Geminate carbon monoxide rebinding to a c-type haem

International audienceA chemically modified form of cytochrome c (cyt. c), termed carboxymethyl cytochrome c (cm cyt. c), possesses a vacant sixth coordination site to the haem iron that is available to bind external ligands. We present data on the rapid flash photolysis of CO from the ferrous haem iron of cm cyt. c and describe the kinetics and spectral transitions that accompany the recombination. This was achieved using 30-femtosecond laser pulses and a white light continuum to monitor spectral transitions. Whereas the photo-dissociation quantum yield is close to 1, the yield of CO escape from the protein (the apparent quantum yield, φ) relative to myoglobin (φ = 1) is small due to rapid geminate recombination of CO. On ligand photo-dissociation the haem undergoes a spin-state transition from low-spin ferrous CO bound to penta-coordinate high-spin. Subsequently the system reverts to the CO bound form. The data were fitted with a minimum number of exponentials using global analysis. Recombination of CO with the haem iron of cm cyt. c is multiphasic (τ = 16 ps, 120 ps and 1 ns), involving three spectrally distinct components. The fraction of haem (0.11) not recombining with CO within 4 ns is similar to the value of φ (0.12) measured on the same preparation by the "pulse method" (M. Brunori, G. Giacometti, E. Antonini and J. Wyman, Proc. Natl. Acad. Sci. USA, 1973, 70, 3141-3144, ). This implies that no further geminate recombination occurs at t > 4 ns. This unusually efficient CO-haem geminate recombination indicates the sterically hindered ("caged") nature of the distal haem pocket in cm cyt. c from which it is difficult for CO to escape. The large geminate phase may be contrasted with the behaviour of myoglobin in which geminate recombination is small. This is in general agreement with the well-documented extensive structural dynamics in myoglobin that allow ligand passage, and a higher structural rigidity in cyt. c imposed by the restraints of minimising reorganisation energy for electron transfer (M. Brunori, D. Bourgeois and D. Vallone, J. Struct. Biol., 2004, 147, 223-234, ). The high pH ferrous form of cm cyt. c is a low-spin species having a lysine bound to the central iron atom of the haem (M. Brunori, M. Wilson and E. Antonini, J. Biol. Chem., 1972, 247, 6076-6081; G. Silkstone, G. Stanway, P. Brzezinski and M. Wilson, Biophys. Chem., 2002, 98, 65-77, ). This high pH (pH 8) form of deoxy cm cyt. c undergoes photo-dissociation of lysine (although the proximal histidine is possible) after photo-excitation. Recombination occurs with a time constant (τ) of 7 ps. This is similar to that observed for the geminate rebinding of the Met80 residue in native ferrous cyt. c (τ 6 ps) following its photo-dissociation (S. Cianetti, M. Negrerie, M. Vos, J.-L. Martin and S. Kruglik, J. Am. Chem. Soc., 2004, 126, 13932-13933; W. Wang, X. Ye, A. Demidov, F. Rosca, T. Sjodin, W. Cao, M. Sheeran and P. Champion, J. Phys. Chem., 2000, 104, 10789-10801, )

Single-use duodenoscopes compared with reusable duodenoscopes in patients carrying multidrug-resistant microorganisms:a break-even cost analysis

Background and study aims:Single-use duodenoscopescan prevent transmission of microorganisms through contaminated reusable duodenoscopes. Concerns regardingtheir economic and environmental impact impede the transition to single-use duodenoscopes. This study investigatedthe costs associated with two scenarios in which single-useduodenoscopes are used in patients carrying multidrug-resistant microorganisms (MDROs).Methods:Break-even costs for single-use duodenoscopeswere calculated for two scenarios in which patients werescreened for MDRO carriage before undergoing endoscopicretrograde cholangiopancreatography (ERCP). Only directcosts related to the endoscopy were taken into consideration. In Scenario 1, patients were screened through microbiological culturing with a lag time in receiving the test result. In Scenario 2, screening was performed using GeneXpert analysis providing a rapid read-out. Calculations wereperformed using data from a Dutch tertiary care centerand also with US healthcare data.Results:In the Dutch situation, single-use duodenoscopesneeded to be priced at a maximum of € 140 to € 250 tobreak-even. In the US analyses, break-even costs variedwidely, depending on the duodenoscope-associated infection costs used, ERCP volume, and infection risk. Thebreak-even costs in Scenario 1 ranged between $78.21 and$2,747.54 and in Scenario 2, between $248.89 and$2,209.23.Conclusions:This study showed that a crossover scenarioin which single-use duodenoscopes are only used in patients carrying MDROs could be an economically viable alternative to a complete transition to single-use duodenoscopes. In the Dutch setting, single-use duodenoscopesneed to be priced much lower than in the United States toreach a per-procedure cost that is comparable with a scenario using reusable duodenoscopes exclusively

Ultrafast photochemistry of the bc₁ complex

We present a full investigation of ultrafast light-induced events in the membraneous cytochrome bc 1 complex by transient absorption spectroscopy. This energy-transducing complex harbors four redox-active components per monomer: heme c 1 , two 6-coordinate b-hemes and a [2Fe-2S] cluster. Using excitation of these components in different ratios under various excitation conditions, probing in the full visible range and under three well-defined redox conditions, we demonstrate that for all ferrous hemes of the complex photodissociation of axial ligands takes place and that they rebind in 5-7 ps, as in other 6-coordinate heme proteins, including cytoglobin, which is included as a reference in this study. By contrast, the signals are not consistent with photooxidation of the b hemes. This conclusion contrasts with a recent assessment based on a more limited data set. The binding kinetics of internal and external ligands are indicative of a rigid heme environment, consistent with the electron transfer function. We also report, for the first time, photoactivity of the very weakly absorbing iron-sulfur center. This yields the unexpected perspective of studying photochemistry, initiated by excitation of iron-sulfur clusters, in a range of protein complexes

Tipping elements in the human intestinal ecosystem

Data available at http://datadryad.org/resource/doi:10.5061/dryad.pk75dPeer reviewe

The circularly permuted globin domain of androglobin exhibits atypical heme stabilization and nitric oxide interaction

In the decade since the discovery of androglobin, a multi-domain hemoglobin of metazoans associated with ciliogenesis and spermatogenesis, there has been little advance in the knowledge of the biochemical and structural properties of this unusual member of the hemoglobin superfamily. Using a method for aligning remote homologues, coupled with molecular modelling and molecular dynamics, we have identified a novel structural alignment to other hemoglobins. This has led to the first stable recombinant expression and characterization of the circularly permuted globin domain. Exceptional for eukaryotic globins is that a tyrosine takes the place of the highly conserved phenylalanine in the CD1 position, a critical point in stabilizing the heme. A disulfide bond, similar to that found in neuroglobin, forms a closed loop around the heme pocket, taking the place of androglobin's missing CD loop and further supporting the heme pocket structure. Highly unusual in the globin superfamily is that the heme iron binds nitric oxide as a five-coordinate complex similar to other heme proteins that have nitric oxide storage functions. With rapid autoxidation and high nitrite reductase activity, the globin appears to be more tailored toward nitric oxide homeostasis or buffering. The use of our multi-template profile alignment method to yield the first biochemical characterisation of the circularly permuted globin domain of androglobin expands our knowledge of the fundamental functioning of this elusive protein and provides a pathway to better define the link between the biochemical traits of androglobin with proposed physiological functions

A High Cell-Bearing Capacity Multibore Hollow Fiber Device for Macroencapsulation of Islets of Langerhans

Macroencapsulation of islets of Langerhans is a promising strategy for transplantation of insulin-producing cells in the absence of immunosuppression to treat type 1 diabetes. Hollow fiber membranes are of interest there because they offer a large surface-to-volume ratio and can potentially be retrieved or refilled. However, current available fibers have limitations in exchange of nutrients, oxygen, and delivery of insulin potentially impacting graft survival. Here, multibore hollow fibers for islets encapsulation are designed and tested. They consist of seven bores and are prepared using nondegradable polymers with high mechanical stability and low cell adhesion properties. Human islets encapsulated there have a glucose induced insulin response (GIIS) similar to nonencapsulated islets. During 7 d of cell culture in vitro, the GIIS increases with graded doses of islets demonstrating the suitability of the microenvironment for islet survival. Moreover, first implantation studies in mice demonstrate device material biocompatibility with minimal tissue responses. Besides, formation of new blood vessels close to the implanted device is observed, an important requirement for maintaining islet viability and fast exchange of glucose and insulin. The results indicate that the developed fibers have high islet bearing capacity and can potentially be applied for a clinically applicable bioartificial pancreas

Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule

Monitoring Radiographic Brain Tumor Progression

Determining radiographic progression in primary malignant brain tumors has posed a significant challenge to the neuroncology community. Glioblastoma multiforme (GBM, WHO Grade IV) through its inherent heterogeneous enhancement, growth patterns, and irregular nature has been difficult to assess for progression. Our ability to detect tumor progression radiographically remains inadequate. Despite the advanced imaging techniques, detecting tumor progression continues to be a clinical challenge. Here we review the different criteria used to detect tumor progression, and highlight the inherent challenges with detection of progression