Monday, November 25, 2024
11/25/2024
Oxidoreductases use metallocofactors, organic molecules and four types of amino acids (tyrosine, tryptophan, cysteine and glycine) to perform electron transfer (ET) and proton-coupled electron transfer (PCET) reactions. The four amino acids serve as one-electron redox mediators in biocatalytic and multistep electron/hole transfer (radical transfer) processes some of which are essential to life on earth. There is also a sinister side to amino- acid ET/PCET since these reactions can be induced at oxidative stress conditions and cause significant cellular damage. It is extremely challenging to experimentally resolve the thermodynamic and kinetic redox properties of a single amino-acid residue. The highly oxidizing and reactive nature of amino-acid radicals increases the barrier for experimental characterization even further. Consequently, key information to support the understanding of one of nature's essential redox tools is missing. This research project is based on a family of well-structured model proteins with the unique capability of providing both reversible reduction potentials (E°'s) and detailed PCET kinetic/mechanistic information on tyrosine (Y) and tryptophan (W) oxidation- reduction reactions. The so-called a3X proteins are based on a three-helix bundle (a3) with an aromatic residue at interior position 32 (X32). The a3X system will be developed along three connected paths to gain novel information on the fundamental factors that govern Y/W multistep electron/hole transfers. (1) DE°'s and solution NMR structures obtained on the current a3X family will be analyzed using density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) methods. The goal is to obtain a robust computational protocol for quantitatively predicting DE°'s of Y and W residues. The computational protocol will be used to identify W analogs with significantly shifted potentials relative to the potential of native W. Selected analogs will be inserted at site 32 in a3W and their E°'s measured by protein film voltammetry. Iterative prediction/test cycles will refine and validate the computational protocol. (2) a3 proteins containing one (a3Y & a3W) and two (a3YX & a3WX, X = Y, W & analogs) redox-active aromatic residues and a tethered ruthenium (Ru) photosensitizer will be made. These proteins will be used to: (a) Determine the rate of radical formation in Ru-a3Y and Ru-a3W as a function of the PCET driving force to determine the reorganization energies associated with the Y32·/Y32 and W32·/W32 redox pairs. This is a fundamental parameter (l) in ET and PCET rate constant expressions and thus far unknown for protein Y·/Y and W·/W redox pairs. (b) Generate a comprehensive description of Y/W multistep electron/hole transfers based on rigorously determined structural (electron donor/acceptor & proton donor/acceptor spatial organization), thermodynamic (DE°' & DpKa), and kinetic (kPCET & l values) parameters. (3) Advanced NMR and EPR methods will be used to determine the hydration state of the X32 site in reduced and oxidized a3Y and a3W. Fast sidechain fluctuations essential for radical formation and infrequent larger-scale fluctuations controlling the radical decay will also be examined.
HHS’ Tracking Accountability in Government Grants System (TAGGS) website provides access to detailed descriptions of grants, loans, aggregated direct payments and other types of financial assistance awarded by the HHS Operating Divisions (OPDIVs) and the Office of the Secretary Staff Divisions (STAFFDIVs).
