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.

The Keyes to Reclaiming the Racial History Of the Roberts Court

This article advocates for a fundamental re-understanding about the way that the history of race is understood by the current Supreme Court. Represented by the racial rights opinions of Justice John Roberts that celebrate racial progress, the Supreme Court has equivocated and rendered obsolete the historical experiences of people of color in the United States. This jurisprudence has in turn reified the notion of color-blindness, consigning racial discrimination to a distant and discredited past that has little bearing to how race and inequality is experienced today. The racial history of the Roberts Court is centrally informed by the context and circumstances surrounding Brown v. Board of Education. For the Court, Brown symbolizes all that is wrong with the history of race in the United States - legal segregation, explicit racial discord, and vicious and random acts of violence. Though Roberts Court opinions suggest that some of those vestiges still exits, the bulk of its jurisprudence indicate the opposite. With Brown’s basic factual premises as its point of reference, the Court has consistently argued that the nation has made tremendous strides away from the condition of racial bigotry, intolerance, and inequity. The article accordingly argues that the Roberts Court reliance on Brown to understand racial progress is anachronistic. Especially as the nation’s focus for racial inequality turned national in scope, the same binaries in Brown that had long served to explain the history of race relations in the United States (such as Black-White, North-South, and Urban-Rural) were giving way to massive multicultural demographic and geographic transformations in the United States in the years and decades after World War II. All of the familiar tropes so clear in Brown and its progeny could no longer fully describe the current reality of shifting and transforming patterns of race relations in the United States. In order to reclaim the history of race from the Roberts Court, the article assesses a case that more accurately symbolizes the recent history and current status of race relations today: Keyes v. School District No. 1. This was the first Supreme Court case to confront how the binaries of cases like Brown proved of little probative value in addressing how and in what ways race and racial discrimination was changing in the United States. Thus, understanding Keyesand the history it reflects reveals much about how and in what ways the Roberts Court should rethink its conclusions regarding the history of race relations in the United States for the last 60 years.