title: A temperature-dependent conformational change of NADH oxidase from Thermus thermophilus HB8.
abstract: 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°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.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, Washington.
authors.forename: Eric D
authors.initials: ED
authors.lastname: Merkley
authors.forename: Valerie
authors.initials: V
authors.lastname: Daggett
authors.forename: William W
authors.initials: WW
authors.lastname: Parson
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D002942:Circular Dichroism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D008027:Light; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D016963:Thermus thermophilus / Q000201:enzymology*; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
chemical_list: D009097:Multienzyme Complexes; D005182:Flavin-Adenine Dinucleotide; D014443:Tyrosine; D014364:Tryptophan; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases
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