Mechanistic Importance of Redox Potentials and Conformational Flexibility in Electron-Transferring Flavoproteins

The mitochondrial matrix flavoproteins electron transfer flavoprotein (ETF) and electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) are responsible for linking fatty acid β-oxidation with the main mitochondrial respiratory chain. Electrons derived from flavoprotein dehydrogenases are transferred sequentially through ETF and ETF-QO to ubiquinone and then into the respiratory chain via complex III. In this study, the effects of changes in ETF-QO redox potentials on its activity and the conformational flexibility of ETF were investigated. ETF-QO contains one [4Fe-4S]2+,1+ and one flavin adenine dinucleotide (FAD). In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. FAD redox potentials were measured by potentiometric titration probed by electron paramagnetic resonance (EPR) spectroscopy. The N338T and N338A mutations lowered the midpoint potentials, which resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF1e- catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone, but not in electron transfer from ETF to ETF-QO. Therefore it is proposed that the iron-sulfur cluster is the immediate acceptor from ETF. It has been proposed that the αII domain of ETF is mobile, allowing promiscuous interactions with structurally different partners. Double electron-electron resonance (DEER) was used to measure the distance between spin labels at various sites and an enzymatically reduced FAD cofactor in Paracoccus denitrificans ETF. Two or three interspin distance distributions were observed for spin-labels in the αI (A43C) and βIII (A111C) domains, but only one is observed for a label in the βII (A210C) domain. This suggests that the αII domain adopts several stable conformations which may correspond to a closed/inactive conformation and an open/active conformation. An additional mutation, E162A, was introduced to increase the mobility of the αII domain. The E162A mutation doubled the activity compared to wild-type and caused the distance distributions to become wider. The DEER method has the potential to characterize conformational changes in ETF that occur when it interacts with various redox partners.

Identification of geostationary satellites using polarization data from unresolved images

In order to protect critical military and commercial space assets, the United States Space Surveillance Network must have the ability to positively identify and characterize all space objects. Unfortunately, positive identification and characterization of space objects is a manual and labor intensive process today since even large telescopes cannot provide resolved images of most space objects. Since resolved images of geosynchronous satellites are not technically feasible with current technology, another method of distinguishing space objects was explored that exploits the polarization signature from unresolved images. The objective of this study was to collect and analyze visible-spectrum polarization data from unresolved images of geosynchronous satellites taken over various solar phase angles. Different collection geometries were used to evaluate the polarization contribution of solar arrays, thermal control materials, antennas, and the satellite bus as the solar phase angle changed. Since materials on space objects age due to the space environment, it was postulated that their polarization signature may change enough to allow discrimination of identical satellites launched at different times. The instrumentation used in this experiment was a United States Air Force Academy (USAFA) Department of Physics system that consists of a 20-inch Ritchey-Chrétien telescope and a dual focal plane optical train fed with a polarizing beam splitter. A rigorous calibration of the system was performed that included corrections for pixel bias, dark current, and response. Additionally, the two channel polarimeter was calibrated by experimentally determining the Mueller matrix for the system and relating image intensity at the two cameras to Stokes parameters S0 and S1. After the system calibration, polarization data was collected during three nights on eight geosynchronous satellites built by various manufacturers and launched several years apart. Three pairs of the eight satellites were identical buses to determine if identical buses could be correctly differentiated. When Stokes parameters were plotted against time and solar phase angle, the data indicates that there were distinguishing features in S0 (total intensity) and S1 (linear polarization) that may lead to positive identification or classification of each satellite.