Respiratory Complex I in Bos taurus and Paracoccus denitrificans Pumps Four Protons across the Membrane for Every NADH Oxidized.

Respiratory complex I couples electron transfer between NADH and ubiquinone to proton translocation across an energy-transducing membrane to support the proton-motive force that drives ATP synthesis. The proton-pumping stoichiometry of complex I (i.e. the number of protons pumped for each two electrons transferred) underpins all mechanistic proposals. However, it remains controversial and has not been determined for any of the bacterial enzymes that are exploited as model systems for the mammalian enzyme. Here, we describe a simple method for determining the proton-pumping stoichiometry of complex I in inverted membrane vesicles under steady-state ADP-phosphorylating conditions. Our method exploits the rate of ATP synthesis, driven by oxidation of NADH or succinate with different sections of the respiratory chain engaged in catalysis as a proxy for the rate of proton translocation and determines the stoichiometry of complex I by reference to the known stoichiometries of complexes III and IV. Using vesicles prepared from mammalian mitochondria (from Bos taurus) and from the bacterium Paracoccus denitrificans, we show that four protons are pumped for every two electrons transferred in both cases. By confirming the four-proton stoichiometry for mammalian complex I and, for the first time, demonstrating the same value for a bacterial complex, we establish the utility of P. denitrificans complex I as a model system for the mammalian enzyme. P. denitrificans is the first system described in which mutagenesis in any complex I core subunit may be combined with quantitative proton-pumping measurements for mechanistic studies

Proton translocation in proteins

The active transport of protons across the low dielectric barrier imposed by biological membranes is accomplished by a plethora of proteins that span the ca. 40 Å of the phospholipid bilayer. The free energy derived from the proton electrochemical potential established by the translocation of these protons can subsequently be used to drive vital chemical reactions of the cell, such as ATP synthesis and cell locomotion. Membrane-bound proton translocating proteins have now been found for a variety of organisms and tissues (1). The driving force for proton pumping in these proteins is supplied by numerous mechanisms, including light absorption (e.g. bacteriorhodopsin) (2a,b), ligand binding (e.g. ATPase) (3), and electrochemistry (e.g. electron transfer through cytochrome c oxidase) (4). Thus nature has devised a variety of methods for supplying the energy required for proton pumping by these proteins. Such diversity notwithstanding, the proteins most likely share some common elements of structure and mechanism that allow them to function as proton pumps. A number of theoretical mechanisms have been put forth for both general proton translocation (5-7) and for energy coupling in specific proton pumps. However, despite almost three decades of intensive research, the details of the mechanism(s) and structural requirements for proton pumping remain largely unresolved. To some extent this is the result of the paucity of structural information available for integral membrane proteins. This situation may soon improve as a result of advances in protein methodologies that have allowed several integral membrane proteins to be successfully crystalized (8), and the increased use of genetic engineering to obtain recombinant proton translocating proteins that will offer an opportunity to assess the importance of specific amino acids for the proton translocation process (9)

Real-time observation of intramolecular proton transfer in the electronic ground state of chloromalonaldehyde: An ab initio study of time-resolved photoelectron spectra

The authors report on studies of time-resolved photoelectron spectra of intramolecular proton transfer in the ground state of chloromalonaldehyde, employing ab initio photoionization matrix elements and effective potential surfaces of reduced dimensionality, wherein the couplings of proton motion to the other molecular vibrational modes are embedded by averaging over classical trajectories. In the simulations, population is transferred from the vibrational ground state to vibrationally hot wave packets by pumping to an excited electronic state and dumping with a time-delayed pulse. These pump-dump-probe simulations demonstrate that the time-resolved photoelectron spectra track proton transfer in the electronic ground state well and, furthermore, that the geometry dependence of the matrix elements enhances the tracking compared with signals obtained with the Condon approximation. Photoelectron kinetic energy distributions arising from wave packets localized in different basins are also distinguishable and could be understood, as expected, on the basis of the strength of the optical couplings in different regions of the ground state potential surface and the Franck-Condon overlaps of the ground state wave packets with the vibrational eigenstates of the ion potential surface

Importance of charge capture in interphase regions during readout of charge-coupled devices

The current understanding of charge transfer dynamics in charge-coupled devices (CCDs) is that charge is moved so quickly from one phase to the next in a clocking sequence and with a density so low that trapping of charge in the interphase regions is negligible. However, simulation capabilities developed at the Centre for Electronic Imaging, which includes direct input of electron density simulations, have made it possible to investigate this assumption further. As part of the radiation testing campaign of the Euclid CCD273 devices, data have been obtained using the trap pumping method, a method that can be used to identify and characterize single defects within CCDs. Combining these data with simulations, we find that trapping during the transfer of charge among phases is indeed necessary to explain the results of the data analysis. This result could influence not only trap pumping theory and how trap pumping should be performed but also how a radiation-damaged CCD is readout in the most optimal way

Postirradiation behavior of p-channel charge-coupled devices irradiated at 153 K

The displacement damage hardness that can be achieved using p-channel charge-coupled devices (CCD) was originally demonstrated in 1997, and since then a number of other studies have demonstrated an improved tolerance to radiation-induced CTI when compared to n-channel CCDs. A number of recent studies have also shown that the temperature history of the device after the irradiation impacts the performance of the detector, linked to the mobility of defects at different temperatures. This study describes the initial results from an e2v technologies p-channel CCD204 irradiated at 153 K with a 10 MeV equivalent proton fluences of 1.24×109 and 1.24×1011 protons cm-2. The dark current, cosmetic quality and the number of defects identified using trap pumping immediately were monitored after the irradiation for a period of 150 hours with the device held at 153 K and then after different periods of time at room temperature. The device also exhibited a flatband voltage shift of around 30 mV / krad, determined by the reduction in full well capacity