A temperature-dependent conformational change of NADH oxidase from Thermus thermophilus HB8

作者：Merkley Eric D; Daggett Valerie; Parson William W^*

来源：Proteins: Structure, Function, and Genetics , 2012, 80(2): 546-555.

DOI：10.1002/prot.23219

• thermophiles
• thermophilic enzymes
• Thermus thermophilus
• flavoproteins
• NADH oxidase
• fluorescence
• fluorescence quenching
• electron transfer
• temperature effects
• molecular dynamics

Using molecular dynamics simulations and steady-state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). The enzyme's far-UV circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75 degrees C. However, the fluorescence of the tightly bound FAD cofactor increased approximately fourfold over this temperature range. This effect appears not to be due to aggregation, unfolding, cofactor dissociation, or changes in quaternary structure. We therefore attribute the change in flavin fluorescence to a temperature-dependent conformational change involving the NOX active site. Molecular dynamics simulations and the effects of mutating aromatic residues near the flavin suggest that the change in fluorescence results from a decrease in quenching by electron transfer from tyrosine 137 to the flavin. Proteins 2012.

• 出版日期2012-2