Urea and temperature dependence of the physical and spectroscopic properties of the NADH oxidase from Thermus thermophilus HB8

This dissertation focuses on the properties of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). Thermophilic enzymes are active at the high temperatures where their host organisms thrive, but relatively inactive at more moderate temperatures. The "corresponding states" hypothesis attributes this difference to the greater conformational rigidity of thermophilic proteins. By means of molecular dynamics simulations, we have found that NOX and a related mesophilic protein (Escherichia coli nitroreductase, NTR) have similar flexibility on the nanosecond time scale. However, NOX maintains its native conformation better than NTR at high temperatures. NOX has previously been reported to experience a 2.5-fold enzymatic activation and a 50% decrease in intrinsic tryptophan fluorescence in the presence of ∼1 M urea. We were unable to reproduce these findings. Instead, we observe ∼1.7-fold increase of the enzymatic rate and a marginal decrease in fluorescence. We also found that the flavin fluorescence of NOX increases ∼4-fold from 25°C to 75°C. Mutagenesis studies suggest that the increase in flavin fluorescence with temperature may be due to dimished electron transfer from Y137 at high temperature. We have also carried out a molecular dynamics study of bovine pancreatic ribonuclease A, in which we describe a conformational change seen in the simulations that could represent the thermal pretransition.