptor agonist, respectively, and tested for antinociception. Systemic administration of ACEA-1328 alone increased the tail flick latencies with an ED50 of approximately 45 mg kg-1. Concurrent administration of ACEA-1328 with morphine, or U50,488H, at doses that did not affect tail flick latencies, potentiated the antinociceptive effect of the opioid analgesics and vice versa. Naloxone, an opioid receptor antagonist, while not modifying the effect of ACEA-1328, did block the augmentation, suggesting that opioid receptors might be involved in the latter effect. 5-Aza-7-chloro-4-hydroxy-3-(m-phenoxyphenyl)quinoline-2(1H)-one (ACEA-0762), a selective NMDA receptor/glycine site antagonist, also showed enhancement of the antinociceptive effect of morphine and U50,488H. However, concurrent administration of 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzol[f]quinoxaline (NBQX), a selective non-NMDA receptor antagonist, with morphine did not alter the antinociceptive potency of the opioid analgesic. Overall, the data suggest that ACEA-1328 may increase the potency of the opioid analgesics by antagonising the glycine site associated with the NMDA receptor.

1H and (31)P NMR and EPR of Pentaammineruthenium(III) Complexes of Endocyclically Coordinated Nucleotides, Nucleosides, and Related Heterocyclic Bases. Autoxidation of [(Guokappa(N7))(NH(3))(5)Ru(III)] (Guo = Guanosine). Crystal Structure of [7MeGuakappa(N9)(NH(3))(5)Ru]Cl(3).3H(2)O.

The (1)H-NMR spectra of complexes involving the paramagnetic metal center [(NH(3))(5)Ru(III)] coordinated at ring nitrogens have been examined with pyridine, purine, nucleoside, and nucleotide ligands along with (31)P-NMR of the nucleotide complexes and EPR of representative complexes. Variations in the spectra have been investigated as a function of the coordination site and pH. Pseudocontact and contact shifts have been calculated for various protons, and an attempt has been made to correlate sugar conformations in coordinated 5'GMP, 5'IMP, Guo, and Ino with paramagnetically induced shifts. The compound [(7MeGuakappa(N9))(NH(3))(5)Ru]Cl(3).3H(2)O crystallizes in the orthorhombic space group Pna2(1) with cell parameters a = 25.375(4) Å, b = 11.803(4) Å, c = 6.958(2) Å, Z = 4, and R = 0.042. The autoxidation of [L(NH(3))(5)Ru(III)], where L = Guo, dGuo, and 1MeGuo, to the corresponding 8-oxo complexes under atmospheric oxygen is first order in the complex and [OH(-)]. For L = Guo, k = 6.6 x 10(-5) M(-1) s(-1), DeltaH = 58 kJ/mol, and DeltaS = -124 J/(mol K).

Does an SVC electrode further reduce DFT in a hot-can ICD system?

Pectorally implanted ICDs that defibrillate with the RV electrode and the ICD housing have gained clinical acceptance. However, it is still debatable whether adding an SVC electrode connected to the housing will further reduce the threshold of defibrillation (DFT). This study utilized eight pigs. DFTs were measured with a 50 V step-down protocol starting at 650 V (20 J). Shock strength for 50% success (E50) was estimated with the average of three reversals. In addition to alpha dummy device, Lead I (Pacesetter Models 1558 and 1538) or Lead II (Endotak 72) were used. Leads I are active fixation, true bipolar sensing with 5-cm shocking coils. Lead II has an integrated bipolar sensing with a 4.7-cm RV and 6.9-cm SVC shocking coils. A 95 microF defibrillation system was used to deliver a 44% tilt tuned biphasic 1.6/2.5 ms waveform, and to measure lead impedance. The RV electrode was the anode during phase I. With Lead IRV-->CAN the DFT was 531 +/- 75 V (13.6 +/- 3.8J) and the E50 was 496 +/- 89 (12 +/- 4.3 J). These were not significantly (NS) different than the DFT for RV-->CAN and SVC which was 518 +/- 84 V (13 +/- 4.2 J) or the E50 which was 476 +/- 84 V (11 +/- 3.9 J). Similar results were obtained with Lead II. Despite a decrease in lead impedance there was no apparent benefit from the addition of the SVC electrode. Lead I provided equivalent DFT performance to Lead II.

1H NMR, EPR, UV-Vis, and Electrochemical Studies of Imidazole Complexes of Ru(III). Crystal Structures of cis-[(Im)(2)(NH(3))(4)Ru(III)]Br(3) and [(1MeIm)(6)Ru(II)]Cl(2).2H(2)O.

Comparisons of the spectroscopic properties of a number of Ru(III) complexes of imidazole ligands provide methods of distinguishing between various types of bonding that can occur in proteins and nucleic acids. In particular, EPR and (1)H NMR parameters arising from the paramagnetism of Ru(III) should aid in determining binding sites of Ru(III) drugs in macromolecules. Electrochemical studies on several imidazole complexes of ruthenium suggest that imidazole may serve as a significant pi-acceptor ligand in the presence of anionic ligands. Crystal structures are reported on two active immunosuppressant complexes. cis-[(Im)(2)(NH(3))(4)Ru(III)]Br(3) crystallizes in the triclinic space group P&onemacr; (No. 2) with the cell parameters a = 8.961(2) Å, b = 12.677(3) Å, c = 7.630(2) Å, alpha = 98.03(2) degrees, beta = 100.68(2) degrees, gamma = 81.59(2) degrees, and Z = 2 (R = 0.044). [(1MeIm)(6)Ru(II)]Cl(2).2H(2)O crystallizes in the monoclinic space group P2(1)/n (No. 14) with the cell parameters a = 7.994(2) Å, b = 13.173(4) Å, c = 14.904(2) Å, beta = 97.89(1) degrees, and Z = 2 (R = 0.052). The average Ru(II)-N bond distance is 2.106(8) Å.

Active site structure of Rieske-type proteins: electron nuclear double resonance studies of isotopically labeled phthalate dioxygenase from Pseudomonas cepacia and Rieske protein from Rhodobacter capsulatus and molecular modeling studies of a Rieske center.

Continuous wave electron nuclear double resonance (CW ENDOR) spectra of [delta-15N,epsilon(-14)N]histidine-labeled phthalate dioxygenase (PDO) from Pseudomonas cepacia were recorded and found to be virtually identical to those previously recorded from [delta,epsilon-15N2]histidine-labeled protein [Gurbiel, R. J., Batie, C. J., Sivaraja, M., True, A. E., Fee, J. A., Hoffman, B. M., & Ballou, D. P. (1989) Biochemistry 28, 4861-4871]. Thus, the two histidine residues, previously shown to ligate one of the irons in the cluster [cf. Gurbiel et al. 1989)], both coordinate the metal at the N(delta) position of their imidazole rings. Pulsed ENDOR studies showed that the "remote", noncoordinating nitrogen of the histidine imidazole ring could be observed from the Rieske protein in a sample of Rhodobacter capsulatus cytochrome bc1 complex uniformly labeled with 15N but not in a sample of PDO labeled with [delta-15N,epsilon-14N]histidine, but this atom was easily observed with a sample of Rh. capsulatus cytochrome bc1 complex that had been uniformly labeled with 15N; this confirmed the conclusion from the CW ENDOR studies that ligation is exclusively via N(delta) for both ligands in the PDO center. Modifications in the algorithms previously used to simulate 14N ENDOR spectra permitted us to compute spectra without any constraints on the relative orientation of hyperfine and quadrupole tensors. This new algorithm was used to analyze current and previously published spectra, and slightly different values for the N-Fe-N angle and imidazole ring rotation angles are presented [cf. Gurbiel et al. (1989) Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., and Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. This analysis has permitted us to refine the proposed structure of the [2Fe-2S] Rieske-type cluster and rationalize some of the properties of these novel centers. Although the spectra of cytochrome bc1 complex from Rh. capsulatus are of somewhat lower resolution than those obtained with samples of PDO, our analysis nevertheless permits the conclusion that the geometry of the cluster is essentially the same for all Rieske and Rieske-type proteins. Structural constraints inferred from the spectroscopic results permitted us to apply the principles of distance geometry to arrive at possible three-dimensional models of the active site structure of Rieske protein from Rh. capsulatus. Results from this test case indicate that similar procedures should be generally useful in metalloprotein systems. We also recorded the pulsed and CW ENDOR spectra of 57Fe-labeled PDO, and the resulting data were used to derive the full hyperfine tensors for both Fe(III) and Fe(II) ions, including their orientations relative to the g tensor. The A tensor of the ferric ion is nominally isotropic, while the A tensor of the ferrous ion is axial, having A(parallel) > A(perpendicular); both tensors are coincident with the observed g tensor, with A(parallel) of the ferrous ion lying along the maximum g-value, g1. These results were examined using refinements of existing theories of spin-coupling in [2Fe-2S]+ clusters, and it is concluded that current theories are not adequate to fully describe the experimental results.

Multi-frequency EPR evidence for a binuclear CuA center in cytochrome c oxidase: studies with a 63Cu- and 65Cu-enriched, soluble domain of the cytochrome ba3 subunit II from Thermus thermophilus.

We have recorded multi-frequency EPR spectra of 63Cu- and 65Cu-labeled, water-soluble CuA-protein from the cytochrome ba3 of T. thermophilus. The spectrum taken at the highest frequency (34.03 GHz) shows no hyperfine structure and is nominally axial with apparent gz approximately 2.18 and gxy approximately 2.00. The spectrum taken at the lowest frequency (3.93 GHz) shows a rich hyperfine structure. Analyses of the spectra show that the observed splitting arises from an interaction of the unpaired electron with two Cu nuclei and support the notion that CuA is a mixed-valent [Cu(II)/Cu(I)] complex in which the unpaired electronic spin is distributed evenly over the two Cu ions.