Long-Time Oxygen Localization in Electron Transfer Flavoprotein

Abstract

Reactive oxygen species (ROS) exert a wide range of biological effects from beneficial regulatory function to deleterious oxidative stress. The electron transfer flavoprotein (ETF) is ubiquitous to life and is associated with aerobic metabolism and ROS production due to its location in the mitochondria. Quantifying oxygen localization within the ETF complex is critical for understanding the potential for electron transfer and radical pair formation between flavin adenine dinucleotide (FAD) cofactor and superoxide during ROS formation. Our study employed all-atom molecular dynamics simulations and identified several novel, long-lived oxygen binding sites within the ETF complex that appear near the FAD cofactor. Site locations, the local electrostatic environment, and characteristic oxygen binding times for each site were evaluated to establish factors that may lead to possible charge transfer reactions and superoxide formation within the ETF complex. The study revealed that some oxygen binding sites are naturally linked to protein domain features, suggesting opportunities to engineer and control ROS production and subsequent dynamics.

Long-Time Oxygen and Superoxide Localization in Arabidopsis thaliana Cryptochrome

“…In contrast to the case for ETF, , the values of the RDFs for both the low- and high-oxygen-concentration simulations are in close agreement, except for the peak arising at 0.49 nm. Black crosses and red plus signs represent RDF values at particular distances and values from bootstrapped simulation data, generated by splitting all of the data into five equal subsets.…”

“…In contrast, site 381M, identified below as the longest-lived binding site, contains a single, compact domain. In the case of O 2 binding in the ETF, the most stable binding sites were identified as single, compact volumes, while the least stable site was composed of several distinct locations . Here, the term compact does not indicate size but rather refers to the shape of the site and indicates that it has a large volume-to-surface-area ratio, indicating single, sphere-like volumes.…”

“…In order to relax and equilibrate the simulated systems, a protocol similar to the one used previously was employed. , An initial 4 ns simulation was performed with all protein and cofactor atom positions fixed in order to relax the water molecules and ions. A subsequent 6 ns simulation was performed with the protein backbone atom positions harmonically restrained, followed by a 2 ns simulation with no atom positions harmonically restrained.…”

“…Today, MD simulations can routinely model microsecond time scales and quantify processes on the millisecond time scale. For small molecules like oxygen, even significantly shorter time scales on the order of 10–100 ns are often sufficient to identify and characterize potential binding sites. ,,− Hence, MD is also well-suited for characterizing protein–oxygen interactions.…”

“…Given that protein conformational dynamics often occur on time scales longer than those achievable in MD simulations, it is helpful to outline the time scales and rates relevant to ROS production. Previous studies of electron-transfer flavoprotein (ETF) found characteristic oxygen unbinding times on the order of 1–40 ns, times readily achieved by MD. , Comparisons between At CRY1 and ETF are particularly useful, as both are known to produce superoxide, likely as a result of electron transfer from the fully reduced FAD cofactor. Estimates of superoxide production rates show that for electron-transfer rates significantly lower than the inverse unbinding time, the superoxide production rate is proportional to the unbinding time itself .…”