Electron Transfer Studies Of Bacterial Cytochrome And Quinol Oxidases Using Time-Resolved Optical Absorption Spectroscopy
- Author(s): Kittredge, Clive Tristan;
- Advisor(s): Einarsdottir, Olof;
- et al.
Insight into the mechanism of intramolecular electron transfer and ligand access in the heme-copper oxidases during O2 reduction is fundamental to understanding how the protein environment modulates the functions of these enzymes. To address this issue, several projects were pursued. The reactions of O2 and NO with fully reduced E. coli bo3 ubiquinol oxidase were investigated using photolabile O2 and NO carriers and the CO flow-flash method. The O2/NO binding step was found to be 3.9 x 107 M-1s-1 in both the absence and presence of CO. The O2 binding was followed by the formation of an oxyferryl F intermediate, and not the P intermediate observed in the aa3 oxidases. Mutation of the two tryptophan "constriction" residues in the ligand channel of bo3 to corresponding smaller residues in Thermus thermophilus ba3 did not affect the O2/NO binding, suggesting that the ligand pathway to the active site in bo3 is different than in the aa3 and ba3 oxidases.
The CO flash-photolysis and recombination dynamics of the fully reduced Rhodobacter sphaeroides (Rs) cbb3 holoenzyme and truncated enzyme (without subunit CcoP, which reportedly contains one of the c-type hemes capable of CO binding) in the presence and absence of partially solubilized soybean lipids was investigated. CO rebinding to both heme b3 and a c-type heme was observed for both the truncated enzyme and holoenzyme without lipids. This suggests more complex CO binding dynamics in cbb3 than previously reported, with CO possibly binding to c-type hemes in the two subunits in addition to the b3-type heme. Preliminary second-order rate constant of ~2 x 107 M-1 s-1 was observed for NO and O2 binding in cbb3.
In an intramolecular electron transfer study, a positively charged dye, 1-thiouredopyrene-3,6,8-trisulfonyl dimethylethylenediamine (DTUPS), was synthesized and characterized for the direct attachment to a single cysteine Rs aa3 mutant. No intramolecular electron transfer was observed, which was attributed to the low photolytic yield of the photolabile dye. A proton-coupled electron transfer study of a biomimetic tyrosine-containing peptide showed that the lifetime of the tyrosyl radical generated upon UV photolysis was significantly affected by its interaction with a cross-strand histidine.